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Flow cytometry analysis and sorting of chromosomes following hybridization with fluorescent probes that… Brind'Amour, Julie 2011

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FLOW CYTOMETRY ANALYSIS AND SORTING OF CHROMOSOMES FOLLOWING HYBRIDIZATION WITH FLUORESCENT PROBES THAT TARGET SPECIFIC DNA REPEAT SEQUENCES  by  Julie Brind’Amour  B.Sc., Université Laval, 2003 M.Sc., Université Laval, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate Studies  (Medical Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (VANCOUVER)   July 2011  © Julie Brind’Amour, 2011 ii    Abstract  Traditional cytogenetic approaches allow analysis of the chromosomal composition (karyotype) of mitotic cells fixed on slides cells by microscopy. The combination of karyotyping and Fluorescence In Situ Hybridization (FISH) enables the detection of specific target sequences on individual chromosomes. Disadvantages are that traditional cytogenetic approaches are very labor and time consuming and that chromosome specific information from only a few dozen cells has poor statistical power. An alternative is flow karyotyping, a method to analyze chromosomes in suspension by flow cytometry. For flow karyotyping, the DNA composition of specific chromosomes in suspension is measured based on the DNA-specific dyes Hoechst 33258 (HO) and Chromomycin A3 (CA3). My thesis work has focused on the development of a new method to analyze and sort chromosomes using FISH with labeled peptide nucleic acid (PNA) probes on chromosomes in suspension. I found that, following FISH, flow karyotyping can be used to detect and quantify repetitive DNA sequences within individual chromosomes.  Using chromosome flow FISH (CFF), chromosomes isolated from cells of various species were hybridized to PNA probes and analyzed by flow cytometry. CFF was used to detect a variety of repeats; interstitial telomeric sequences in Chinese Hamster chromosomes, major satellite in mouse chromosomes and D18Z1 alpha satellite repeats in human chromosomes. Quantitative measurements of repeat length by CFF were validated by comparison with measurements obtained using Q-FISH. We found that parental homologs of human chromosome 18 with different D18Z1 satellite repeat array size could be purified using CFF and Fluorescence Activated Cell Sorting (FACS). Illumina short read sequencing of libraries built from these purified chromosomes enabled us to determine, with a high resolution, the allelic phasing of each homolog over the entire chromosome 18. Finally, CFF was modified to study sister iii    chromatids separately. Using a cell model with inducible separation of sister chromatids, flow karyograms were generated. Using chromosome orientation FISH (CO-FISH) in suspension, we could identify sister chromatids according to the presence of DNA template strands. We anticipate that this approach will allow the purification of sister chromatids to study epigenetic differences between sister chromatids defined on the basis of DNA template strands.     iv    Preface   Chapter 2 is equivalent to the manuscript entitled “Analysis of repetitive DNA in chromosomes by flow cytometry”, accepted for publication in Nature Methods (March 29th, 2011). I designed all the experiments included in this manuscript with the help of my supervisor. I performed all the experiments, analyzed the data and wrote the manuscript with the support of my supervisor. Dr. Barbara Trask, Sandra Vanderbyl and Cam Smith advised us on chromosome preparation protocols and Elizabeth Chavez, on FISH procedures. Lindsey Marmolejo, Wenbo Xu and Gary de Jong from the Flow Core Facility at the TFL set up the instrument prior to analysis and sorting and advised us on flow cytometry.  In chapter 3, we are exploring CFF as a tool for chromosome and allele-specific sequencing. I executed all the chromosome purification experiments that were designed with the help of my supervisor. Libraries for Illumina short read sequencing were prepared either at the Genome Sciences Center (Vancouver) for the first section (chromosome 18 sequencing) or in collaboration with Ester Falconer, a postdoctoral fellow in the laboratory (second section, allele- specific chromosome 18 sequencing). Sequencing was also done at the Genome Sciences Center. The sequence analysis was done by myself with help from Mark Hills and Steven Poon, respectively a postdoctoral fellow and a research associate in the laboratory.  In chapter 4, we describe the method we are developing to purify sister chromatids on the basis of DNA template strands. For this project we are using a modification of CFF; chromosome orientation flow FISH. HT1080 cells with inducible (over-) expression of Separase-1 were cloned by my supervisor from B49.5 cells (Wendy Bickmore, Edinburg) using an FKBPCreERT2 construct (Patrick McDonel, Cambridge) and cDNA encoding a non-phosphorylatable S1126A mutant Separase-1 (Steven Taylor, Manchester) which was cloned in between lox sites of an expression vector. I performed the characterization of the clones and optimized the induction of v    chromatids and their isolation. Elizabeth Chavez, a technician in the laboratory, helped me with the development of protocols for CO-FISH on chromosomes and chromatids in suspension. vi    Table of contents  Abstract ....................................................................................................................................... ii Preface ........................................................................................................................................ iv Table of contents ....................................................................................................................... vi List of tables ............................................................................................................................... ix List of figures .............................................................................................................................. x List of abbreviations ................................................................................................................. xii Acknowledgements ................................................................................................................. xiv Dedication .................................................................................................................................. xv Chapter 1 General introduction ................................................................................................. 1 1.1 Cytogenetics ................................................................................................................... 2 1.1.1 Molecular cytogenetics ............................................................................................ 3 Fluorescent in situ hybridization (FISH) .................................................................. 5 1.1.2 Flow cytogenetics .................................................................................................. 10 Critical steps in flow cytogenetics .......................................................................... 14 Chromosome isolation ..................................................................................................... 14 Chromosome staining ...................................................................................................... 17 Flow Cytometry ................................................................................................................ 17 1.2 Flow cytometry and FISH ............................................................................................. 22 1.2.1 Satellite DNA ......................................................................................................... 23 Interstitial telomeric sequences in Chinese Hamsters ........................................... 24 Human alpha-satellite DNA ................................................................................... 25 Mouse major satellite DNA .................................................................................... 26 1.3 Next generation sequencing ......................................................................................... 27 1.3.1 The Illumina sequencing technology .......................................................................... 27 1.4 Thesis objectives .......................................................................................................... 31 Chapter 2 Analysis of repetitive DNA in chromosomes by flow cytometry ........................ 33 2.1 Introduction ................................................................................................................... 35 vii    2.2 Results and discussion ................................................................................................. 36 2.2.1 Preservation of flow karyograms following FISH ................................................... 36 2.2.2 Chromosome-specific measurement of interstitial telomere repeats in CHO chromosomes ...................................................................................................................... 37 2.2.3 Chromosome-specific detection of major satellite DNA in mouse cell lines from various backgrounds ............................................................................................................ 40 2.2.4 Discrimination of parental homologs of chromosome 18 in a human cell line ....... 40 2.3 Conclusion ......................................................................................................................... 43 2.4 Material and methods ........................................................................................................ 44 2.4.1 Cell culture and chromosome isolation ................................................................. 44 2.4.2 PNA FISH on suspension chromosomes .............................................................. 45 2.4.3 Chromosome sorting and analysis ........................................................................ 46 2.4.4 Q-FISH on metaphase and sorted chromosomes ................................................. 47 2.4.5 Statistical analyses ................................................................................................ 48 2.5 Supplementary Note ..................................................................................................... 49 2.5.1 Optimization of hybridization conditions ................................................................ 49 2.5.2 Optimization of flow cytometry settings ................................................................. 50 2.5.3 Correlation of between measurements by CFF and Q-FISH................................. 51 Chapter 3 Sorting of parental homologs of human chromosome 18 for sequencing ........ 60 3.1 Introduction ................................................................................................................... 61 3.2 Material and methods ................................................................................................... 63 3.2.1 Cells and cell culture ............................................................................................. 63 3.2.2  Chromosome isolation ........................................................................................... 63 3.2.3 FISH on sorted chromosomes ............................................................................... 63 3.2.4  FISH on suspension chromosomes ...................................................................... 64 3.2.5 Flow Cytometry and chromosome sorting ............................................................. 64 3.2.6  Library construction for Next Generation Sequencing ........................................... 64 3.3 Results .......................................................................................................................... 67 3.3.1  Purification of human chromosome 18 for Solexa Illumina sequencing ............... 67 3.3.2 Single nucleotide variant frequencies on chromosome 18 .................................... 68 3.3.3 Purification of parental homologs of human chromosome 18 and Illumina library construction ......................................................................................................................... 70 3.3.4 Individual Illumina sequencing of human chromosome 18 parental homologs ..... 71 viii    3.3.5 Determination of allelic combinations over the entire chromosome 18 ................. 73 3.4 Discussion and conclusions ......................................................................................... 76 Chapter 4 Purification of sister chromatids based on DNA template strands by chromosome orientation flow FISH ........................................................................................ 82 4.1 Introduction ................................................................................................................... 83 4.2 Materials and methods ................................................................................................. 86 4.2.1 Cells and cell culture ............................................................................................. 86 4.2.2  Cloning (Lox Separase system) ............................................................................ 86 4.2.3 Chromosomes and chromatids isolation ............................................................... 88 4.2.4  FISH on suspension chromosomes and chromatids ............................................. 88 4.2.5  CO-FISH on suspension chromosomes ................................................................ 88 4.2.6 Cell cycle analysis following Cre-mediated overexpression of Separase S1126A 89 4.2.7 Flow Cytometry and chromatid sorting .................................................................. 90 4.3 Results .......................................................................................................................... 91 4.3.1  Generation of chromatids from a human cell line conditionally expressing Separase S1126A ................................................................................................................ 91 4.3.2 Chromosome orientation flow FISH to identify DNA template strands by flow cytometry ............................................................................................................................. 94 4.4 Discussion and conclusions ......................................................................................... 97 Chapter 5 General discussion ............................................................................................... 101 5.1 Summary of thesis findings ........................................................................................ 102 5.2 CFF as an analysis tool: potential applications and drawbacks ................................. 103 5.3 Sequencing libraries from CFF purified populations: finding epigenetic differences in genetically identical regions of parental homologs ................................................................ 107 5.4 Sequencing template strands: are sister chromatids functionally equivalent? ........... 109 5.5 Concluding remarks .................................................................................................... 110 Bibliography ............................................................................................................................ 112 Appendix 1 Hardware setup for flow cytometry analysis and sorting of chromosomes . 128 Appendix 2 Excitation and emission spectra of fluorochromes used for one and two probes CFF .............................................................................................................................. 129 Appendix 3 Characterization of reads mapping outside chromosome 18 ........................ 130  ix    List of tables  Chapter 5  5-1  Comparison between different FISH-based methods to measure telomere length ........... 106      x    List of figures  Chapter 1  1-1  Peptide nucleic acid (PNA) structure ..................................................................................... 8 1-2  Telomere measurements using quantitative FISH (Q-FISH) ................................................. 9 1-3  Main steps of chromosome preparation for flow karyotyping .............................................. 16 1-4  Main components of a flow cytometer ................................................................................. 21 1-5  Main steps of paired-end sequencing by Illumina technology ............................................. 30  Chapter 2  2-1  Preservation of flow karyograms following FISH enables chromosome specific detection of interstitial telomere repeats in CHO chromosomes  ................................................................... 39 2-2  Chromosome and allele-specific analysis of satellite DNA in mouse and human cell lines  42 S2-1  Preservation of flow karyograms following FISH ............................................................... 52 S2-2  Hybridization specificity of PNA probes by CFF ................................................................ 53 S2-3  Parameters affecting Cy5 fluorescence signal for quantitative measurements of  telomere repeats ........................................................................................................................................ 54 S2-4  Improvements on Cy5 signal resolution and detection above background after optimization ................................................................................................................................. 55 S2-5  Comparison of quantitative fluorescence measurements of telomere repeats by CFF to measurements of sorted CHO chromosomes using Q-FISH ...................................................... 56 S2-6  Distinct hybridization patterns of major satellite probe in mouse chromosomes ............... 57 S2-7  Improved definition of poorly resolved chromosome populations in mouse flow  .................. karyograms ................................................................................................................................. 58 S2-8  Main steps of paired-end sequencing by Illumina technology ........................................... 59  Chapter 3  3-1  Paired-end sequencing of purified human chromosome 18 ................................................ 69 3-2  Sorting of parental homologs of chromosome 18 for paired-end sequencing ..................... 72 3-3  Allelic frequencies of single nucleotide variants on parental homologs of chromosome 18 ....  .................................................................................................................................................... 75 xi      S3-1  Reads coverage in chromosome 18 sequencing libraries ................................................. 78  Chapter 4  4-1  Strategy for conditional induction of separated chromatids ................................................. 85 4-2  Identification of template strands by flow cytometry on separated chromatid  following CO- FISH ............................................................................................................................................ 87 4-3  Conditional induction of separated sister chromatids in the HT1080 cell line ...................... 93 4-4  CO-FISH in suspension for purification of template strands by flow cytometry ................... 96 4-5  Models of outcomes for chromosome orientation flow FISH ............................................. 100  Chapter 5  5-1  Increasing sample purity to build sequencing libraries answering different biological questions ................................................................................................................................... 111  xii     List of abbreviations   4OHT    4-hydroxytamoxifen BAC    bacterial artificial chromosome bp    base pair(s) BrdU    5-bromo-2’-deoxyuridine CA3    chromomycin A3 cDNA    complementary DNA CENP    centromeric protein(s) ChIP    chromatin immunoprecipitation CIB    chromosome isolation buffer CO-FISH   chromosome orientation FISH DAPI    4'-6-diamidino-2-phenylindole DNA    deoxyribonucleic acid EB    DNA extraction buffer ER    estrogen receptor FACS    fluorescence activated cell sorting FISH    fluorescent in situ hybridization FU    fluorescence unit(s) HO    Hoechst 33258 ISH    in situ hybridization ITS    interstitial (or intrachromosomal) telomeric sequences kb    kilobase(s) LPA    linear polyacrylamide Mb    megabase(s) xiii    MESF    Molecules of Soluble Fluorochrome MNAse   micrococcal nuclease mW    milliwatts NGS    Next Generation Sequencing nm    nanometer PAGE    polyacrylamide gel electrophoresis PCR    polymerase chain reaction PI    propidium iodide PNA    peptide nucleic acid RNA    ribonucleic acid SNV    single nucleotide variant(s) UV    ultraviolet xiv    Acknowledgements    I would first like to thank my supervisor, Peter Lansdorp, for his mentorship. He gave me the freedom to try new things, along with some guidance back in the right direction when it was required. I am very grateful for the incomparable learning experience.  I would also like to thank all the members of the Lansdorp laboratory, past and present, with everyone bringing a different expertise and way of thinking. The combination of personalities made the lab a great place to exchange ideas or just hang out. I would like to specially acknowledge Geraldine, Mark and Ester, for guidance and mentorship throughout my journey. I would also like to thank Liz, for sharing some of her chromosome wisdom with me.  I owe a special thanks to my family, for being understanding and for making sure that the distance with them was only a physical one. I would also like to thank my extended “Vancouver family”, for making sure that I lived for the moment and that the time I spent completing my Ph.D. was not just something in the interim.  I would like to acknowledge my great friends, who never complained of the sometimes (very) long intervals between phone calls and emails.  And finally, a special mention to Sam, who against all odds made the tail end of my journey the sanest of it all.   xv     Dedication Pour ma famille. 1      Chapter 1  General introduction 2      1.1 Cytogenetics  Cytogenetics is an area of genetics that is focused on chromosomes, their structure and their relationship to cell structure and function. Professor Tao-Chiuh Hsu, one of the most prominent figures in mammalian cytogenetics, has divided the development of traditional cytogenetics into four major periods according to major discoveries that enabled refining the visualization of chromosomes (Hsu 1979): the pre-hypotonic era, the development of hypotonic treatments, the chromosome banding era and the modern period (reviewed in (van der Ploeg 2000)).  During the pre-hypotonic era, chromosomes were often studied in paraffin sections, a type of preparation that offered very little resolution of individual chromosomes and where it was hard to be certain that complete cells were studied. Minor improvements were implemented with the use of cell squashes, a method where pressure was applied to flatten samples, thus spreading and enlarging the chromosomes. Only a slight improvement on the resolution of paraffin sections, cell squashes still did not allow separation of all chromosomes from the mitotic plate and thus the actual number of chromosomes present in human diploid cells remained under debate (reviewed in (van der Ploeg 2000)). In the 1930’s, the introduction of mitotic spindle poisons such as colchicine (Blakeslee, Avery 1937, Levan 1938) allowed to significantly increase the mitotic index of harvested cultured cells while favoring chromosome condensation. It is only after the discovery of the structure of deoxyribonucleic acid (DNA), with the introduction of hypotonic treatment that permitted cell swelling and a better spreading of the chromosome preparations (Hughes 1952, Hsu 1952, Makino, Nishimura 1952) that, after years of debate, the human diploid chromosome number was finally established at 46 (Tijo, Levan 1956, Ford, Hamerton 1956). The next big step forward in the traditional cytogenetics field came with the 3      development of chromosome banding methods by Caspersson in the 1960’s (Caspersson, Farber et al. 1968, Caspersson, Castleman et al. 1971), which allowed a more detailed view of chromosome structure. Banding methods are based on staining of chromosomal preparations with a DNA dye, quinacrine mustard, in the case of Caspersson’s studies, which binds differentially to guanine-cytosine-rich or adenine-thymine-rich DNA. The resulting alternating dark and light bands reflects nucleotide content along specific chromosomes and thus allows visualizing of chromosomal aberrations in better detail. Further refinements in chromosome preparation and spreading methods, in combination with other banding stains, such as Giemsa G or R-banding (Arrighi, Hsu 1971, Drets, Shaw 1971, Dutrillaux, Lejeune 1971, Seabright 1971, Patil, Merrick et al. 1971, Eiberg 1974) and high resolution banding (Yunis, Lewandowski 1983) have brought down the resolution limit of detection of aberrations to the equivalent of 1-5 megabases (Mb) of DNA.  A few subspecialties, such as molecular cytogenetics and flow cytogenetics, have been added to the array of chromosome preparation and staining techniques since the traditional methods described above, in further efforts to improve the resolution, speed and statistical power of cytogenetic analysis.  1.1.1 Molecular cytogenetics  Traditional cytogenetic methods have evolved alongside the discovery of DNA structure and the development of diverse molecular biology techniques, to be merged into a prominent subspecialty, molecular cytogenetics.  4      Although the various banding methods allowed for a better characterization of chromosome structure, the discovery that chromosomal rearrangements such as fusion or breaks can be associated with specific human diseases increased the demand for resolution below the megabase level for detection of finer aberrations. At around the same time as the banding methods were developed, the first in situ hybridization (ISH) experiments were shown to visualize specific nucleic acid sequences with increased sensitivity and resolution over banding methods (Gall, Pardue 1969, John, Birnstiel et al. 1969). ISH was a very attractive method based on the base pair complementarity between a labeled nucleotide probe and the target sequence that was visualized directly on the chromosome preparation, allowing its physical mapping. During initial experiments, radiolabeled ribonucleic acid (RNA) or DNA probes were hybridized on specimens and signal was detected by autoradiography (Gall, Pardue 1969), allowing the detection of abundant sequences such as satellite DNA (Pardue, Gall 1969) or integrated virus (Evans, Baluda et al. 1974) in a variety of species or types of cell preparations. Developments in both chromosome preparations and isotopic ISH made it a very sensitive tool, even allowing the detection of signal from single copy sequences in metaphase spreads (Gerhard, Kawasaki et al. 1981, Harper, Saunders 1981) using RNA or DNA probes. Detection of radioactive signal often meant that, in addition to various issues related to the use of isotopes, long exposure times were required in order to detect specific signals. Furthermore, the grain of autoradiography paper available at the time limited the signal resolution sometimes below that of a single chromosome band.    5 Fluorescent in situ hybridization (FISH)  Progress in molecular biology and in utilizing fluorescent molecules soon brought a new means of detecting specific targets by microscopy that were far less time consuming and offered a far greater resolution than isotopic ISH. The first fluorescent detection of a hybridized probe was done by indirect immunofluorescence, using an antibody that was raised against DNA-RNA hybrids (Rudkin, Stollar 1977). Fluorescence microscopy and non isotopic ISH became very attractive means to circumvent the low resolution of autoradiography, replacing autoradiography paper with sensitive cameras. The use of RNA probes directly labeled with a fluorochrome allowed for instantaneous visualization of abundant sequences (Bauman, Wiegant et al. 1980) and correlated to a karyogram using the DNA dye 4'-6-diamidino-2-phenylindole (DAPI) to obtain a banding pattern (Lin, Comings et al. 1977). As for isotopic ISH, FISH could be used to detect and map highly represented DNA such as satellite DNA (Manuelidis, Langer-Safer et al. 1982), RNA gene clusters (Van Prooijen-Knegt, van der Ploeg 1982) or the amplified DNA sequences of polytene DNA (Langer-Safer, Levine et al. 1982). Refinements in probe labeling, hybridization conditions and detection strategies improved the sensitivity of FISH and enabled the detection of chromosome or locus specific probes (Lichter, Cremer et al. 1988, Pinkel, Landegent et al. 1988, Landegent, Jansen in de Wal et al. 1987, Wiegant, Ried et al. 1991) or, using differentially labeled probes, allowed the simultaneous detection of multiple targets (Hopman, Wiegant et al. 1986).  The versatility of FISH has provided a platform for diversification into a wide array of procedures (reviewed in Volpi, Bridger 2008), from large-scale physical mapping to support the human 6      genome project to high resolution mapping of fiber-FISH, where probes are hybridized on stretched single DNA molecules (Michalet, Ekong et al. 1997). FISH can be used to localize or quantify specific sequences in interphase nuclei or metaphase chromosomes and fluorescence signal can be detected by microscopy or flow cytometry, depending on the application. Three specialized FISH procedures, Quantitative FISH (Q-FISH), Flow FISH and chromosome orientation FISH (CO-FISH) will be discussed here.  Q-FISH Quantitative FISH (Q-FISH) is a method that enables the measurement of repetitive DNA by FISH. It has been developed to measure telomere length using (CCCTAA)3 peptide nucleic acid (PNA) oligonucleotide probes directly labeled with fluorescent dyes (Lansdorp, Verwoerd et al. 1996). PNA are synthetic oligonucleotide analogs where purines and pyrimidines are attached to a polypeptide backbone through methylene carbonyl links to mimic single stranded DNA (Egholm, Buchardt et al. 1993) (Figure 1-1). DNA and RNA probes are negatively charged, but the uncharged N-(2-amino ethyl)-glycine backbone of PNA probes allows them to bind their target DNA in absence of electrostatic repulsion and, as a result, with higher affinity than the corresponding equivalent DNA/DNA or DNA/RNA duplexes (Egholm, Buchardt et al. 1993) (Figure 1-2). PNA can be directly labeled with fluorescent or hapten reporters and used in FISH techniques similar to traditional synthetic DNA oligonucleotide probes. The binding of PNA probes to their target DNA is more sensitive to mismatches than the binding of DNA (Egholm, Buchardt et al. 1993) or RNA probes (Jensen, Orum et al. 1997), making those more discriminating than equivalent labeled synthetic DNA or RNA oligonucleotides. Consequently, shorter, 15 to 18 nucleotide long probes are often sufficient to discriminate between very similar sequences (Chen, Hong et al. 1999, Taneja, Chavez et al. 2001). Shorter probes translate to 7      brighter signals when probing repetitive DNA as more fluorescent molecules can be loaded per kb of repeats. The strong interaction between PNA and complementary DNA or RNA sequences can be exploited to select stringent conditions for hybridization (Orum, Nielsen et al. 1995) and washes in FISH to obtain a quantitative relationship between fluorescently labeled PNA probes and the length of repetitive target DNA (Figure 1-2). Using digital microscopy and appropriate calibration slides (plasmids with telomeric DNA inserts of known length) and controls, telomere length can be calculated by conversion of fluorescence units into the length of telomere repeats in kilobases (Poon, Martens et al. 1999). Q-FISH has proven to be a very powerful method to quantify telomere length in a chromosome specific manner (Lansdorp, Verwoerd et al. 1996, Martens, Zijlmans et al. 1998) or in interphase cells (de Pauw, Verwoerd et al. 1998).                         Figure 1-1 Peptide nucleic acid (PNA) structure Comparison of the structure of PNA to that of DNA. The deoxyribose backbone of DNA is replaced by an uncharged pseudopeptide backbone in PNA. DNA-PNA heteroduplex formation. B: purine or pyrimidine base   8             Figure 1-2 Telomere measurements using quantitative FISH (Q-FISH) DNA is denatured to allow the PNA probe to bind to its complementary single stranded target DNA. At low ionic strength, only PNA-DNA heteroduplexes can form, resulting in PNA probes to the target DNA in a quantitative manner. The intensity of the fluorescent signal is proportional to the number of probes bound to the target DNA (telomeres). The absolute telomere length can also be calculated from calibration using plasmids containing known telomere stretches. >: telomere repeat unit. ——: PNA oligomer. : fluorescent marker  9      10      1.1.2 Flow cytogenetics  Flow karyotyping was developed in the mid-seventies (Gray, Carrano et al. 1975b, Stubblefield, Cram et al. 1975) and is based on the combination of a method to isolate mitotic chromosomes in suspension (Wray, Stubblefield 1970) and flow cytometry, the then recently developed technology used to analyze and sort cells from heterogeneous populations. The first commercially available fluorescence flow cytometers (Van Dilla, Trujillo et al. 1969, Dittrich, Gohde 1969) were developed only a few years before the beginnings of flow cytogenetics, and the field has evolved alongside the technological development of flow cytometry.  Early experiments were of Chinese hamster chromosome suspensions that were stained with ethidium bromide and analyzed on a flow cytometer, where individual chromosomes emitted fluorescence proportionally to their DNA content (Gray, Carrano et al. 1975b, Stubblefield, Cram et al. 1975). Fluorescence peak patterns, or flow karyograms, representing both chromosome size and relative representation were established for Chinese hamster cells and could be compared with that of aberrant clones to detect cytogenetic differences (Gray, Carrano et al. 1975a). Rapidly, this new method was also extended to the analysis of human chromosomes (Gray, Carrano et al. 1975a) and promised to accelerate traditional cytogenetic research by providing a fast, yet sensitive and highly quantitative means of measuring small variations in the DNA content in individual chromosomes. In addition to ethidium bromide, other DNA intercalating agents, such as Hoechst 33258 (HO) (Carrano, Gray et al. 1979), chromomycin A3 (CA3) or propidium iodide (PI) were used to improve the resolution or obtain different peak patterns on monovariate flow karyotypes (Jensen, Langlois et al. 1977). Monovariate flow karyograms have been used to study normal chromosome variability (for example: Green, 11      Fantes et al. 1984, Harris, Boyd et al. 1985, Harris, Cooke et al. 1987a) and purify specific chromosome populations (Harris, Boyd et al. 1985), but the use of a single dye to stain chromosomes did not allow the resolution of every single chromosome as a single peak and so limited the analytical power of flow karyotyping (Gray, Carrano et al. 1975b, Stubblefield, Cram et al. 1975, Gray, Carrano et al. 1975a).  The introduction of dual beam cytometry and the possibility to analyze DNA content in cells using two different DNA dyes (Dean, Pinkel 1978, Langlois, Jensen 1979) further increased the resolution power of flow karyotyping (Langlois, Carrano et al. 1980, Langlois, Yu et al. 1982, Young, Ferguson-Smith et al. 1981, Gray, Langlois et al. 1979). Bivariate flow karyotyping is based on the use of two DNA dyes with different base composition preferences. Various studies have been made on the use of various DNA dyes (Langlois, Jensen 1979, Langlois, Carrano et al. 1980, Latt, Sahar et al. 1980), but the combination of HO and CA3 is the most commonly used.  HO is a synthetic dye with a preference for adenine/thymine-rich DNA (Latt, Wohlleb 1975) and CA3 is an antibiotic isolated from Streptomyces griseus with fluorescent properties and a preference for guanine/cytosine-rich DNA (Ward, Reich et al. 1965). HO and CA3 have different excitation/emission spectra, with maxima at respectively 360/461 nm and 430/570 nm, enabling their independent analysis by dual beam flow cytometry. The combination of HO and CA3 for the analysis of chromosomes in suspension therefore allows quantitative measurements not only of DNA content and relative representation, but also of relative base pair composition of each chromosome population. Using bivariate flow karyotyping, the number of resolved chromosome populations in Chinese hamster went from 10 to 16 and in humans, from 12 to 20 when switching from monovariate to bivariate flow cytometry (Gray, Langlois et al. 1979). The DNA content and relative base composition of a chromosome population can be 12      derived by determining the length and slope of a line between the center of the population and the origin of a HO/CA3 bivariate plot (Trask, van den Engh et al. 1989b).  Bivariate flow karyotyping has since been used to quantify human chromosomes (Young, Ferguson-Smith et al. 1981, Gray, Langlois et al. 1979), normal variation between individuals (Langlois, Yu et al. 1982, Trask, van den Engh et al. 1989b, Harris, Cooke et al. 1987b, Trask, van den Engh et al. 1989a, van den Engh, Trask et al. 1988, Boschman, Rens et al. 1991, Mefford, van den Engh et al. 1997), the detection of deletions, aneuploidy, translocations or other chromosomal abnormalities (Lebo, Anderson et al. 1986, Gray, Trask et al. 1988, Cooke, Gillard et al. 1988, Cooke, Tolmie et al. 1989a, Cooke, Tolmie et al. 1989b, Bartholdi, Parson et al. 1990, Trask, van den Engh et al. 1990, Boschman, Manders et al. 1992, Boschman, Rens et al. 1992, Boschman, Buys et al. 1993). Flow karyotyping has been combined with fluorescence activated cell sorting (FACS) to purify specific chromosome populations. Sorted chromosomes were used to generate whole chromosome paints (examples: (Blennow, Telenius et al. 1992, Carter, Ferguson-Smith et al. 1992, Gribble, Ng et al. 2004, Rabbitts, Impey et al. 1995, Telenius, Pelmear et al. 1992, Carter 1994, Howarth, Blood et al. 2008)), for sequence analysis (Boschman, Buys et al. 1993, Lebo, Chakravarti et al. 1983, Lebo, Golbus et al. 1986) (Lebo, Anderson et al. 1986) or to generate chromosome specific DNA libraries (Harris, Boyd et al. 1985, Davies, Young et al. 1981, Krumlauf, Jeanpierre et al. 1982, Lalande, Kunkel et al. 1984, Fuscoe, Clark et al. 1986) that were used for alignment and mapping during the initial stages of the human genome project (Van Dilla, Deaven 1990). Requirements for very large amounts of sorted chromosomes, sometimes necessitating several days of sorting, were lessened by advances in polymerase chain reaction (PCR) protocols that allowed library construction from as little as a single sorted chromosome (VanDevanter, Choongkittaworn et al. 1994). 13      Despite improvements both in chromosome preparation protocols (van den Engh, Trask et al. 1988, Ng, Carter 2006, Ng, Yang et al. 2007) and flow cytometry (Ng, Carter 2010) to obtain high resolution chromosome profiles, it is still impossible to distinguish and sort every individual chromosome population on a bivariate flow karyogram. Incorporation of the thymine analog 5- bromo-2’-deoxyuridine (BrdU) during DNA synthesis is often used to identify the newly formed DNA strand. BrdU has been demonstrated to quench HO fluorescence (Latt 1973), a property that is used to differentially label the newly synthesized DNA strand. BrdU incorporation has been used in combination with flow karyotyping to estimate the replication kinetics of specific chromosomes (Severin, Ohnemus 1982, Cremer, Gray 1983) or to improve the discrimination of late-replicating chromosome populations (Cremer, Gray 1983). There have also been attempts at developing protocols for FISH on chromosomes in suspension (Dudin, Cremer et al. 1987, He, Deng et al. 2001, Ma, Lee et al. 2005, Macas, Dolezel et al. 1995, Nguyen, Lazzari et al. 1995), but to our knowledge, only polyamides binding to the minor groove of DNA without a requirement for denaturation of the target have been successfully combined with bivariate flow karyotyping (Gygi, Ferguson et al. 2002).  One of the goals of the work described in this thesis was to combine molecular cytogenetics with flow cytometry and explore if FISH procedures developed for cells in suspension (Baerlocher, Vulto et al. 2006) could be used to develop CFF.  14 Critical steps in flow cytogenetics  Chromosome isolation  Resolution of flow karyograms is highly dependent on the quality and type of the chromosome preparations. A few of the methodological variables for chromosome preparation will be described here. For a good review, see (Trask 1989). Figure 1-3 summarizes the main steps involved in the isolation of chromosomes for flow karyotyping.  Good quality chromosome preparations start with healthy, exponentially growing cell cultures. Presence of dead, unhealthy cells or contamination will lead to the presence of debris that can decrease resolution and contaminate fractions of purified chromosomes. To achieve the high mitotic index required to prepare chromosomes in suspension, mitotic spindle poisons such as Colchicine or Colcemid are usually added to the cultures prior to harvesting. Prior to the release of the chromosomes in suspension, cells need to be expanded in hypotonic buffer to both separate chromosomes and make the cell membrane more fragile. The choice of hypotonic buffer can vary depending on the cell type and can sometimes contain DNA stabilizing agents to prevent DNA uncoiling if some cell membranes are disrupted.  Chromosomes in suspension prepared using various chromosome isolation buffers (CIB) have been successfully used to generate monovariate or bivariate flow karyograms. In general, the cell type used, the intended use and potential time of storage will direct the appropriate choice of CIB. The purpose of CIBs is to stabilize chromosomes in suspension, i.e to maintain 15      chromosomes intact and prevent uncoiling or clumping. For example, Tris/MgCl2 (Otto, Oldiges et al. 1980) and Hepes/MgSO4 (van den Engh, Trask et al. 1984) CIBs are based on the use of divalent cations to stabilize DNA structure and keep chromatin in a condensed state at low ionic strength. DNA intercalating agents such as propidium iodide are also known to stabilize DNA and can be used in CIBs (Carrano, Gray et al. 1979), with the combined advantage and convenience that it doubles as a DNA stain for subsequent flow cytometry analysis (Aten, Buys et al. 1987). Chromosome isolation buffers using cationic polyamines such as spermine and spermidine (Sillar, Young 1981) are also popular because of the quality of the preparations obtained and of the possibility to add chelators (EDTA, EGTA or sodium citrate) to inhibit nuclease activity and thus enable long term storage. Detergents such as digitonin or Triton X- 100 are added to most CIBs in order to create pores in the cell membranes prior to disruption. Chromosomes are released in suspension by mechanical disruption, usually gentle, of the membrane by syringing, vortexing or sonication.            Figure 1-3 Main steps of chromosome preparation for flow karyotyping a. Mitotic arrest using microtubule disrupting agents and mitotic cells harvesting by mitotic shake off (adherent cells only). b. Hypotonic treatment of cells. c. Release of chromosomes in suspension and d. staining prior to e. analysis by flow cytometry. f. Standard metaphase preparations are generally fixed prior to be processed for microscopy analysis.   16      17      Chromosome staining  Depending on the chromosome isolation procedure, the type of analysis and the flow cytometer utilized, various DNA dyes can be used alone or in combination. For monovariate flow karyotyping, DNA intercalating agents that bind DNA in a stoichiometric manner such as propidium iodide or ethidium bromide are usually preferred (Gray, Carrano et al. 1975b, Stubblefield, Cram et al. 1975), but HO (Carrano, Gray et al. 1979) or CA3 have also successfully been used. For bivariate flow karyotyping, as described above (p.11-12), two DNA dyes with non-overlapping emission spectra and different base pair affinity are used in combination. The most commonly used pair of dyes is CA3, and HO, with a preference for GC and AT-rich DNA, respectively. HO exhibits a quenched fluorescence signal on chromosomes that have incorporated BrdU (Buys, Mesa et al. 1986), a property that has been used in many traditional and flow cytogenetics applications to study replication timing of specific regions or chromosomes. DAPI has a similar fluorescence spectra and base pair preference than HO (Lin, Comings et al. 1977) and has been successfully used in bivariate flow karyograms (Meyne, Bartholdi et al. 1984, Lebo, Gorin et al. 1984, Hutter, Stoehr 1985). Its fluorescence is not affected by BrdU incorporation in the same manner as HO in suspension chromosomes (Buys 1986), a characteristic that can be advantageous to stain chromosomes for CO-FISH.  Flow Cytometry  Flow cytometry is a method that enables the analysis of single cells or other particles such as isolated chromosomes that are moving through one or multiple light sources in a liquid stream. It is a rapid and quantitative way to measure multiple parameters simultaneously, either intrinsic to 18      the particle (size, light refraction) or through the use or antigenic or biochemical markers (fluorescent antibodies, DNA dyes). In addition to its analytical power, flow cytometry also enables the purification or separation of specific cells and particles by FACS. The main components of a fluorescence flow cytometer (Figure 1-4), the fluidics, the optics and the electronics will be described here briefly. For good reviews, see (Shapiro 1985, Cram 2002).   Fluidics The fluidics system is responsible for the transport and alignment of the particles through the flow cytometer at the interrogation point (see Optics). The main components of the fluidics system are the sample and the sheath fluid, which are both differentially pressurized to be injected into the flow cell. The sample is injected with a higher pressure than the sheath buffer, creating a process called laminar flow, where the sample travels at the center core without the fluids mixing. The particles are required to travel one by one at a steady flow rate through the interrogation point (see Optics), situated in the flow cell, in order to obtain information for individual particles. The alignment of particles and their focusing at the center of the stream is achieved by a process called hydrodynamic focusing, where a flow width proportional to the particle size is obtained by forcing a larger volume of liquid from the sample chamber into the smaller space of the flow cell. Consequently, the particles to be analyzed are positioned like beads on a string at the center of the flow stream, enabling uniform illumination at the interrogation point. Faster measurements (high number of events/second) are achieved by creating a wide flow stream using a high sample/sheath injection pressure ratio, while a narrow flow stream, generated through a low sample/sheath injection/pressure ratio will provide a more uniform focusing of particles in the light beam(s) and thus, more precise measurements.  19      Optics The optical system of a flow cytometer is what will determine the nature and number of fluorochromes that can be analyzed simultaneously. Its purpose is to provide uniform and specific illumination of the particles to be interrogated and to separate emitted wavelengths into specific optical detectors (see Electronics). The optics system comprises excitation light sources, most commonly red, blue, green and violet lasers (633 nm, 488 nm, 514 nm and 405 nm, respectively) and a combination of mirrors and filters. The combination of fluorescent markers used to characterize the sample being interrogated is selected according to the availability of the excitation light source(s) and for their potential to be detected with a minimal spectral overlap. A combination of prisms, mirrors and filters is responsible to align and focus all excitation photons of different wavelengths onto the interrogation points, the area of the flow cell where the laser beams intersect the sample stream, illuminate the particles and collect the fluorescence and scattered light from particles into specific detectors. Filters (long pass, short pass or band pass) are arranged in a way to only permit transmission of specific light wavelengths, and dichroic mirrors are used to split emitted wavelengths and direct them into different directions for collection by separate photomultiplier tubes (see Electronics).  Electronics Photomultiplier tubes (PMTs) are the main component of the electronic system of the flow cytometer. The fluorescence signal emitted by the particles is directed to the PMTs as photons, which are converted into photo electrons, focused and amplified before being converted into an electronic signal proportional to the intensity of the transmitted light.  The photoelectronic signal is then transmitted to the computer that collects and stores the information prior to analysis. 20      Depending on the dynamic range of the signal being detected, amplification of the photomultiplier signals can be done in linear or logarithmic mode. For DNA content analysis, such as for flow karyotyping or cell cycle analysis, a linear mode of amplification is sufficient and desirable and provides good visual resolution of signal within a narrow range. When quantifying signal with several orders of magnitude of dynamic range, such as quantification of specific proteins by immunofluorescence, logarithmic amplification is often preferred.  FACS Sorting of specific cell or particle populations by flow cytometry is made possible using electrostatic deflection. The particles to be purified are selected according to a combination of parameters analyzed on a flow cytometer, for example DNA content or the presence of specific cell surface markers. A charge is pulsed at the break-off point, the precise area where the selected particle is formed into a droplet. Two charged deflection plates situated below the break-off point deflect the charged droplets containing the particles of interest towards a collection tube, and the uncharged droplets are collected into a waste tube. Timing and synchronization of charge pulse and droplet formation need to be precisely adjusted, and factors that could perturb the stream, such as the presence of debris or clumps, need to be eliminated. The purity of sorted particle populations can often be increased at the cost of speed, by increasing the ratio of empty droplets between droplets that contain particles.   Figure 1-4 Main components of a flow cytometer Simplified view of the main fluidic, optic and electronic components of a fluorescence flow cytometer.  21      22      1.2 Flow cytometry and FISH  The large number of events that can be analyzed in a relatively short period of time provides distinct advantages to flow cytometers over fluorescence microscopes equipped with digital cameras dedicated to the analysis of images of interest. Whereas traditional cytogenetic and FISH analysis takes up to a week to analyze up to a hundred events, thousands of particles can be analyzed in a manner of seconds on a flow cytometer. As a result, the statistical power of flow cytometry is much higher than what can be obtained using digital image cytometry.  After the development of Q-FISH (Lansdorp, Verwoerd et al. 1996) to measure telomere length using specific PNA probes on metaphase spreads analyzed by digital image cytometry (section, efforts were made to exploit PNA probes to measure the average length of telomeres in cells in suspension using flow cytometry (Rufer, Dragowska et al. 1998). Flow FISH has proven to be a very powerful technique with all kinds of applications. For example, blood samples from patients suspected of heritable telomerase defects in genes involved in telomere maintenance can be screened using flow FISH, as the telomeres in the nucleated blood cells from such patients are invariably very short (for example: (Alter, Baerlocher et al. 2007, Calado, Regal et al. 2009a, Calado, Regal et al. 2009b, Sasa, Ribes-Zamora et al. 2011)). Flow FISH can also be used to compare telomere length in patients to that of their relatives to distinguish heritable familial syndromes with variable penetrance from idiopatic disease and to exclude relatives from patients as donors for bone marrow transplantation. In addition, flow FISH can be combined with antibody staining to obtain telomere length measurements that are specific for various leukocytes subsets (Baerlocher, Lansdorp 2003). Cell type specific telomere length is useful to investigate if specific hematopoietic cell subsets are more severely affected than 23      others bone marrow failure syndromes (for example, (Goldman, Aubert et al. 2008)) or to follow variations in telomere erosion rates during normal aging (Halaschek-Wiener, Vulto et al. 2008). Clinical telomere length meausurement services are provided by Repeat Diagostics Inc, a spin- off company from the BC Cancer Agency with a laboratory in North Vancouver.  Telomere repeats, derived from a RNA template by reverse transcription, are highly conserved and highly abundant, typically over several kb at every human chromosome end, making them ideal sequences for quantitative FISH using PNA probes. Following FISH, thousands of PNA probes will typically be bound specifically to target sequences in each cell, giving rise to good signal to noise ratios for detection by either microscopy or flow cytometry. A limitation of PNA FISH is that few repetitive sequences are as abundant or ubiquitous as telomere repeats. Detection of rare or unique DNA sequences would represent major costs, as hundreds of expensive PNA probes would have to be synthesized. However, certain repetitive sequences, such as the ones presented below, are sufficiently abundant to be analyzed conveniently using PNA probes and of potential interest for chromosome specific analysis by flow cytometry.   1.2.1 Satellite DNA  Satellite DNA, composed of head-to-tail, tandemly repeated sequences, is a component of centromeric and pericentromeric DNA (reviewed in Plohl, Luchetti et al. 2008). Its association with centromere proteins (CENPs) triggers the formation of centromere-specific heterochromatin, essential for mitotic spindle attachment and proper distribution of genetic material in daughter cells during mitosis. While centromeric chromatin shows a very high degree of conservation amongst species, centromeric DNA is highly variable. Interstitial telomeric 24      sequences (ITSs) in Chinese Hamsters, alpha-satellite DNA in humans and mouse major satellite DNA will be briefly touched in the next chapters and introduced here. Interstitial telomeric sequences in Chinese Hamsters  ITSs are blocks of TTAGGG repeats found at non telomeric regions of chromosomes in a wide variety of species (Meyne, Baker et al. 1990) that have been hypothesized to have arisen as a result of chromosomal rearrangements during genome evolution (Meyne, Baker et al. 1990, IJdo, Baldini et al. 1991, Slijepcevic 1998). In most cases, those (TTAGGG)n regions are found either within or at the margin of constitutive heterochromatin (Meyne, Baker et al. 1990). They can be classified into four main categories: short ITSs, subtelomeric ITSs, fusion ITSs and large ITSs. Large ITSs are long stretches of TTAGGG tandem repeats that are found mainly at the pericentromere and only occasionally in the interstitial region of chromosomes (Faravelli, Azzalin et al. 2002).  Chinese hamster chromosomes contain large, mainly pericentromeric ITSs that can span hundreds of kilobases (kb) (Meyne, Baker et al. 1990, Faravelli, Moralli et al. 1998) and represent the major component of their satellite DNA. The vertebrate consensus (TTAGGG)n telomeric sequence is also found at Chinese Hamster chromosomes terminal ends, but represents only a minor fraction of the total signal, with an average total telomere length of 38kb in vivo (Slijepcevic, Hande 1999). In the Chinese Hamster ovary (CHO) cell line, no FISH signal can be detected at chromosome ends, suggesting that the telomere repeat length falls below the 1kb detection threshold of the method (Slijepcevic, Bryant 1995, Bolzan, Paez et al. 2001). Nineteen different ITSs observed by telomere FISH have been reported on a typical CHO 25      metaphase spread; seventeen are located in the pericentromeric region (fifteen strong and two weaker signals), one in the interstitial region and one located near the subtelomeric region (Sanchez, Bianchi et al. 2010). Human alpha-satellite DNA  Alpha-satellite DNA is a primate-specific type of satellite repeat (Willard 1991) that is the main component of human centromeric DNA. Human alphoid DNA is characterized by stretches of AT-rich, 171 bp monomeric units (Manuelidis 1978b) that are found at all human chromosomes (Manuelidis 1978a). Individual monomers are not identical and have between 20 and 40% variability (Waye, Willard 1987). These slightly diverged monomer units are themselves organized into higher-order repeat structures that form the 33 alphoid subfamilies identified so far (Choo, Vissel et al. 1991) that are classified according to their organization. Alphoid subfamilies can be found on single or small groups of chromosomes and each human centromere is composed of one or several alphoid subfamilies (Willard, Waye 1987). This high diversity in human alpha satellite DNA makes it an attractive target to design chromosome specific oligonucleotide FISH probes. For example, in this thesis, we are using a probe (L1.84) against the D18Z1 alpha satellite to specifically detect chromosome 18 (Devilee, Slagboom et al. 1986, Devilee, Cremer et al. 1986). Furthermore, because satellite DNA is tandemly repeated, chromosome specific oligonucleotide probes designed against specific alphoid families have been used to determine DNA orientation using a strand specific detection method called Chromosome Orientation FISH (CO-FISH) (Bailey, Meyne et al. 1996, Bailey, Goodwin et al. 1996). 26 Mouse major satellite DNA  Two main satellite units, the minor and the major (γ) satellite are found at the centromere and on the centromeric heterochromatin of mouse chromosomes, respectively (Pietras, Bennett et al. 1983, Vig, Latour et al. 1994). The major satellite is estimated to account for 5 to 10% of the entire mouse genome (Kit 1961, Vissel, Choo 1989) and is found on all chromosomes, with the notable exception of the Y chromosome (Pietras, Bennett et al. 1983, Pardue, Gall 1970). Its consensus sequence is a 234 bp repeat unit (Horz, Altenburger 1981) arranged in tandem arrays that range from 240 to 2000 kb (Vissel, Choo 1989).The size of the arrays varies between species and probes targeted against the major satellite have been used to identify chromosome origin in mouse crosses (Siracusa, Chapman et al. 1983, Matsuda, Chapman 1991). Due to their tandem organization, probes against the major satellite have also been used to identify DNA template strands in cell pairs using CO-FISH (Falconer, Chavez et al. 2010a). Note that it is the less abundant minor satellite that is found at the centromeres and contains CENP-B binding sites and thus is thought to be the active component of mouse centromeres (Kipling, Mitchell et al. 1995).   27      1.3 Next generation sequencing   Sanger sequencing (Sanger, Nicklen et al. 1977) for the Human Genome Project in the 1990s to early 2000s has been a costly endeavor that took several years, hundreds of scientists and sequencing machines as well as several billion dollars to complete (International Human Genome Sequencing Consortium 2004). In the last few years, however, with the development of the so-called Next Generation Sequencing (NGS) technologies, it has become possible to sequence a whole human genome in about a week for a fraction of the cost (reviewed in Metzker 2010).  Most next-generation sequencers currently available on the market mainly use clonal amplification of immobilized DNA fragments for simultaneous sequencing of billions of individual fragments, with variations in template preparation, sequencing and imaging methodologies (reviewed in Metzker 2010). In our project, the Illumina Genome Analyzer II sequencing instrument was used.  1.3.1 The Illumina sequencing technology  The Illumina technology (Bentley, Balasubramanian et al. 2008). (Figure 1-3) uses universal Y- shaped adapters bound onto fragmented double-stranded DNA, allowing the insertion of different sequences on the 5' and on the 3’-end of the DNA fragment and thus enabling to determine the directionality of the sequencing. Adapters also contain sequences necessary for the annealing of sequencing primers and barcoding primers during sequencing. Once denatured, the fragments are anchored to a flow cell, a glass slide coated with primers 28      complementary to either end of the Y-shaped adapters. The flow cell is used as a support for solid-phase amplification by a process called bridge amplification, where DNA fragments are amplified into clusters using primers complementary to either end of the adapters.  Sequencing is performed by a process called cyclic reversible termination, where four-color fluorescent nucleotides with a 3’ patented reversible terminator moiety are incorporated into each of the clusters. Fluorescence images are captured after incorporation of every nucleotide and the image sequence is used to create parallel, individual sequence reads corresponding to each of the clusters present on the flow cell (Guo, Xu et al. 2008).  Early versions of the genome analyzer could only perform short reads of approximately 30 base pairs (bp), but rapid technological developments now enable reads of up to 150 bp. Although it is possible to create de novo sequences using Illumina short reads, it is usually required to align the reads to an already existing reference sequence (constructed from Sanger sequencing libraries) when working with complex genomes (Trapnell, Salzberg 2009, Chaisson, Brinza et al. 2009). The number of reads aligning to a specific area of the genome is referred to as “coverage” and will be an indicator of the ability to detect rare events.  Despite the relatively recent introduction of the NGS platforms on the market, several specific applications and refinements have already been developed. For example, RNA-Seq is a method where complementary DNA (cDNA) is sequenced to study gene expression at the whole transcriptome level (Wang, Gerstein et al. 2009). Various strategies for targeted genomic enrichment have also been developed in order to reduce the complexity of the material to be sequenced and thus obtain the deeper coverage required to detect rare mutations and variants 29      in re-sequencing experiments (reviewed in (Mamanova, Coffey et al. 2010)). High resolution epigenetic profiling, previously limited to known genes, is now currently possible at the genome- wide level using ChIP-seq (Johnson, Mortazavi et al. 2007), a combination of chromatin immunoprecipitation (ChIP) using antibodies that are specific to modified histones (Barski, Cuddapah et al. 2007) or DNA modifications (Pomraning, Smith et al. 2009) and sequencing. Alternatively, genome-wide bisulfite sequencing can also be performed to map imprinting regions in an allele-specific manner (Meissner, Mikkelsen et al. 2008, Smith, Gu et al. 2009, Lister, Pelizzola et al. 2009, Cokus, Feng et al. 2008).   Figure 1-5 Main steps of paired-end sequencing by Illumina technology (a) Libraries are constructed by ligated Y-shaped adapter to A-tailed fragmented DNA. (b) DNA is denatured and bound to a glass slide coated with oligonucleotides (ι ι) complementary to either end of the adapters for solid-state bridge amplification and generation of clusters of identical fragments. (c) Sequencing is performed by cyclic reversible termination: nucleotide incorporation, fluorescence imaging and cleavage of the inhibiting group and fluorescent dye from the incorporated nucleotide. • • • •: fluorescently labeled nucleotides. (d) For complex genomes, reads are generally aligned to a reference genome.  30      31       1.4 Thesis objectives    Early in the course of my work in the Lansdorp lab, I was involved in a project that aimed to study telomere biology at the chromosome specific level. It became rapidly clear that Q-FISH, although very sensitive and the only available method at the time to measure telomere length at all chromosome ends, was a very demanding method that had a limited statistical power. I hence sought to develop a new method for analysis of telomere repeats that had equivalently high statistical power to flow FISH, but also offered resolution at the individual chromosome level. The main goal of my thesis was therefore to develop a method based on a combination of flow karyotyping and FISH in suspension to reliably measure repetitive DNA of individual chromosomes.  My first objective was to develop a protocol to perform FISH on chromosomes in suspension that was compatible with analysis by flow cytometry. The development of CFF to study repetitive DNA such as interstitial telomere repeats in Chinese Hamster chromosomes, major satellite DNA in mouse chromosomes and a specific alpha-satellite (D18Z1) in human chromosomes (currently in press at Nature Methods) is presented in chapter 2.  During the course of my research, the introduction of next generation sequencing technologies opened additional area of possible investigations in all areas of biology. My second objective consisted of exploiting the methods developed in my first objective and apply CFF to purify specific chromosome populations for sequencing. The use of CFF to individually purify parental 32      homologs of human chromosome 18, followed by library construction and sequencing using the Illumina sequencing technology is presented in chapter 3.  More recently, a new focus of interest in our laboratory has been directed to questions regarding the functional equivalence and inheritance of sister chromatids. My third objective was to purify specific DNA template strands by FACS using a modified protocol of CFF. The generation and characterization of a model to induce separated sister chromatids in human cells, and the development of chromosome orientation flow FISH to purify unidirectional DNA template strands by FACS is described in chapter 4.  Finally, a summary and discussion of results from my work so far as well as a description of potential novel applications of CFF and flow (CO) FISH will be described in chapter 5.   33      Chapter 2  Analysis of repetitive DNA in chromosomes by flow cytometry                         From: BRIND'AMOUR, J. and LANSDORP, P.M., 2011. Analysis of repetitive DNA in chromosomes by flow cytometry. Nature methods. DOI 10.1038/nmeth.1601 34      We developed a flow cytometry method, chromosome flow fluorescence in situ hybridization (FISH), called CFF, to analyze repetitive DNA in chromosomes using FISH with directly labeled peptide nucleic acid (PNA) probes. We used CFF to measure the abundance of interstitial telomeric sequences in Chinese hamster chromosomes and major satellite sequences in mouse chromosomes. Using CFF we also identified parental homologs of human chromosome 18 with different amounts of repetitive DNA.  35      2.1 Introduction  The chromosomal makeup of cells can be studied with various methods. The most common technique, karyotype analysis, uses chromosomes that are fixed onto slides. In combination with fluorescence in situ hybridization (FISH) this cytogenetic approach is used to detect deletions, translocations (Bentz, Cabot et al. 1994) and copy number variations (Kallioniemi, Visakorpi et al. 1996) on specific chromosomes. One can also study chromosomes in suspension using flow cytometry. Flow karyotyping uses fluorescent dyes specific for DNA on isolated chromosomes to cluster specific chromosomes based on their size and DNA content. Flow karyotyping has been used to study variations between human chromosomes (Mefford, van den Engh et al. 1997), to detect chromosomal anomalies, to map genes (Van Dilla, Deaven 1990), and to generate chromosome-specific libraries (Davies, Young et al. 1981). Ideally, one would also like to use flow cytometry to study specific DNA sequences in chromosomes. However, there are very few reports using this approach. Polyamide probes that bind to the major groove of double stranded DNA have been used to improve the discrimination of chromosome populations in bivariate flow karyograms (Gygi, Ferguson et al. 2002). It was also reported that hybridization of specific DNA probes on chromosome suspensions followed by microscopy enabled the detection of chromosomal rearrangements (Dudin, Cremer et al. 1987). However, FISH on isolated chromosomes followed by flow cytometry has not been reported, most likely because chromosome suspensions are believed to poorly tolerate the harsh denaturation and wash steps required for in situ hybridization.  Synthetic oligonucleotide probes allow expansion of the scope of FISH beyond qualitative analyses. For example, quantitative FISH (Q-FISH) is used to measure telomere length by FISH 36      using fluorescently labeled 5’-(CCCTAA)3-3’ peptide nucleic acid (PNA) oligonucleotide probes (Lansdorp, Verwoerd et al. 1996). The uncharged peptide backbone of PNA permits annealing to complementary target sequences without the electrostatic repulsion inherent to negatively charged DNA or RNA probes, resulting in higher binding affinity for complementary single stranded DNA and a quantitative relationship between the number of bound fluorescent probes and the length of repetitive DNA target sequences. Q-FISH can be used to quantify telomere length in a chromosome-specific manner (Lansdorp, Verwoerd et al. 1996), but acquisition and image data analysis from even a few dozen cells is labor-intensive. Flow FISH (Baerlocher, Vulto et al. 2006, Rufer, Dragowska et al. 1998), a method for quantitative measurement of telomere repeats in cells by flow cytometry, has a greater statistical power than Q-FISH, but can only be used to measure the average telomere length in cells without chromosome-specific information. Here we report a chromosome-specific, quantitative FISH method that is more rapid than Q-FISH with the high statistical power of flow FISH.  2.2 Results and discussion     2.2.1 Preservation of flow karyograms following FISH  We based our protocol on the polyamine chromosome isolation procedure (Sillar, Young 1981), with minor modifications to suit particular cell types. First, we compared bivariate flow karyotypes of chromosome suspensions to that of chromosomes taken through the denaturation and wash steps of FISH before flow analysis using the standard HO and CA3 dyes (Fig. 2-1a and Supplementary Fig. 2-1). Most chromosome clusters in bivariate plots of HO versus CA3 37      fluorescence could still be recognized after FISH, albeit with some loss of resolution in larger Chinese Hamster and human chromosomes. The resolution of mouse flow karyograms appeared to improve after denaturation (Supplementary Fig. 2-1). Taken together, these data suggest that FISH is compatible with flow karyotyping, enabling FISH analysis of specific chromosomes with PNA probes by flow cytometry.  2.2.2 Chromosome-specific measurement of interstitial telomere repeats in CHO chromosomes   Next, we used Chinese hamster ovary (CHO) cells to optimize the various steps involved in CFF. Hamster chromosomes contain regular (TTAGGG)n telomeric sequences as well as interstitial (TTAGGG)n sequences (Meyne, Baker et al. 1990) of variable size that are difficult to measure using traditional methods such as Q-FISH. Hybridization of a fluorescently labeled (Cy5-(CCCTAA)3) PNA probe on metaphase chromosomes on slides (Fig. 2-1b) or in suspension (Fig. 2-1c) revealed fluorescence signals corresponding to both telomeric and intrachromosomal (TTAGGG)n sequences. Compared to hybridization without probe, flow cytometric analysis of chromosomes hybridized with the telomere probe showed a wide range of Cy5 signal intensities, corresponding to different chromosome clusters (Fig. 2-1d). We further confirmed the specificity of the signal by hybridizing chromosome suspensions with a Cy5- labeled probe to (TTAGGC)n repeats of Caenorhabditis elegans telomeres, which displayed no signal on metaphase spreads (data not shown), but had weak fluorescence proportional to chromosome size by flow cytometry, most likely reflecting non-specific probe binding (Supplementary Fig. 2-2). In a series of experiments (described in Supplementary Note) we optimized the detection of specific fluorescence in CHO chromosomes (Figure 2-1d and 38      Supplementary Figs. 2-3a and 4) to maximize the distinction of the chromosome clusters by CFF. We further validated CFF fluorescence measurements by direct comparison to telomere repeat measurements using Q-FISH (described in Supplementary Note and Supplementary Fig. 2-5). We obtained an excellent correlation between CFF and Q-FISH, albeit one that was not linear for very bright fluorescence signals. We presume that the very bright fluorescence from the longest internal repeats in some CHO cell chromosomes was poorly resolved using Q- FISH because of limitations in the dynamic range of our digital image cytometry setup.                Figure 2-1 Preservation of flow karyograms following FISH enables chromosome-specific detection of interstitial telomere repeats in CHO chromosomes. (a) HO and CA3 bivariate flow karyograms of chromosomes isolated from CHO cells using the polyamine method before (Standard) and after denaturation of the sample in 70% formamide at 80°C for 5 minutes (FISH). (b) Hybridization of a telomere probe (5’-Cy5- (CCCTAA)3-3’) on CHO cell metaphase spreads (b) or on chromosomes in suspension (c). Scale bars, 20 μm (b) and 5 μm (c). (d) Chromosome-specific hybridization pattern compared to the background fluorescence in chromosomes hybridized without probe (green background) before (non optimized) and after (optimized) selection of optimal hybridization conditions and instrument configuration.  39      40      2.2.3 Chromosome-specific detection of major satellite DNA in mouse cell lines from various backgrounds  We next tested whether we could transfer conditions optimized for CHO cell chromosomes to different species and PNA probes. We had previously shown that unidirectional, highly repetitive major satellite sequences are present in all mouse chromosomes except the Y chromosome (Falconer, Chavez et al. 2010b). We studied the array size variability of major satellite sequences on chromosomes isolated from mouse cell lines of different genetic background: 3T6 (Swiss-albino) fibroblasts, C166 (NMRI-GSF × CD-1) endothelial cells and C1 (129/S) embryonic stem cells. Using a PNA probe specific for major satellite DNA, we observed chromosome-specific fluorescence patterns that varied with the mouse genetic background (Fig. 2-2a and Supplementary Fig. 2-6). Detection of satellite sequences by CFF improved the resolution of chromosome populations that were poorly resolved in the bivariate flow karyogram (Supplementary Fig. 2-7). As major satellite DNA repeat expansion patterns differ in mouse chromosomes from distinct backgrounds, CFF appears an attractive method to both analyze and sort chromosomes from hybrid cells. Alternatively, CFF could provide more resolution than in-gel analysis for the detection of satellite DNA instability.  2.2.4 Discrimination of parental homologs of chromosome 18 in a human cell line  Finally, we tested the specificity and the sensitivity of CFF by hybridizing human chromosomes with a PNA probe to chromosome 18-specific centromeric alpha satellite D18Z1 (Devilee, 41      Slagboom et al. 1986). Hybridization of L1.84 PNA on chromosomes isolated from HT1080 human fibrosarcoma cells identified two populations with distinct Cy5 fluorescence, corresponding to the two parental homologs in those cells (Fig. 2-2b). Chromosome 18 Cy5 intensity peaks were present at a 2:1 ratio, suggesting a trisomy (Fig. 2-2c). Hybridization of the same probe onto metaphase spreads confirmed both the trisomy and the D18Z1 repeat array size difference between the parental homologs (Fig. 2-2d). The size and sequence diversity of alpha satellite arrays in human chromosomes (Choo, Vissel et. al. 1991) suggests that CFF using various specific alpha satellite probes could enable the purification of parental chromosomes homologs by flow cytometry. Sequencing of libraries built from such purified chromosome populations could be useful to establish haplotypes.    Figure 2-2 Chromosome and allele-specific analysis of satellite DNA in mouse and human cell lines. (a,b) Bivariate flow karyograms of HO versus CA3 (top) and Cy5 versus CA3 (bottom) fluorescence of C166 mouse chromosomes hybridized with a major satellite PNA probe (5′-Cy5- GACGTGGAATATGGCAAG-3′; a) and of chromosomes from HT1080 human fibrosarcoma cells hybridized with L1.84 PNA probe to D18Z1 satellite DNA (5′-Cy5- GAGAATTGAACCACCG-3′; b). Chromosome 18 data are shown in red. (c) Cy5 fluorescence intensity histogram of chromosomes shown in b. Above background fluorescence (*), the two peaks correspond to the two chromosome 18 populations (marked ** and ***). Median Cy5 fluorescence and event frequencies are listed. (d) Hybridization of the L1.84 probe on HT1080 metaphase spreads. Copies with dull Cy5 fluorescence (**) and one with brighter fluorescence (***) are marked. Scale bar, 15 μM.    42      43      2.3 Conclusion  CFF enabled rapid, chromosome-specific measurements of specific DNA repeats. The approach appears promising for quantification of telomere repeats per chromosome and for detection of rare events that can be missed when analyzing limited cell numbers by Q-FISH. We expect that CFF will find many applications for analysis and sorting of chromosomes based on specific DNA repeat sequences. Simultaneous use of multiple probes labeled with different, nonoverlapping fluorochromes is expected to increase the analytical power of CFF.    44      2.4 Material and methods  2.4.1 Cell culture and chromosome isolation  Chinese Hamster Ovary (CHO) cells (ATCC) were grown in MEM-α (Gibco) supplemented with 10% fetal calf serum (Hyclone), 200 µM L-Glutamine and penicillin streptomycin (Gibco). C166 mouse epithelial cells (ATCC), 3T6 mouse fibroblasts (ATCC) and HT1080 human fibrosarcoma cells were grown in DMEM (StemCell Technology Inc.) supplemented with 10% fetal calf serum, 200 µM L-Glutamine and penicillin streptomycin. C1 mouse embryonic stem cells (Ding, Schertzer et al. 2004) were grown in DMEM (StemCell) supplemented with 20% fetal calf serum, 100 ng/ml leukemia inhibitory factor (LIF), non-essential amino acids (Gibco), sodium pyruvate, L-Glutamine and penicillin streptomycin (Invitrogen). Chromosome suspensions were prepared using a modified polyamine-based method (Carrano, Gray et al. 1979, Sillar, Young 1981) to obtain good quality flow karyotypes that can be stored up to a few weeks without degradation (Trask 1989). Briefly, exponentially growing cultures were blocked with 0.1 µg/ml of Colcemid (Gibco) for 3-4 hours and mitotic cells were collected by mitotic shake off. The cells were centrifuged at 350g for 5 minutes at room temperature (18–25°C). The pellets were resuspended in hypotonic solution (55 mM KCl and 20 mM HEPES; pH 7.4) for 3 to 15 minutes, depending on the cell type. We used 5 ml of hypotonic solution when working with ~200,000 or fewer isolated mitotic cells and 10 ml when more cells were collected. CHO cells were treated in hypotonic solution for 15 minutes, C166 and 3T6 for 5 minutes, C1 for 3 minutes and HT1080 for 12 minutes. The swollen cells were then spun down at 350g for 3 minutes at room temperature and resuspended in freshly prepared ice-cold chromosome isolation buffer (2 mM EDTA, 0.5 mM EGTA, 15 mM Tris-HCl, 80 mM KCl, 20 mM NaCl, 0.1% (vol/vol) 2- 45      mercaptoethanol, 0.1% Triton X-100, 0.2 mM spermine and 0.5 mM spermidine). Approximately 1 ml of chromosome isolation buffer per 200,000 collected mitotic cells was used. After 15 minutes of incubation on ice, the chromosomes were liberated by vigorously vortexing for 75 seconds. The number of chromosomes obtained per isolation was estimated by counting the number of collected cells and multiplying this number with the estimated mitotic index and the known ploidy. The chromosome suspensions were stored at 4°C for up to 3 weeks before use, and we stained them with 40 μg/ml CA3 and 2 µg/ml HO (Sigma) at least 4 hours before analysis.  2.4.2 PNA FISH on suspension chromosomes  We pretreated 1–10 million isolated chromosomes per condition with 100 U/ml of RNase T1 (Sigma) for 20 minutes at room temperature. After testing variables as described in Supplementary Note, the following hybridization and wash conditions were used. The chromosomes were centrifuged at 350g for 5 minutes at 4°C and resuspended in 100 μl of hybridization solution (70% deionized formamide (EMD), 0.25% blocking reagent (PerkinElmer), 4.1 mM Na2HPO4, 0.45 mM citric acid, 1 mM MgCl2 and 10 mM Tris-HCl; pH 7.4) prewarmed at 80 °C. The PNA probes 5′-Cy5-(CCCTAA)3-3′ (vertebrate telomere probe), 5′-Cy5-(GCCTAA)3- 3′ (C. elegans telomere probe) and 5′-Cy5-GAGAATTGAACCACCG-3′ (L1.84 satellite probe) were a gift from Boston Probes (now Applied Biosystems). The 5′-Cy5- GACGTGGAATATGGCAAG-3′ (mouse major satellite DNA) PNA probe was obtained from Panagene. PNA probes were used at a concentration of 0.3 μg/ml (telomere probes), 0.2 μg/ml (L1.84 probe) and 0.75 μg/ml (major satellite probe). Non optimized conditions for hybridization of the telomere probe on CHO cell chromosomes were 3 μg/ml probe, 5 minutes denaturation 46      time and two washes after hybridization. The DNA in chromosome suspensions was denatured at 80°C for 5 minutes and allowed to hybridize at room temperature for 60 minutes. Excess probe was washed twice in 0.5 ml prewarmed hybridization solution for 5 minutes at 37°C. Chromosome pellets were resuspended in 350 μl of chromosome isolation buffer, and stained with CA3 and HO overnight at 4°C. Chromosome preparations were filtered using 35 μm cell strainer cap tubes (BD Biosciences) to remove any large clumps that may have been created during the hybridization procedure.  2.4.3 Chromosome sorting and analysis  Chromosome sorting and fluorescence analysis was performed using a BD Influx cell sorter (BD Cytopeia) equipped with two Coherent I305C argon lasers. The first laser was tuned to emit UV light (351.1 nm) for HO excitation, and the second laser was tuned to 457.9 nm for CA3 excitation. Both lasers were used at 200 mW power. Initial experiments (data presented in Fig. 2-1d and Supplementary Figs.2-2 and 4) used a Coherent Radius 30 mW 635 nm diode laser for Cy5 excitation, which was replaced by a 125 mW 642 nm diode laser (Melles Griot 56CRH/52796) for ‘optimized’ experiments. Cy5 excitation power was varied (Supplementary Note) using the 125 mW 642 nM laser as follows: 10 mW and 50 mW were obtained using neutral density filters, 117 mW was delivered without any filters and 180 mW was obtained using a higher-voltage power supply. Excitation power intensity was measured with a laser power meter. Non optimized conditions for the detection of Cy5 fluorescence were 30 mW 635 nM laser with 60 mW of PMT amplification. Optimized conditions for the detection of Cy5 fluorescence were 125 mW 642 nM laser with 50 mW of PMT amplification. HO, CA3 and Cy5 emission were collected with a 460/50 bandpass filter, a 470 nm long-pass filter and a 670/40 47      bandpass filter (Semrock), respectively. HO was used as the trigger signal for acquisition. Data on pulse width, HO, CA3 and forward scatter were collected for 50,000–100,000 events per condition. Flow cytometry data were acquired with the Software Spigot (BD Cytopeia) and results were analyzed with FlowJo version 8.8.6 software (Tree Star). Debris and clumps were excluded from the flow karyograms by gating on high CA3 and low pulse width (Supplementary Fig. 2-8).  2.4.4 Q-FISH on metaphase and sorted chromosomes  Q-FISH (Lansdorp, Verwoerd et al. 1996) was performed on CHO cell and HT1080 cell metaphase spreads as well as on sorted CHO cell chromosome populations. Metaphase spreads were prepared according to standard procedures. Briefly, exponentially growing cells were blocked using 0.1 μg/ml of colcemid (Gibco) for 3–4 hours, collected and swollen in hypotonic solution (55 mM KCl and 20 mM HEPES; pH 7.4) for 10 minutes and fixed 3 times in methanol:acetic acid 3:1. Cells were dropped on slides to obtain metaphase spreads that were taken through the Q-FISH protocol. CHO cell chromosome populations (Supplementary Note) were sorted directly onto a microscope slide using the BD Influx cell sorter, fixed for 20 minutes in 4% formaldehyde and air-dried overnight before being processed through Q-FISH. For the Q- FISH procedure, the slides were fixed with 4% formaldehyde, treated with 1 mg/ml pepsin (Sigma), dehydrated in 70%, 90% and 100% ethanol and air-dried. Chromosomal DNA was denatured for 3 minutes at 80°C in hybridization mixture (70% formamide, 0.25% blocking reagent (PerkinElmer), 4.1 mM Na2HPO4, 0.45 mM citric acid, 1 mM MgCl2 and 10 mM Tris-HCl; pH 7). Probes were used at a concentration of 0.2 μg/ml of Cy5-L1.84 PNA (HT1080 metaphase spreads), 0.5 μg/ml of Cy5-telomere PNA (CHO cell metaphase spreads) or 0.5 μg/ml of Cy3– 48      telomere PNA (sorted CHO cell chromosome populations). Hybridization was performed at room temperature for 1 hour before slides were washed with 70% formamide and 10 mM Tris- HCl (pH 7.2) twice for 15 minutes each, and with a solution of 0.05 M Tris-HCl (pH 7.2), 0.15 M NaCl and 0.05% Tween-20 three times for 5 minutes each. Slides were dehydrated, air-dried, counterstained with 0.2 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) and mounted in antifade solution Vectashield (Vector Laboratories). Fluorescence signal was visualized on a Zeiss Axioplan 2 microscope equipped with an X-Cite 120 high-pressure metal lamp (EXFO) using a 63× oil-immersion objective (Carl Zeiss) and the images were acquired with an Axiocam MRM digital camera controlled by Isis version 5 software (Metasystems). The following filters (Chroma) were used in our microscope setup: 31000 DAPI (350/50 excitation, 400 LP dichroic and 460/50 emission); 41007 Cy3 (535/50 excitation, 565 LP dichroic and 610/75 emission); 41008 Cy5 (620/60 excitation, 660 LP dichroic and 700/75 emission). Separate DAPI and Cy3 images were analyzed with the TFL-TELO V.2 software (Poon, Martens et al. 1999) for quantification of interstitial telomere repeats on sorted CHO cell chromosomes (Supplementary Fig. 2-5).  2.4.5 Statistical analyses  The Q-FISH frequency histograms as well as measured median telomere length, 25th and 75th percentiles (Supplementary Fig. 2-5c) were obtained using Origin software version 5.0 (MicroCal). The CFF frequency histograms and median Cy5 value, 25th and 75th percentiles (Supplementary Fig. 2-5c) were obtained using FlowJo version 8.8.6 software (Tree Star). Pearson coefficient correlation (alpha = 0.05) between CFF (median Cy5) and Q-FISH measurements (median telomere length) for the seven XY pairs corresponding to the six sorted 49      chromosome populations (Supplementary Note and Supplementary Fig. 2-5) was calculated using GraphPad Prism 5 software (GraphPad Software, Inc.).  2.5 Supplementary Note  2.5.1 Optimization of hybridization conditions  We optimized conditions to detect specific fluorescence from telomere repeats in CHO chromosomes as follows. We selected chromosome clusters with low (red) and high (blue) fluorescence with the Cy5 labeled telomere probe (Supplementary Fig. 2-3a and b) and plotted their fluorescence (Supplementary Fig. 3c) to ensure that increased fluorescence was not associated with decreased specificity. Additionally, we calculated the ratio of Cy5 fluorescence from chromosomes with dim telomere signals over the background fluorescence (no probe control) to select conditions favoring reproducible detection of dim fluorescence (Supplementary Fig. 2-3c). Similarly, we optimized the signal resolution by selecting conditions that maximize the ratio of Cy5 signal emitted by chromosomes with bright telomere fluorescence over the signal emitted by chromosomes with dim fluorescence. Using this approach, we evaluated various parameters involved in the hybridization process, including the number and volume of post hybridization washes, the probe concentration and the denaturation time (Supplementary figure 2-3c). We selected hybridization conditions that showed good specificity and resolution. For example, when optimizing probe concentration, using more probe increases the dim signal over background ratio (bottom histogram), but the increase in dim fluorescence signal is not proportional to the increase in probe concentration (top curve), indicating that the fluorescence signal increase is due to non specific binding of the probe. We 50      determined the best conditions to be 5 minutes heat denaturation at 80°C in the presence of 0.3 µg/ml telomere Cy5 PNA probe, followed by hybridization for 60 minutes at room temperature (18-25°C) and two 0.5 ml post hybridization washes at 37°C.  2.5.2 Optimization of flow cytometry settings  We used similar criteria to determine the optimal instrument configuration and setting for the flow cytometer. To investigate if CFF measurements were limited by the number of excitation photons, we measured telomere fluorescence at different laser power using different lasers and neutral density filters. The ability to distinguish dim fluorescence from background fluorescence improved with increased laser power for excitation of the Cy5 dye in the telomere probe (Supplementary Fig. 2-3b and c), in agreement with a previous study that demonstrated an improved resolution of chromosome clusters in HO versus CA3 bivariate flow karyograms with increasing laser power (Ng, Carter 2010). We obtained optimal excitation at 117mW of 642nm light, significantly more than generated by the most common lasers used to excite Cy5 in flow cytometers. Changes in the photomultiplier tubes (PMT) settings had no effect on the resolution of bright versus dim signals (Supplementary Fig. 2-3c), indicating that fluorescence detection is not limited by PMTs on our instrument in the power range tested. It is important to note that increasing PMT power is an amplification procedure that will also increase the background fluorescence (Supplementary Fig. 2-3c, top curve). We determined an excitation power of 117 mW and a PMT power of 50 mW to be optimal to detect dim Cy5 fluorescence signal above background while preserving the resolution of bright signals. When using a lower laser excitation power, PMT power can be further increased to distinguish chromosome clusters with 51      dim fluorescence intensities from background, but at the cost of resolution between clusters of similar fluorescence intensities (Supplementary Fig. 2-4b).  2.5.3 Correlation of between measurements by CFF and Q-FISH  We validated the potential of CFF for quantitative measurements of telomere repeats by comparing CFF to Q-FISH, the current standard for chromosome specific telomere length measurements. We sorted six specific chromosome clusters by fluorescence activated cell sorting (FACS) (Supplementary Fig. 2-5a) and fixed them onto slides. Representative Q-FISH images for each of the six sorted chromosome clusters are presented in Supplementary Figure 2-5b and individual telomere measurement histograms for each of these sorted clusters are available in Supplementary Figure 2-5c. Note that the sixth sorted cluster (cluster F) corresponds to two distinct chromosomes (Fa and Fb) which cannot be discriminated on the bivariate HO versus CA3 flow karyogram, but which are easily identifiable by microscopy (Supplementary Fig. 2-5b) and CFF (Supplementary Fig. 2-5a). CFF measurements correlated with Q-FISH measurements (Pearson’s = 0.9847) (Supplementary Fig. 2-5d), highlighting the potential of CFF for quantitative analysis of repeat numbers on specific chromosomes. However, note that the dimmest fluorescence signals from CHO chromosomes are near the limit of resolution of our current CFF method (Supplementary Fig. 2-5d).          Supplementary figure 2-1 Preservation of flow karyograms following FISH. HO and CA3 bivariate flow karyograms of chromosomes isolated from murine (C166) and human (HT1080) cell line cells using the polyamine method before (Standard) and after denaturation in 70% formamide at 80°C for 5 minutes (FISH).  52                 Supplementary figure 2-2 Hybridization specificity of PNA probes by CFF. Chromosome specific hybridization of telomere probe (Cy5-(CCCTAA)3) hybridized to CHO chromosomes compared to the background fluorescence of chromosomes hybridized without probe or with a non specific probe (Cy5- (GCCTAA)3).          53        Supplementary figure 2-3 Parameters affecting Cy5 fluorescence signal for quantitative measurements of telomere repeats. (a) Bivariate flow karyograms of CHO chromosomes hybridized in suspension with a Cy5- labeled PNA telomere probe (Cy5- (CCCTAA)3). Chromosomes with a low (red) and a high (blue) number of interstitial telomere sequences are shown. (b) Histograms representing background (black), low (red) and high (blue) Cy5 fluorescence at variable power (in mW) of the 642 nM laser used to excite Cy5. The red asterisk represents the selected condition. (c) Selection of hybridization conditions for quantitative measurements of telomere repeats using discrimination between background, low and high Cy5 fluorescence at different protocol parameters; number of post-hybridization washes, concentration of probe, denaturation time, PMT power and excitation power. The red bar highlights the selected, optimal condition. 54       Supplementary figure 2-4 Improvements on Cy5 signal resolution and detection above background after optimization. (a) Specific and non-specific fluorescence of CHO chromosomes following hybridization with telomere probe compared to the background fluorescence (no probe) before (Non optimized) and after (Optimized) selection of optimal hybridization conditions and instrument configuration. The green box represents the range of background fluorescence. Non optimized conditions were 3 µg/ml probe, 5 minutes denaturation time and no post hybridization wash steps, 30mW 635 nm laser and 60 mW PMT amplification. Optimized conditions were 0.3 µg/ml probe, 5 minutes denaturation time and 2 post hybridization washes, 125mW 642 nm laser power and 50 mW PMT amplification. (b) Detection of human chromosome 18 in HT1080 human chromosomes using a probe against the D18Z1 alpha satellite sequence using optimized hybridization conditions with a 30 mW laser and 60 mW PMT amplification (left) or a 125 mW laser and 50 mW PMT amplification (right). 55          Supplementary figure 2-5 Comparison of quantitative fluorescence measurements of telomere repeats by CFF to measurements of sorted CHO chromosomes using Q-FISH. (a) The six indicated CHO chromosome clusters (A-F, red circles) were sorted on slides by FACS for telomere length measurements by Q-FISH. (b) Representative telomere Q-FISH images of the sorted chromosome clusters. The sixth chromosome cluster (F) consists of two chromosomes (Fa and Fb) which are indistinguishable in the Hoechst versus chromomycin flow karyogram but have different amounts of interstitial telomeric sequences by CFF and Q-FISH (c) Telomere length measurements of the sorted chromosome clusters measured by Q-FISH (median telomere length in kb) and CFF (median fluorescence in arbitrary units). (d) Correlation between the values obtained by Q-FISH and CFF for the same chromosome cluster. The range of telomere fluorescence obtained with murine (red box) and human (blue box) chromosomes is shown for comparison. FU: arbitrary fluorescence units. ¡: Median, -: Cy5 FU 25/75th percentiles, +: telomere length (kb) 25/75th percentiles. 56             Supplementary figure 2-6 Distinct hybridization patterns of major satellite probe in mouse chromosomes HO and CA3 bivariate flow karyograms and Cy5 versus CA3 plots of chromosomes isolated from mouse cell lines from different backgrounds and hybridized with a major satellite PNA probe (Cy5-GACGTGGAATATGGCAAG). 3T6 (Swiss-albino fibroblasts), C166 (NMRI-GSF × CD-1 endothelial cells) and C1 (129/S embryonic stem cells).      57               Supplementary figure 2-7 Improved definition of poorly resolved chromosome populations in mouse flow karyograms HO and CA3 bivariate flow karyogram of C166 (mouse) chromosomes hybridized with a major satellite PNA probe (Cy5-GACGTGGAATATGGCAAG). Cy5 fluorescence intensity histograms can resolve two poorly defined clusters (red and blue) into three and two distinct populations respectively.      58        Supplementary figure 2-8 Gating strategy to remove debris from analysis plots Gated and ungated HO and CA3 bivariate flow karyogram of HT1080 (human) chromosomes containing debris. Gating is performed on a chromomycin/pulse width plot (debris present in the lower left corner).   59      60      Chapter 3  Sorting of parental homologs of human chromosome 18 for sequencing    61      3.1 Introduction  Massively parallel sequencing, such as paired-end sequencing with the Illumina technology (Bentley, Balasubramanian et al. 2008), has made detailed studies of the genetic makeup of a growing number of normal and diseased cell types accessible to an increasing number of laboratories. Along with the enormous possibilities and potential offered by short-read sequencing come a few drawbacks. Massively parallel sequencing is not a favored method for de novo sequencing of large genomes due to the difficulty to assemble short reads over large genomic regions. For complex genomes, such as the mammalian genome, paired-end sequencing has mainly been used as a re-sequencing tool, where sequence reads are aligned to a reference sequence that has previously been built using long-read Sanger sequences and restriction maps of bacterial artificial chromosome (BAC) libraries (Trapnell, Salzberg 2009, Chaisson, Brinza et al. 2009). Another limitation of using short read sequencing includes the presence of gaps, miscalled bases or misalignments, in addition to an inadequate determination of large scale variations such as the presence and size of repetitive elements or the allelic combinations present on each of the parental homolog of a chromosome.  Human chromosomes display some degree of size heterogeneity that can be observed between normal individuals. Traditional (Geraedts, Pearson 1974, McKenzie, Lubs 1975) and flow cytogenetics (Harris, Cooke et al. 1987a, Langlois, Yu et al. 1982, Trask, van den Engh et al. 1989b, Trask, van den Engh et al. 1989a, van den Engh, Trask et al. 1988, Boschman, Rens et al. 1991, Mefford, van den Engh et al. 1997, Trask 1989) have been used to detect and measure those differences, mainly attributed to variations in the array size of tandemly repeated DNA present in heterochromatic and satellite regions that are inherited in a Mendelian fashion 62      (Trask, van den Engh et al. 1989a). In chapter 2, section 2.2.4, complete resolution of two chromosome 18 parental homologs on a flow karyogram is achieved by CFF using a probe for the alpha satellite D18Z1, suggesting that parental homologs of a chromosome may be purified and sequenced separately.  In this chapter, we describe sequencing of human chromosome 18 purified from the HT1080 fibrosarcoma cell line with the Illumina technology, providing a targeted, deeper coverage of this specific chromosome compared to whole genome sequencing. Moreover, we show that we can use CFF to identify and purify the two parental homologs of chromosome 18 according to the array size of their D18Z1 satellite DNA. We also show that individual, high quality Illumina sequencing libraries can be built from a few hundred chromosomes, and that paired-end sequencing of purified parental homologs enables the allelic phasing of each homolog without requirement for additional familial information.   63      3.2 Material and methods  3.2.1 Cells and cell culture  HT1080 (Rasheed, Nelson-Rees et al. 1974) cells with a multimeric lacO array integrated on chromosome 4q (B49.5 cells, described previously (Chubb, Boyle et al. 2002) were a kind gift from Wendy Bickmore (Edinburgh). They were cultured in Dulbecco’s modified Eagle’s medium (DMEM, STEMCELL Technologies) + 10% FCS (Hyclone) supplemented with 200 µM L- glutamine (STEMCELL Technologies) and penicillin and streptomycin (STEMCELL Technologies). Cells were maintained in a humidified atmosphere of 95% air and 5% CO2.     3.2.2  Chromosome isolation  Isolation of chromosomes from HT1080 cells was performed as described in chapter 2, section 2.4.1.   3.2.3 FISH on sorted chromosomes  FISH on chromosomes sorted on slides was performed as described in chapter 2, section 2.4.4, with the following modification: instead of the telomere specific probe, a Cy3-labeled PNA (L1.84)  probe against the D18Z1 α-satellite, specific to chromosome 18 (Devilee, Slagboom et al. 1986) (O-GAG-AAT-TGA-ACC-ACC-G-CONH2) was used at a 0.5 µg/ml concentration.  64      3.2.4  FISH on suspension chromosomes  FISH on isolated chromosomes was performed essentially as described in chapter 2, section 2.4.2, with the following modifications: instead of the telomere specific probe, a Cy5-labeled L1.84 PNA probe against the D18Z1 α-satellite, specific to chromosome 18 (Devilee, Slagboom et al. 1986) (O-GAG-AAT-TGA-ACC-ACC-G-CONH2) was used at a 0.2 µg/ml concentration.    3.2.5 Flow Cytometry and chromosome sorting  Analysis by flow cytometry of chromosomes after FISH was carried as described in chapter 2, section 2.4.3. Appendix 2 illustrates the flow cytometry setup for chromosome analysis. For construction of the human chromosome 18 library, 100,000 chromosomes were sorted following gating according to the CA3/HO DNA profile. For construction of allele-specific chromosome 18 libraries, 150-2,500 chromosomes per library were sorted according to their Cy5 fluorescence level as well as their CA3/HO DNA profile.   3.2.6  Library construction for Next Generation Sequencing  Human chromosome 18 DNA extraction and library construction (section 3.3.1) was performed at the Genome Sciences Center (Vancouver) using standardized procedures for paired-end sequencing. Briefly, DNA was sonicated to obtain fragments of approximately 200bp. After end- repair and A-tailing, Illumina pair-end primers were ligated on the sheared DNA and amplified for 15 rounds by PCR. Fragments of the appropriate size (250 bp ± 50 bp) were purified from a polyacrylamide gel and used for paired-end sequencing (one lane, 50 bp reads). 65       For allele-specific sequencing of human chromosome 18, DNA extraction and library construction were performed with a modified library construction protocol standardized in our laboratory (Ester Falconer) that has been optimized for DNA from single cells. Briefly, DNA was fragmented by a short micrococcal nuclease (MNAse) digestion immediately following chromosome sorting in chromosome isolation buffer (150 to 2,500 sorted chromosomes). DNA was extracted with phenol chlorophorm using linear polyacrylamide (LPA) as a co-precipitating agent. End-repairing, A-tailing and pair-end primer ligation were performed according to standard procedures in low volumes. Samples were barcoded so as to run a pool of multiple samples on a single sequencing lane. After fifteen rounds of PCR amplification, constructed libraries were quality checked by visualization on an Agilent High Sensitivity DNA chip to ensure proper fragment size and yield. A total of fourteen libraries were pooled for sequencing, including the five homolog 18.1 libraries and the four homolog 18.2 libraries that are being discussed in this chapter. The pooled barcoded libraries sent to the Genome Sciences center for polyamide gel electrophoresis (PAGE) size purification (200-350 bp) followed by sequencing (one lane, 76 bp paired-end sequencing reads).  3.2.7 Alignment and single nucleotide variant analysis  Sequencing data was aligned to the human reference genome and analyzed as described below. Single nucleotide variants (SNVs) were used as identification markers for respective chromosome 18 alleles. In order to identify  and map these SNVs, the raw sequence files were aligned to the human genome (NCBI build 36) using the Byrrows-Wheeler aligner (bwa) (Li, Durbin 2010), compressed in BAM format and processed using SAMtools (Li, Handsaker et al. 66      2009). Files were first sorted by coordinates, and then converted to pileup format in SAMtools, generating values for read and alignment mapping quality scores together with read depth at every nucleotide. Files were then sequentially filtered, first by excluding all reads mapping to chromosomes other than chromosome 18, then by excluding all covered positions in which the reference and reads were identical (non-SNV positions). The resultant list of chromosome 18 SNVs was further filtered on high mapping and base calling quality scores. To ensure that only high-quality SNVs were included in the analysis, a phred-scaled mapping quality of >10 was selected (at least 90 % probability that the read alignment is correct) and a phred-scaled base quality of >20 was used (at least 99 % probability that a SNV was correctly called). A read depth of at least 20 (section 3.3.2) or 5 (section 3.3.5) was selected in order to ensure adequate coverage of both alleles. Subsequently, regions where the read depth was over 100 were excluded, as these tended to represent regions of repetitive DNA such as centromeres. All resulting candidate SNVs were converted to Sam enhanced format to include the frequencies of each allele, and the major and minor allelic variant frequencies were determined at all heterozygous sites within the bulk chromosome 18 library (sections 3.3.1 and 3.3.2). 67      3.3 Results    3.3.1  Purification of human chromosome 18 for Solexa Illumina sequencing  Chromosomes isolated from the human HT1080 fibrosarcoma cell line were stained with HO and CA3 and bivariate flow karyotyping with a high resolution of individual chromosome populations was performed (Figure 3-1a). The chromosome 18 cluster was identified and 100,000 chromosomes were sorted in chromosome isolation buffer for DNA extraction and library construction. FISH on a fraction of the sorted chromosomes fixed on a slide was performed with a probe specific to chromosome 18 to determine the purity, which was found to be >90% (Figure 3-1b).  Paired-end sequencing (Illumina) was performed as described (Bentley, Balasubramanian et al. 2008) in collaboration with the Genome Sciences Center (Vancouver). No amplification step was required prior to library construction. 32 million, 50 base pairs reads were obtained from one lane of paired-end sequencing. 22 million (69%) of those reads mapped back to the human genome and 15.2 million (70% of the human reads), mapped to chromosome 18, for an average coverage depth of 13X. Reads mapping outside chromosome 18 include centromeric regions of other chromosomes (Supplementary fig. 3-1a), potentially at regions containing repetitive DNA families closely related to sequences present on chromosome 18 (Appendix 3). Reads mapping outside chromosome 18 also include portions of chromosomes 12 and 8p. These reads likely originate from a translocated chromosome which clusters near our gated chromosome 18 population on a bivariate flow karyogram (Figure 3-1a, black arrow). 68       3.3.2 Single nucleotide variant frequencies on chromosome 18  We used loci that were heterozygous (SNV) in our bulk chromosome 18 sequenced library to identify markers that would be specific for each parental homolog. In total, we found 2664 SNV with at least 20X coverage. The distribution of these SNV, along with the frequency of each allele present at these variant loci is presented in Figure 3-1c. The relative spread of SNVs is relatively uniform across the length of chromosome 18, with the exception of the centromeric region, which contains degenerate repeat regions where reads could map inappropriately. Relative allelic representation at variant loci does not appear to follow a normal distribution centered at 50% as would be expected when there is an equivalent representation of both parental homologs (Figure 3-1c and d). In contrast, variants appear to follow a bimodal distribution, in agreement with results from the previous chapter (section 2.2.4 and Figure II-1b- d) that determined based on the array size of the D18Z1 satellite, that our HT1080 clone had a chromosome 18 trisomy, with a 2 to 1 ratio of small to large satellite array size homolog. Calculating and mapping the frequency of the each allele (major and minor) at variant loci of our sequenced chromosome 18 data confirms both the presence and the relative representation of the two different parental homologs. The average representation frequency of the major variant is 66.75% and, for the minor variant at the same loci, 33.25%.  SNV alleles, therefore, manifests on average a 2 to 1 ratio, which strongly suggests that one allele is derived from the two copies of chromosome 18 that have a smaller D18Z1 array, and is represented twice as much as the other allele, which is derived from the single chromosome 18 copy that has a larger D18Z1 array.      Figure 3-1 Paired-end sequencing of purified human chromosome 18 (a) Chromosome 18 gating on the HO/CA3 bivariate karyogram for fluorescence activated cell sorting (FACS). Black arrow: possible t(8;12). (b) FISH on sorted chromosomes fixed on a slide with the chromosome 18 specific D18Z1 satellite DNA probe L1.84 to confirm purity of the sorted chromosome population (>90%). (c) Distribution of single nucleotide variants (SNVs) along chromosome 18 in our bulk chromosome 18 sequenced library. (d) Relative frequency of allelic variants representation at in our chromosome 18 sequenced library. 69      70      3.3.3 Purification of parental homologs of human chromosome 18 and Illumina library construction   As described in Chapter 2 (section 2.2.4 and Figure II-2), HT1080 cells have a chromosome 18 trisomy that can be distinguished by the array size of their D18Z1 satellite by both FISH on metaphase spreads or by chromosome flow-FISH. Here we therefore sought to use FACS to purify the parental homologs of chromosome 18 to generate sequencing libraries.  We hybridized chromosomes isolated from HT1080 cells with a PNA probe against the D18Z1 satellite. As described previously, two populations of chromosome 18 are clearly defined and can be easily gated on a CA3/Cy5 fluorescence density plot (Figure 3-2a). The two populations of chromosome 18 (named 18.1 and 18.2 for dim and bright gated Cy5 populations, respectively) were sorted in Eppendorf tubes containing either 10 µl of DNA extraction buffer (EB, Qiagen) or 10 µl of polyamine chromosome isolation buffer (CIB) and immediately transferred on ice to be processed for library construction. As anticipated, the chromosome 18.1 and 18.2 populations were recovered at a 2 to 1 ratio. Aliquots of 150 to 2,500 chromosomes were used to optimize DNA fragmentation and library construction parameters.  Using a modification of the standard Illumina procedure optimized for single cell library construction, we were able to generate barcoded sequencing libraries for samples between 150 and 2,500 sorted chromosomes, orders of magnitude smaller than our original chromosome 18 bulk library. Chromosomes sorted into CIB yielded higher quality libraries than sorting them into EB (Figure 3-2b), with a narrower range of fragment size and higher yield of recovery, most likely due to a better conservation of nucleosome structure in CIB. 71      3.3.4 Individual Illumina sequencing of human chromosome 18 parental homologs   Paired-end sequencing (76 bp reads) of pooled barcoded libraries generated in our laboratory generated 76 million reads in one sequencing lane, 80% of which mapped back to the human genome. The libraries generated from FACS sorted parental homologs of chromosome 18 generated a total of 6.1 million reads for the chromosome 18.1 libraries and 4.7 million reads for the chromosome 18.2 libraries. Libraries for chromosomes 18.1 and 18.2 showed a marked enrichment of chromosome 18 sequences (Figure 3-2c) compared to the rest of the human genome, with 77% and 46% of the reads mapping back to chromosome 18 for an average coverage of 6X and 5X, respectively. The amplification step included in the library construction procedure only gave rise to a minimal number of PCR duplicate reads in our sequencing. Reads mapping outside chromosome 18 appeared to mainly align to centromeric and pericentromeric regions (Supplementary fig. 3-1b and c), suggesting that these reads mostly consist of repetitive sequences present on several human chromosomes that are unspecifically aligned (Appendix 3).     Figure 3-2 Sorting of parental homologs of chromosome 18 for paired-end sequencing (a) FACS purification of parental homologs 18.1 (blue gate) and 18.2 (red gate) from HT1080 chromosomes hybridized with a Cy5-labeled L1.84 PNA probe to D18Z1 satellite. (b) Typical Agilent High Sensitivity DNA assay of Illumina libraries constructed from parental chromosome 18 populations sorted in either DNA extraction buffer (Qiagen, EB) or chromosome isolation buffer (CIB). (c) Mapping of reads to the human genome shows enrichment of chromosome 18 reads in purified chromosome libraries. Blue: 18.1 library, red: 18.2 library, black dashed lines: expected frequency, calculated from the relative chromosome size compared to the whole human genome.  72      73      3.3.5 Determination of allelic combinations over the entire chromosome 18  We then sought to determine the allelic ratio of SNVs in chromosome 18.1 and 18.2 reads and comparing it to our bulk chromosome 18 reads (sections 3.3.1 and 3.3.2) to determine if we obtained homolog specific libraries from CFF sorted chromosomes. Figure 3-3a depicts the combined allelic ratios at 4619 SNVs that were sequenced in all three libraries, with at least 10X coverage in the bulk chromosome 18 library and 10X combined coverage in 18.1 and 18.2 libraries. Allelic ratios were calculated by determining the proportion of reads belonging to one allele over the total covered reads. As described in section 3.3.2, our bulk chromosome 18 sequence library harbors two different alleles present at a 2 to1 ratio, as illustrated in figure 3-1d by an allelic ratio of 66.75% for the major variant and 33.25% for the minor variant at the same loci. Sequenced libraries from purified parental homologs of chromosome 18 show a further bias, with over 70% of chromosome 18.1 and 95% of chromosome 18.2 SNV harboring a single variant at heterozygous SNV loci (figure 3-3a, major variant). The 18.2 homolog appears to have been better purified, with a large majority of unique (100%) allelic representation at variant sites. When present, the minor variant generally displayed around 10% allelic frequency for both homologs (Figure 3-3a).  We next used the information obtained from purified sequenced libraries from both homologs to determine the allelic combinations unique to each. In figure 3-3b, variants (or major variants) have been attributed to either homolog and are illustrated in blue (18.1) or red (18.2). Both purified homolog reads show a mainly uniform distribution pattern. When a second variant is present, it almost consistently corresponds to the allele present on the other homolog, indicating some level of contamination from that second homolog during the sorting process. Finally, when 74      comparing our homolog specific libraries to reads obtained from our bulk chromosome 18 library, the allele present on the homolog 18.1 mostly corresponds to the major variant and is present in about two thirds of the reads, while the alleles attributed to the homolog 18.2 represent the other third of the reads (Figure 3-3b), further confirming that our HT1080 cell line contains two copies of the 18.1 homolog for one copy of the 18.2 homolog.    Figure 3-3 Allelic frequencies at single nucleotide variants on parental homologs of chromosome 18 (a) Frequency plots illustrating allelic representation of the major and minor allele present in each library at 4619 SNV sites on the entire chromosome 18. Gray: 18 bulk, blue: 18.1, red: 18.2. (b) Allelic representations of 18.1 (blue) or 18.2 (red) major variants at SNV sites in chromosome 18 libraries. SNV between base pairs 18,446,791 and 34,273,957 at low (4619 variants), medium (500 variants) and high (50 variants) resolution. Dark: A/T, light: G/C. 75       76      3.4 Discussion and conclusions  Application of the basic principle of simultaneous sequencing of hundreds of thousands of short sequences by imaging has enabled the sequencing of individual genomes faster and cheaper than with the traditional Sanger method. Sequencing depth, along with the ability to detect rare events, decreases with genome complexity due to the spreading of the reads over a larger genome portion. For example, human chromosome 18 represents approximately 2.5% of the human genome, so 40 times more reads are required to cover the entire genome. The use of flow sorted chromosomes to generate sequencing libraries therefore provides the means for deeper coverage over the region of interest. Here, we used flow sorted chromosomes to generate a chromosome 18 specific sequencing library. Paired-end sequencing of this library using the Illumina technology enabled a 13X average coverage over the entire chromosome 18. Covering the entire human genome only once would have required 60 million 50 bp reads, nearly twice as many total reads as we obtained from one sequencing lane of purified chromosome 18. Calculating allelic frequencies at SNV that are heterozygous between parental homologs our in cell line shows a 2 to 1 allelic frequency, confirming our previous observation of trisomy 18 in section 2.2.4.  We also tested the generation of sequencing libraries for each parental homolog of chromosome 18 with starting DNA amounts several orders of magnitude below what is normally required to obtain sequencing libraries with adequate coverage. Coverage of chromosome 18 appeared unbiased, with the exception of reads aligning to the repetitive centromeric and pericentromeric regions, as expected. Analysis of SNVs on the chromosome 18 of our HT1080 77      clone shows a clear bias towards one allele or the other, with the vast majority of loci displaying only one allele in our homolog specific libraries.  Mapping back to the variants associated to homolog 18.1 or 18.2 back to our bulk chromosome 18 sequenced library confirms that the alleles present on the 18.1 population represent approximately two thirds of the allelic fraction at variant loci, and that the alleles associated to the 18.2 population represent the other third. These results indicate that the generation of sequencing libraries from flow-sorted parental homologs of chromosomes detected by CFF can provide a means to determine the allelic combinations associated to each homolog separately. Other methods permitting the phasing of entire chromosomes without family information involve isolating single chromosomes using microdissection or, more recently, microfluidic devices (Fan, Wang et al. 2011). Although microfluidics promises to become a very powerful tool to isolate single parental homologs, disadvantages associated with this single cell methodology such as poor statistical power and requirements to compensate for cell-to-cell variation greatly reduces its ability to detect rare variants in mosaic cases.  Sequencing of chromosome parental homologs provides the means to obtain very high resolution haplotyping for each homolog. In addition, since homologs can be physically isolated from one another, high resolution epigenetic profiling using ChIP-seq (Johnson, Mortazavi et al. 2007) or genome-wide bisulfite sequencing (Meissner, Mikkelsen et al. 2008, Smith, Gu et al. 2009, Lister, Pelizzola et al. 2009, Cokus, Feng et al. 2008) should also be possible and would enable not only to study the genetic differences, but also how genetically identical regions of chromosome homologs could be differentially regulated. 78      In summary, we demonstrated that we can purify and sequence chromosomes isolated by flow cytometry to obtain a deeper coverage of a specific region. Furthermore, using CFF, we are able to isolate and sequence parental homologs of the same chromosome, providing the means to determine allelic combinations over the span of an entire chromosome.                      Supplementary figure 3-1 Reads coverage in chromosome 18 sequencing libraries Chromosome specific coverage of sequenced reads for (a) bulk chromosome 18, (b) 18.1 and c. 18.2 libraries shows enrichment and uniform coverage of the chromosome 18. Bin size: 200 kb. Gray: forward direction, orange: reverse direction, o: saturated regions, —: centromeres. Peaks A, B, C and D mapping outside chromosome 18 are further characterized in Appendix 3.  79       80       81       82      Chapter 4  Purification of sister chromatids based on DNA template strands by chromosome orientation flow FISH    83      4.1 Introduction   For each cell division, genetic material has to be distributed evenly between the two daughter cells. Prior to mitosis, duplicated chromosomes, consisting of two genetically identical sister chromatids, are aligned on the metaphase plate, where sister chromatids are attached to opposite spindle poles and recruited to each daughter cell. Although it is generally believed that sister chromatids are functionally identical, it is not clear and perhaps even unlikely that all chromatin and other epigenetic marks on sister chromatids are also identical. There is evidence that the segregation of sister chromatids does not always occur randomly in cells that undergo asymmetric division (for example: Falconer, Chavez et al. 2010b, Potten, Owen et al. 2002, Smith 2005, Shinin, Gayraud-Morel et al. 2006, Armakolas, Klar 2006). Epigenetic differences between sister chromatids with or without non-random distribution of sister chromatids during asymmetric cell division suggests that sister chromatids could result in differences in gene expression between daughter cells (Lansdorp 2007).  Sister chromatids are genetically equivalent, making the study of their epigenetic state at the time of cell division challenging. However, there are some inherent differences that can be used to identify them. Semi-conservative replication of DNA results in two sister chromatids, each consisting of one parental DNA (template) strand and one newly synthesized DNA strand. Chromosome orientation FISH (CO-FISH) (Goodwin, Meyne 1993) is a method developed to identify (the orientation of) DNA template strands. The CO-FISH procedure is based on selective degradation of the newly synthesized DNA strands with BrdU followed by hybridization of unidirectional FISH probes on metaphase spreads, allowing identification of sister chromatids according to the presence of specific DNA template strands (Meyne, Goodwin 1995). 84        In this chapter, we aimed to develop a method for CO-FISH on isolated chromatids in suspension to purify chromatid template strands according to their orientation by flow cytometry (Figure 4-1). The study of template strands purified according to their orientation could enable us to separately examine the epigenetic status of sister chromatids. Using inducible expression of constitutively active Separase S1126A, previously shown to arrest mitosis by causing premature chromatids disjunction (Holland, Taylor 2006), we were able to conditionally separate sister chromatids in human fibrosarcoma cells. We generated flow karyograms of good quality from chromatids isolated from Separase S1126A expressing cells arrested in mitosis. In addition, we modified our previously developed CFF protocol (described in chapter 2) to perform CO-FISH in suspension chromosomes and chromatids using a unidirectional fluorescent probe (L1.84) to the chromosome 18 specific alpha satellite D18Z1.        Figure 4-1 Strategy for identification of template strands by flow cytometry on separated chromatids following CO-FISH. The thymidine analog BrdU is added to cell cultures for one entire round of DNA replication (s- phase), where it will be incorporated into newly synthesized DNA. DNA strands with incorporated BrdU are less stable than the parental template strands and exposition to ultraviolet (UV) radiation following incorporation of the DNA dye Hoechst selectively breaks the newly synthesized strand. Fragments of broken DNA can be degraded, leaving only the template strands, which can be identified with a strand specific probe against unidirectional tandem repeats. When CO-FISH is performed on separated sister chromatids, it is possible to distinguish and sort template strands from either direction.    85      86      4.2 Materials and methods   4.2.1 Cells and cell culture  B49.5 cells (Chubb, Boyle et al. 2002), a subclone of HT1080 (Rasheed, Nelson-Rees et al. 1974) human fibrosarcoma cells with a multimeric lacO array integrated on chromosome 4q were a kind gift from Wendy Bickmore, MRC Human Genetics Unit, Edinburgh, U.K. Cells were cultured as described in Chapter 2, section 2.4.1.   4.2.2  Cloning (Lox Separase system)  To produce separated sister chromatids from HT1080 cells, we aimed to express Separase conditionally. For this purpose, we electroporated HT1080 cells with a plasmid encoding a FKBP-destabilized (Banaszynski, Chen et al. 2006) CreERT2 (Leone, Genoud et al. 2003) and plasmid DNA containing floxed DNA encoding non-phosphorylatable S1126A mutant Separase (Holland, Taylor 2006) (Figure 4-2). Stable clones were selected in 400 µg/ml hygromycin and 100 µg/ml puromycin, respectively. Induction of Cre recombinase was tested by adding 500 nM of Shield-1 (Clontech, Mountain View, Ca) the stabilizing ligand of the destabilizing domain FKBP and 500 nM 4-Hydroxytamoxifen (4OHT) (H7904, Sigma, St Louis, MO) to the culture medium. Cre expression was monitored using a floxed Cherry-Venus reporter system (Figure 4-2), where Shield-1 and 4OHT treatment led to conversion of Cherry to Venus fluorescence monitored by flow cytometry. The best clone, HT1080 WBCFS11, was selected based its ability to switch from Cherry to Venus fluorescence and to accumulate in mitosis upon Cre induction by the addition of Shield-1 and 4OHT.       Figure 4-2 Strategy for conditional induction of separated chromatids Plasmid vectors introduced into HT1080 cells for conditional expression of Separase S1126A. The first vector encodes a FKBP domain-destabilized Cre recombinase restricted to the cytoplasm by an ERT2 domain. The second vector encodes a Separase S1126A gene that is situated after a pair of lox sites. A third vector to control for expression and activity of Cre recombinase encodes a floxed Cherry fluorescent protein, followed by a Venus fluorescent protein. Lox sites recombination following induction of Cre recombinase with Shield-1 and 4OHT permits Separase-1 expression and Cherry to Venus conversion. CBA: Chicken β-actin promoter. PGK: phosphoglycerate kinase promoter. CAG: cytomegalovirus early enhancer /chicken beta-actin promoter.   87      88      4.2.3 Chromosomes and chromatids isolation  Isolation of chromosomes from HT1080 cells was performed as described in chapter 2, section 2.4.1. Isolation of chromatids from HT1080 WBCFS11 cells was performed essentially as described in section 2.4.1, with the following modifications: cells were treated with 10 µM Shield- 1 (Cheminpharma) and 50 µM 4-OHT (Sigma) 18-21 hours prior to harvest. Media was changed 3 hours prior to harvest in order to limit the collection of dead cells during mitotic shake-off and no Colcemid treatment was required to arrest the cells in mitosis. For the CO-FISH experiments, 8 µM of BrdU was added to the culture media 15-16 hours prior to mitotic cells harvest.   4.2.4  FISH on suspension chromosomes and chromatids  FISH on isolated chromosomes and chromatids was performed essentially as described in chapter 2, section 2.4.2.  4.2.5  CO-FISH on suspension chromosomes  CO-FISH on suspension chromosomes and chromatids was performed as follows: 1-20 million isolated chromosomes/chromatids labeled with BrdU were pre-treated with 100 U/ml of RNAse T1 for 20 minutes at room temperature, spun down at 1300g for 5 minutes at 4°C and re- suspended gently in a low volume of chromosome isolation buffer containing 10 µg/ml HO. After 15 minutes incubation at room temperature, nicks were created in the BrdU substituted strand by UV irradiation (Stratalinker 1800) of the preparations for 25 minutes. The nicked strand was 89      then degraded by Exonuclease III (New England Biolabs, #M0206) digestion in 1X NEB Buffer 1 for 15 minutes at room temperature. DNA was denatured for 3 minutes at 70°C in hybridization mixture (final concentration: 70% formamide, 0.2 µg/ml Cy5-O-GAG-AAT-TGA-ACC-ACC-G- CONH2 probe, 0.25% blocking agent, 10 mM Tris-HCl and 10 mM MgCl2 buffer). Hybridization was conducted for 1 hour at room temperature. Non-specifically bound probe was removed by two 5 minutes, 37°C washes in wash buffer (70% formamide, 0.1% BSA, 10 mM Tris-HCl and 10 mM MgCl2). Chromosome/chromatid preparations were re-suspended in chromosome isolation buffer containing 0.6 µg/ml DAPI and 40 µg/ml CA3, 6-20 hours prior to analysis.   4.2.6 Cell cycle analysis following Cre-mediated overexpression of Separase S1126A  HT1080 WBCFS11 cells (2X 105/well) were treated with 10 µM Shield-1 and 50 µM 4-OHT for 0, 6, 12, 18 and 24 hours prior to harvest. Cells were fixed in 70% ethanol at 4 degrees Celcius overnight, washed in phosphate buffered saline (PBS), and stained with Propidium Iodide (PI) staining buffer [PBS, 3.8 mM Na-citrate, 0.5 mg/ml RNAse A and 40 µg/ml PI] for 1 hour on ice. DNA content was measured on a FACSCalibur with the acquisition software CellQuest (BD Biosciences) and cell cycle analysis was performed on the FlowJo version 8.8.6 software (Tree Star). 90       4.2.7 Flow Cytometry and chromatid sorting  Analysis of chromosomes and chromatids after FISH and CO-FISH by flow cytometry was carried as described in Chapter 2, section 2.4.3. Appendix 2 illustrates the flow cytometry setup for chromosome analysis.    91      4.3 Results  4.3.1  Generation of chromatids from a human cell line conditionally expressing Separase S1126A  Separase is the cysteine protease responsible for cleaving the cohesin bridges between sister chromatids prior to cell division (reviewed in (Uhlmann 2007)). We decided to use a constitutively active, non-phosphorylatable mutant form of the protein, Separase S1126A (Holland, Taylor 2006), to generate mitotic cells with separated sister chromatids. In order to prevent premature chromatid separation during the cloning and expansion process, Separase S1126A was cloned in a vector that requires Cre-mediated recombination of lox sites situated between the gene and the promoter to induce gene expression (Figure 4-2). HT1080 human fibrosarcoma cells with stable incorporation of the lox Separase S1126A construct and a FKB- destabilized Cre-ERT2 construct were generated by electroporation, followed by puromycin and hygromycin drug selection. To induce Cre-mediated Separase S1126A expression, cells were grown in the presence of Shield-1 and 4-OHT. Following drug treatment, we observed an accumulation of rounded cells similar to mitotic cells arrested using spindle poisons. Cells were fixed at various time points following Cre stabilization, and propidium iodide (PI) staining analysis was used to control for evidence and timing of cell cycle arrest. A G2/M cell cycle accumulation could be observed following Separase S1126A induction, starting as soon as six hours after addition of Shield-1 and 4-OHT, with most cells showing a tetraploid DNA content approximately eighteen hours after drug treatment (Figure 4-3a). Prolonged mitotic arrest in mutant Separase-1 overexpressing cells also appears to cause cell death, as indicated by an accumulation of debris on the low end of the pI fluorescence scale. Metaphase spreads were 92      prepared from cells treated for 24 hours with Shield-1 and 4-OHT to confirm the presence of separated chromatid in the G2/M arrested cells (Figure 4-3b).  We next tested whether we could isolate chromatids in suspension using our Separase S1126A inducible system. Cells were treated with Shield-1 and 4-OHT for 18 to 21 hours or with Colcemid for 4 hours prior to harvest and chromosome isolation. Bivariate flow karyograms were obtained from the stained chromosome or chromatids suspensions (Figure 4-3c). As anticipated, the flow karyotypes of chromosomes prepared from Shield-1 and 4-OHT treated cells consisted of smaller populations, emitting roughly half of the HO and CA3 fluorescence that of chromosomes prepared from Colcemid arrested cultures. Increasing the PMT voltage of our HO and CA3 channels on the flow cytometer resulted in flow karyotypes similar to regular chromosome suspensions (Figure 4-3c), suggesting that chromatids preparations can be obtained upon Separase S1126A induction, and that a chromatid flow karyograms can be generated.      Figure 4-3 Conditional induction of separated sister chromatids in the HT1080 cell line (a) DNA content analysis of HT1080 WBCFS11 cells at various time points after Cre-mediated induction of Separase-1 expression. Colcemid is used as a control to indicate a mitotic arrest. (b) Metaphase preparation of HT1080 WBCFS11 cells before (Colcemid blocked) and 24 hours after induction of Separase-1 (no Colcemid). (c) Low and high voltage analysis of HO/CA3 bivariate flow karyograms of chromosomes (or chromatids) isolated from HT1080 WBCFS11 cells.  93      94      4.3.2 Chromosome orientation flow FISH to identify DNA template strands by flow cytometry  Finally, we sought to modify our CFF protocol to identify DNA template strands using CO-FISH (Goodwin, Meyne 1993) (Figure 4-2). Prior to chromosome or chromatid isolation, we allowed our cells to grow for one round of DNA replication in the presence of the thymine analog BrdU, which gets incorporated in the newly formed DNA during synthesis. Incorporation of BrdU in the newly synthesized DNA quenches HO fluorescence (Latt 1973), so we opted to replace HO by DAPI (Meyne, Bartholdi et al. 1984, Lebo, Gorin et al. 1984, Hutter, Stoehr 1985) to generate bivariate flow karyograms of chromosomes with BrdU substitution (Figure 4-4a), as it has a similar spectral shift upon binding to DNA (Lin, Comings et al. 1977), with negligible fading of fluorescence (Schnedl, Mikelsaar et al. 1977). Hybridization of HT1080 chromosomes in suspension with a Cy5-labeled probe (L1.84) against the tandemly repeated D18Z1 alpha satellite showed two populations with distinct Cy5 fluorescence intensity (Figure 4-4a), corresponding to the parental homologs of chromosome 18. Hybridization of the chromatid suspension with the L1.84 probe also shows two populations with distinct Cy5 intensity (Figure 4-4a, Shield-1 + 4-OHT). The median intensity of each of the L1.84 positive chromatid populations corresponds to approximately half of the Cy5 fluorescence intensity measured in chromosomes (Figure 4-4c), further confirming the presence of separated sister chromatids in preparations obtained from Shield-1 and 4-OHT treated cells. Degradation of the newly synthesized strand for CO-FISH led to a partial deterioration of the quality of flow karyograms (Figure 4-4b). In chromosomes that have gone through the CO-FISH procedure, many populations still remain recognizable, but a lot of debris is created and the definition of the chromosome populations is greatly diminished. The flow karyograms of chromatids that have 95      gone through CO-FISH in suspension displays more degradation than chromosomes, with poorly defined medium and large populations, but with the small populations still mostly recognizable (Figure 4-4b). Following degradation of the newly synthesized DNA two distinct Cy5 positive signal intensities are still detected after hybridization with the unidirectional L1.84 PNA onto chromosome or chromatid suspensions (Figure 4-4b). In chromosome preparations, CO-FISH L1.84-Cy5 signal intensity is lower than after FISH, suggesting a reduction in probe binding due to a degradation of the BrdU substituted DNA. However, the signal is brighter than expected if only the chromosome 18 template strands remained after the CO-FISH procedure (Figure 4-4c). In chromatid suspensions, as expected, the L1.84-Cy5 signal intensity after CO- FISH is similar to the signal obtained after FISH. However, in agreement with the template strands of only half of the chromatids being in the orientation complementary to our unidirectional probe, there is a diminution in the proportion of Cy5 positive chromatids (0.7% after CO-FISH compared to 1.8% after FISH) (Figure 4-4d).      Figure 4-4 CO-FISH in suspension to identify template strands by flow cytometry CA3/DAPI and CA3/Cy5 flow cytometry plots of L1.84 (a) FISH or (b) CO-FISH signal in chromosomes or chromatids (Shield-1 + 4OHT) isolated from HT1080 WBCFS11 cells that have gone through one round of DNA replication in the presence of BrdU. The L1.84 positive population is colored in red. (c) Obtained (gray bars) and expected (red dotted lines) Cy5 fluorescence in chromosomes or chromatids after FISH and CO-FISH in suspension. The relative fluorescence intensity is normalized to the Cy5 signal for FISH on chromosomes in suspension. The absolute Cy5 fluorescence is indicated at the bottom of the bars. Dark bars: 18.2 (D18Z1 short homolog). Light bars: 18.1 (D18Z1 long homolog). (d) Obtained (gray bars) and expected (red dotted lines) Cy5 positive population frequency normalized to the FISH population frequency. The population frequencies (%) are indicated at the bottom of the bars. 96      97      4.4 Discussion and conclusions  In this chapter, we sought to develop a method to purify template strands by flow cytometry. We developed a model of human fibrosarcoma cells where separated chromatids could be generated by conditional expression of constitutively active Separase S1126A. Upon Cre- mediated Separase S1126A expression, we were able to generate and flow karyotype preparations of chromatids in suspension. Chromatid populations on flow karyograms emit approximately half of the fluorescence intensity of the equivalent chromosome populations and can be better resolved by increasing PMT power for the detection of HO and CA3. In addition, bivariate flow karyograms could be generated from chromosomes and chromatids isolated from cells that have gone for one round of DNA replication in the presence of BrdU. As reported in chapter 2, the parental homologs of chromosome 18 in our HT1080 cells are heterozygous for the size of their D18Z1 centromeric alpha satellite array and can be distinguished by CFF on both chromosome and chromatids preparation.  Bivariate flow karyograms could still be generated after degradation of the newly synthesized DNA for chromosome orientation flow FISH, but a lot of debris was created and the chromosome populations were significantly less defined, an effect that was exacerbated in the chromatid preparations, which tend to have more debris in general. This loss of definition is most likely due to DNA damage created by the UV treatment required to nick the newly synthesized DNA.  Compared to CFF, chromosome orientation flow FISH on suspension chromosomes generated a lower emitted Cy5 intensity, in agreement with degradation of the BrdU substituted DNA. The 98      fluorescence, however, was brighter than expected had we obtained a perfect CO-FISH signal. This could be caused by either an incomplete degradation of the BrdU substituted strand, leaving behind some probe binding sites (Figure 4-5a). Alternatively, the same fluorescence readout could be obtained if BrdU was added to the cells after the start of the S-phase, leading to an incompletely substituted newly synthesized DNA strand. Microscopy imaging of flow cytometry sorted chromosome 18 after CO-FISH in suspension would enable us to confirm incomplete degradation or BrdU substitution of the newly synthesized strand. A bright and a dim signal on every chromosome 18 would signify incomplete CO-FISH.  Chromosome orientation flow FISH in chromatids suspensions generated a L1.84-Cy5 signal of intensity similar to CFF, but with a smaller proportion of the chromosome population being Cy5 positive. After degradation of the newly synthesized, BrdU substituted DNA, we expect that half of the sister chromatids will have an intact template strand that is complementary with our unidirectional L1.84 PNA probe and thus have Cy5 signal, while the other half with have a template strand in the opposite direction and therefore show no FISH signal (Figure 4-5b). Although our results indicate a potentially successful CO-FISH in chromatid suspensions, the poor quality of the flow karyograms makes it difficult to estimate with precision the proportion of Cy5 positive chromatids and is only a secondary way to test for degradation of the newly synthesized strand. In order to validate orientation specific template strand purification, future experiments will require the use of a second unidirectional L1.84 probe that is complementary to the opposite strand (RL1.84). With probes complementary to either strand direction labeled with two non overlapping fluorochromes, complete degradation of the newly synthesized strand in separated chromatids should lead to mutually exclusive labeling with one or the other probe (Figure 4-5b, 2-color CO-FISH). 99       In summary, we developed a model for inducible separation of sister chromatids in human fibrosarcoma cells. We demonstrate that CO-FISH in chromosomes and chromatids is possible in suspension, with limited loss of resolution of the flow karyograms. Inclusion of two probes complementary to opposite DNA strands that are labeled with different fluorochromes will enable us to better control for degradation of the newly synthesized DNA and eventually, separate sister chromatids with opposite DNA template strands by flow cytometry.      Figure 4-5 Models of outcomes for chromosome orientation flow FISH Potential fluorescence readouts of FISH or CO-FISH in suspension indicative of the outcome of the newly synthesized DNA in (a) chromosomes or (b) chromatids. Forward strand: dark gray. Reverse strand (light gray). Template strand: full lines. BrdU substituted DNA: dashed lines.L1.84-Cy5 (PNA probe complementary to forward strand): red. RL1.84-Cy3 (PNA probe complementary to reverse strand): pink.      100      101      Chapter 5  General discussion  102      5.1 Summary of thesis findings   In this thesis, I demonstrate that FISH can be performed on isolated chromosomes to analyze repetitive DNA in a chromosome specific manner using flow cytometry. I showed that this approach can be used to purify parental homologs of human chromosome 18 and that CFF can likely be extended to sort sister chromatids based on defined template strands. The technical advances described in this thesis are novel and open up a large number of possibilities for further studies in genetics and epigenetics.  In chapter 2, I describe the development of CFF, a method to perform FISH on chromosomes in suspension using conditions maintaining the integrity of flow karyograms. Using this method, I demonstrate that CFF with fluorescently labeled PNA probes can detect, in a chromosome specific manner, repetitive DNA in mouse, human and Chinese hamster. Detection of interstitial telomeric repeats in Chinese Hamster chromosomes or major satellite DNA in mouse chromosomes enables the detection of chromosome-specific differences that permits distinction between chromosomes otherwise poorly resolved on a bivariate flow karyogram. I also validated the use of CFF to measure repeats in a chromosome specific manner by comparing fluorescent measurements by flow cytometry to Q-FISH, an already established method. I also demonstrate the sensitivity and specificity of CFF by clearly identifying both parental homologs of chromosome 18 using a probe for the alpha satellite D18Z1 in a human chromosome suspension.  In chapter 3, I describe the generation of a sequencing library specific for human chromosome 18 using FACS sorted chromosomes, generating sequence reads with an average coverage of 103      13X over chromosome 18. I also describe the use of CFF to purify and construct sequencing libraries from both parental homologs of chromosome 18, identified according to the different array size of their D18Z1 alpha satellite sequence. Sequencing separately both homolog population of chromosome 18 enabled us to haplotype each parental homolog over the span of the entire 76 Mb chromosome.  In chapter 4, I describe the development and characterization of a cell line that can be used to purify sister chromatids based on their DNA template strands. Using inducible expression of a mutant separase protein, separated sister chromatids in a human cell line were obtained. Isolated chromatids were used to develop chromatid flow FISH, a method that, for the first time, enables the study of possible epigenetic differences between sister chromatids.  5.2 CFF as an analysis tool: potential applications and drawbacks  CFF was originally developed as a new method to measure the average telomere length in individual cell nuclei. I started on a project that aimed to study the effect of different chromatin environments on telomere erosion, maintenance and elongation in specific chromosomes. Prior to my arrival, the observation had been made that the rate of telomere erosion in the inactive X chromosome is much higher than in the active X chromosome (Surralles, Hande et al. 1999). These previous observations were made using Q-FISH, a microscopy based techniques to measure the telomere length at individual chromosome ends (Lansdorp, Verwoerd et al. 1996). Upon my arrival in the Lansdorp laboratory, the two available methods had advantages and disadvantages. Selection of a telomere measurement method inevitably involves a choice between obtaining chromosome specific measurements (Q-FISH) and cell specific 104      measurements (flow FISH).  The idea of CFF evolved as an intermediate with the promise to maintain the high statistical power required to detect small variations or rare events made possible by flow cytometry (see table 5-1).  The sensitivity of CFF for chromosome specific telomere length measurements still remains to be further tested using mouse and human samples with various known telomere lengths. Moreover, although we can estimate the number of available binding sites for our PNA probes, the exact number, as well as the minimum detection threshold of fluorescent molecules that can be detected by CFF is still unknown and will vary depending on the instrument setup, laser power, degree of probe labeling, fluorochromes used and hybridization conditions. The first three parameters should be controlled using calibration beads with known Molecules of Soluble Fluorochrome (MESF) units, which should permit a more precise instrument calibration and comparison between samples analyzed at different times (Schwartz, Gaigalas et al. 2004, Gerena-Lopez, Nolan et al. 2004). Inter-experimental variations in hybridization conditions could be controlled by spiking beads coated with known amounts of telomere repeats.  Trans factors involved in telomere length regulation, for example the role of proteins involved in the Shelterin complex, can be studied using flow FISH or molecular methods such as terminal restriction fragments (Harley, Futcher et al. 1990). Only two methods, Q-FISH and Single Telomere Length Analysis (STELA), enable the study of cis factors involved in chromosome specific telomere dynamics such as replication timing or chromatin environment. Q-FISH, as mentioned before, is the only method that enables the individual measurement of every end of every chromosome and has been useful in demonstrating the heterogeneous nature of telomere length within cells (Lansdorp, Verwoerd et al. 1996). Its labor intensiveness and the small 105      number of metaphase analyzed (15 to 20) limits its statistical power and thus the ability to detect rare events. STELA, a PCR-based molecular method using primers located in the variable subtelomeric region of chromosomes to amplify specific telomeres (Baird, Rowson et al. 2003) has proven very useful in detecting short outliers and can, unlike Q-FISH (and CFF), measure the telomeres of senescent populations. Unfortunately, STELA is a very laborious method which only works on  a fraction of the forty six different p and q chromosome ends (12q, 11q, 2p, 17p and XpYp). So far, STELA has only been used to measure human telomeres which are significantly shorter than those of laboratory mice models. CFF offers a bit less resolution than Q-FISH and STELA, as telomere measurements are for the whole chromosome (p + q), but has the advantage not only to detect but also potentially purify rare chromosome populations with abnormally short (or long) telomeres.  As demonstrated in chapter 2, CFF is not limited to the detection of telomeric repeats and any type of repetitive DNA, provided that a sufficient number of probe binding sites are present, can be detected. We anticipate that CFF can be used to measure and detect satellite DNA instability to test, for example, the effects of cytotoxic agents, providing a great degree of resolution at the chromosome level, as well as the potential to sort by flow cytometry chromosomes where recombination, satellite expansion or large deletion events occurred.  Table 5-1 Comparison between different FISH-based methods to measure telomere length   106      107      5.3 Sequencing libraries from CFF purified populations: finding epigenetic differences in genetically identical regions of parental homologs  The diploid nature of the human genome makes it difficult to determine the allelic combinations inherited together on a single chromosome. Molecular haplotyping methods based on cloning specific genomic regions show a poor capacity to determine haplotypes across large distances. More recently, whole genome haplotyping has become possible using microdissection (Ma, Xiao et al. 2010) or a microfluidic device (Fan, Wang et al. 2011) that enables the physical separation of most chromosomes from a mitotic cell. Both methods are based on whole genome amplification of a sample where only one homolog of each chromosome is present, followed by array-based genotyping. The potential to automate a microfluidics based whole genome haplotyping method is likely to become a very useful tool for mapping disease loci or in evolution genetics. In addition, both groups propose to increase the number of variants identified by performing whole genome sequencing on their amplified homologs.  A drawback is incomplete coverage consequent to a single copy of the starting material (Ma, Xiao et al. 2010).  Haplotyping methods aim to find sequence differences (SNVs) between each parental homolog and the reference genome, but it is also of interest to study epigenetic differences between parental homologs. Physical separation of parental homologs could enable the study of epigenetic differences in genomic regions that are homozygous between parental homologs, indicative of parental imprinting. An advantage of chromosomes purified using CFF over microdissection or microfluidics is that we can physically separate multiple copies of each parental homolog. As demonstrated in the proof-of-principle experiment presented in chapter 3, we were able to obtain entire chromosome coverage without the requirement of an amplification 108      step prior to library construction. In addition, very few sequences were duplicated, suggesting the potential for a deeper coverage than achieved in our experiment, where only a portion of the constructed library was sequenced. We now hope to take advantage of the wide range of applications offered by the Illumina NGS platform to obtain a whole chromosome epigenetic haplotype, starting from bisulfite sequencing of our purified populations to assess for the presence of differential CpG methylation patterns between homologs. Alternatively, methyl-seq, DNA digestion with methylation sensitive restriction enzyme HpaII or its methylation insensitive isoschizomer MspI followed by library construction and sequencing (Brunner, Johnson et al. 2009) could inform us on the methylation status of our chromosome homologs.  Chromosome isolation is reported to preserve nucleosome structure (Kuo 1982), and core histone 2B can be detected by immunofluorescence in isolated chromosomes (Trask, van den Engh et al. 1984). Moreover, in our hands, the retention of mononucleosome fractions by MNAse digestion prior to library construction is indicative that the nucleosome structure is still somewhat intact after the FISH procedure. This leads to the attractive possibility to use ChIP- seq with antibodies specific for histones associated to active (H3K9ac, H3K4me3) or inactive (H3K27me3) chromatin marks to identify regions differentially regulated between parental homologs.  This proof-of-principle experiment was focused on chromosome 18, but, the diversity and presence of alpha satellites at all human centromeres suggests that the method could be applicable to other human chromosomes.   109      5.4 Sequencing template strands: are sister chromatids functionally equivalent?  Although it is generally believed that genetically equivalent sister chromatids are functionally identical, evidence that the segregation of sister chromatids does not always occur randomly (Falconer, Chavez et al. 2010b, Potten, Owen et al. 2002, Smith 2005, Shinin, Gayraud-Morel et al. 2006, Armakolas, Klar 2006) suggest that sister chromatids may harbor some epigenetic differences. A proposed model is that non-random distribution of genetically identical sister chromatids with different epigenetic marks could regulate gene expression in daughter cells during asymmetric cell divisions (Lansdorp 2007).  So far, sister chromatids have mainly been studied using indirect methods. For example, incorporation of a label such as BrdU has been used to track the inheritance of template strands and identifying label-retaining cells. By developing chromosome orientation flow FISH to sort template strands according to their orientation, we aim to use a direct approach to study differences between sister chromatids. Our goal is to build various sequencing libraries from bisulfite converted or immunoprecipitated sister chromatids containing defined DNA template strands in order to study the epigenetic status of sister chromatids separately.  Several challenges still need to be overcome in order to answer the simple question “are sister chromatids functionally equivalent?”. We first need to validate our chromosome orientation flow FISH protocol on isolated chromatids by a pair of differentially labeled fluorescent probes complementary to opposite strands. Sorting chromosomes populations with mutually exclusive FISH signal will enable us to control for proper degradation of the newly synthesized DNA strand. As mentioned in the previous section, our work on chromosome 18 is still a proof of principle and we hope to expand chromosome orientation flow FISH to other human 110      chromosomes. Moreover, we hope to use our inducible Separase S1126A system to generate separated chromatids from cell models with a well characterized differentiation pattern to test the hypothesis that sister chromatids from self-renewing cells are more similar than sister chromatids from differentiating cells.  5.5 Concluding remarks   In recent years, researchers have used NGS technologies to get a closer look at the genetic makeup of normal and diseased cells and thus impacted all areas of biological research. 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High-resolution cytogenetics. Birth defects original article series, 19(5), pp. 11-37.  Appendix 1  Hardware setup for flow cytometry analysis and sorting of chromosomes  128       Appendix 2  Excitation and emission spectra of fluorochromes used for one and two probes CFF       129      Appendix 3  Characterization of reads mapping outside chromosome 18 Alignement of regions containing large amounts of reads outside chromosome 18 in our purified chromosome 18 sequencing library (Supplementary Figure 3-1a, peaks A-D) to various types of repeated elements using the UCSC Genome Browser.  130        131        132      133      


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