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Single-cell whole genome sequencing reveals no evidence for common aneuploidy in normal and Alzheimer’s… van den Bos, Hilda; Spierings, Diana C J; Taudt, Aaron; Bakker, Bjorn; Porubský, David; Falconer, Ester; Novoa, Carolina; Halsema, Nancy; Kazemier, Hinke G; Hoekstra-Wakker, Karina; Guryev, Victor; den Dunnen, Wilfred F A; Foijer, Floris; Colomé-Tatché, Maria; Boddeke, Hendrikus W G M; Lansdorp, Peter M May 31, 2016

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RESEARCH Open AccessSingle-cell whole genome sequencingreveals no evidence for commonaneuploidy in normal and Alzheimer’sdisease neuronsHilda van den Bos1, Diana C. J. Spierings1, Aaron Taudt1,2, Bjorn Bakker1, David Porubský1, Ester Falconer3,Carolina Novoa3, Nancy Halsema1, Hinke G. Kazemier1, Karina Hoekstra-Wakker1, Victor Guryev1,Wilfred F. A. den Dunnen4, Floris Foijer1, Maria Colomé-Tatché1,2, Hendrikus W. G. M. Boddeke5and Peter M. Lansdorp1,3,6*AbstractBackground: Alzheimer’s disease (AD) is a neurodegenerative disease of the brain and the most common form ofdementia in the elderly. Aneuploidy, a state in which cells have an abnormal number of chromosomes, has beenproposed to play a role in neurodegeneration in AD patients. Several studies using fluorescence in situ hybridizationhave shown that the brains of AD patients contain an increased number of aneuploid cells. However, because thereported rate of aneuploidy in neurons ranges widely, a more sensitive method is needed to establish a possible roleof aneuploidy in AD pathology.Results: In the current study, we used a novel single-cell whole genome sequencing (scWGS) approach to assessaneuploidy in isolated neurons from the frontal cortex of normal control individuals (n = 6) and patients with AD(n = 10). The sensitivity and specificity of our method was shown by the presence of three copies of chromosome 21in all analyzed neuronal nuclei of a Down’s syndrome sample (n = 36). Very low levels of aneuploidy were found in thebrains from control individuals (n = 589) and AD patients (n = 893). In contrast to other studies, we observe no selectivegain of chromosomes 17 or 21 in neurons of AD patients.Conclusion: scWGS showed no evidence for common aneuploidy in normal and AD neurons. Therefore, our resultsdo not support an important role for aneuploidy in neuronal cells in the pathogenesis of AD. This will need to beconfirmed by future studies in larger cohorts.Keywords: Aneuploidy, Single-cell sequencing, Alzheimer’s disease, Brain, NeuronsBackgroundAberrant chromosome copy numbers, aneuploidy, hasbeen observed in the developing and adult human brain.However, the reported frequency of neuronal aneuploidyvaries widely (up to 40 %, with an average of ~10 %)[1–3] with some studies reporting no aneuploid cells atall [4, 5]. Since neurons are post-mitotic, the number ofmethods to screen for aneuploidy is limited and mostof the previous studies used interphase fluorescence insitu hybridization (FISH). Interestingly, several recentstudies using single-cell whole genome sequencing(scWGS) consistently found low levels (2–5 %) of aneu-ploid neurons in the human brain [6–8]. Compared tointerphase FISH, which is intrinsically noisy [9], scWGShas three important advantages: (1) all chromosomes ineach single cell can be analyzed (in contrast to a max-imum of four chromosome-specific probes for inter-phase FISH); (2) each chromosome is probed thousands* Correspondence: p.m.lansdorp@umcg.nl1European Research Institute for the Biology of Ageing (ERIBA), University ofGroningen, University Medical Center Groningen, 9713 AV Groningen, TheNetherlands3Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC V5Z 1 L3, CanadaFull list of author information is available at the end of the article© 2016 van den Bos et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.van den Bos et al. Genome Biology  (2016) 17:116 DOI 10.1186/s13059-016-0976-2of times per cell (thousands of unique reads perchromosome representing distinct chromosomal re-gions); and (3) the results are not affected by variableprobe hybridization or artifacts related to tissue sec-tioning or other causes which can result in false-positive or false-negative results. These advantagesmake single-cell sequencing, at least in theory, a morerobust method for detecting aneuploidy.Interestingly, aneuploidy is thought to be involved inthe pathogenesis of Alzheimer’s disease (AD), the mostcommon form of dementia [10]. Several studies havereported an increased level of aneuploid cells in thebrains of AD patients [1, 5, 11–15]. For example, somestudies showed that extra copies of chromosomes 11,17, 18, and 21 were more prevalent in neurons fromAD patients compared to controls [5, 11–13, 15]. Incontrast, other studies reported evidence for selectiveaneuploidy such as a tenfold increase in chromosome21 aneuploidy [12] or a twofold increase in X chromo-some aneuploidy [14]. That extra copies of chromo-some 21 were repeatedly described in AD neurons isinteresting in view of observations that individuals withDown’s syndrome (DS), who also have an extra copy ofchromosome 21, are much more likely to develop ADand at an earlier age than euploid individuals [16].Based on such observations, it was postulated that tri-somy of chromosome 21 and the resulting extra copyof the amyloid precursor protein (APP) gene, locatedon chromosome 21, could contribute to the pathogen-esis of AD. Indeed, mutations in APP are observed inpatients with familial AD and are known to cause earlyonset AD [17]. In contrast, Thomas and Fenech,although finding high levels of aneuploidy in hippo-campal cells for chromosome 17 and 21 (18 % and12 % for chromosomes 17 and 21, respectively), foundno difference in aneuploidy rates from brains of ADand controls [15], questioning the involvement oftrisomy 21 and 17 in the pathogenesis of AD.Since the reported rates of aneuploidy in AD brainsare mostly based on interphase FISH studies and varywidely, we used scWGS to re-examine neuronalkaryotypes in individuals with different stages ofdementia to determine the frequency of aneuploidyin normal and AD brain. We developed a pre-amplification-free library preparation method andvalidated its ability to karyotype single cells by con-firming the presence of three copies of chromosome21 in single DS cells. We found very low levels ofaneuploid neurons in control and AD brains. Also,no aneuploidy was found in non-neuronal cells of acontrol and AD sample. Collectively, these resultsshow that aneuploidy is not common in normal andAD brain and thus unlikely to contribute to thepathogenesis of AD.Results and discussionValidation of the pre-amplification-free method ofpreparing librariesIn this study, we used single-cell sequencing to assessthe presence of aneuploid cells in the frontal cortex ofnormal postmortem brains and brains affected by AD(Braak stage III to VI). The presence of amyloid plaquesin some of the brain samples classified with Braak stagesIII and VI was confirmed by amyloid β (Aβ) staining(Fig. 1). Nuclei were isolated from sections that were dir-ectly adjacent to sections with amyloid plaques. Singleneuronal nuclei were sorted based on the nuclear neur-onal marker NeuN as described previously [18]. scWGSlibraries were prepared without whole genome pre-amplification (Additional file 1: Figure S1), reducingPCR amplification bias and thereby maintaining a moreFig. 1 Examples of beta-amyloid plaque staining. Representativeimages of the area of the frontal cortex from where nuclei forsequencing were isolated of control individual (a) or AD patientswith Braak stage III (b) or VI (c)van den Bos et al. Genome Biology  (2016) 17:116 Page 2 of 9direct correlation between sequence reads and genomecontent. The distribution of reads across the chromo-somes was used as a faithful indicator of the chromo-some copy number. Since there is no pre-amplificationstep, a particular genomic location is expected to be rep-resented in libraries only twice, one from each homologof diploid individuals. Although the genomic coveragewithout pre-amplification is low, losses of genomic DNAduring library preparation were typically found to berandom. As a result, the distribution of reads mappinguniquely to the reference genome is rather even whichallows accurate calls of chromosome copy number.The copy number state of each chromosome was de-termined using an in-house developed algorithm calledAneufinder [19]. Briefly, this algorithm bins the mappedreads and uses a Hidden Markov Model (HMM) to pre-dict the copy number state (i.e. monosomic, disomic,trisomic, etc.) for each bin. The most common state of achromosome was assigned as the copy number for thatchromosome. This means that when the majority of achromosome is lost or gained, it is called monosomic ortrisomic, respectively. Only libraries that passed thestringent quality metrics as determined by Aneufinderwere used for further analysis: out of a total of 2664single-cell libraries prepared for this study, 1632 librar-ies passed quality control (61 %). From these, we ob-tained on average 858,800 reads per library, of which333,000 reads (with MAPQ >10) mapped to a uniquelocation on the genome and library complexity was es-timated to be 950,000 (see Additional file 2: Table S1for more details). Importantly, the relatively shallowsequencing of the single-cell libraries is sufficient todetermine chromosomal copy numbers. Higher cover-age is possible by sequencing longer reads or fewer li-braries per lane.To ensure that our approach faithfully and reprodu-cibly annotates aneuploid events, we first validated ourmethod by sequencing single neuronal nuclei isolatedfrom a fresh frozen postmortem brain sample from anindividual with DS. Indeed, in all 36 single-cell libraries,we detected three copies of chromosome 21, but no fur-ther aneuploidies (Fig. 2). In addition, two copies ofchromosome X were called, as expected from this femaleindividual. In contrast, all single neurons analyzed in ourstudy isolated from male individuals had only one copyof the X-chromosome (Fig. 3a), further validating oursingle-cell-sequencing platform. Finally, scWGS data ofseveral leukemic and solid tumor samples generatedusing this platform, revealed similar overall copy num-ber variation (CNV) patterns as obtained by array com-parative genomic hybridization (CGH) analysis ([19];Paranita et al., personal communication), validatingonce more our approach to enumerate aneuploidy insingle cells.Low level of aneuploidy in normal neuronsTo assess the rate of aneuploidy in normal healthybrains we examined neurons from six control individualswithout dementia. Of the 589 control neurons analyzed,all but four were euploid (Fig. 3a and Additional file 3:Figure S2A; Additional file 4: Table S2 and Additionalfile 5: Table S3). These four aneuploid cells were foundin one control sample (n = 72): the first of which gaineda copy of chromosome 18, the second cell lost a copy ofchromosome 6, the third gained a copy of both chromo-somes 4 and 16, and the fourth gained a copy of 13, 16,21, and 22. Overall, the total prevalence of aneuploidy,cells with loss or gain of one or more chromosomes, inthe control samples was 0.7 % (95 % confidence interval[CI]: 0.2–1.8 %, n = 589). The aneuploidy per chromo-some, cells with loss or gain of a specific chromosome,was in the range of 0–0.34 % in Fig. 3a, Additional file 3:Figure S2, Additional file 4: Table S2, and Additionalfile 5: Table S3. The aneuploidy rates that we find in anormal brain are remarkably lower than reported by mostother studies which used (interphase) FISH to detect an-euploidy [1–5]. For example, when comparing these re-sults with the per chromosome aneuploidy rates reportedby Iourov et al. [12] and Yurov et al. [14], we found sig-nificantly lower aneuploidy rates for all of the chro-mosomes analyzed in these studies (Mann–Whitney–Wilcoxon rank test, p <0.05 for chromosomes 1, 7, 11,14, 17, 18, 21, and X in Iourov et al. [12] and forchromosomes 1, 7, 11, 16, 17, 18, and X in Yurov et al.[14]) (Additional file 5: Table S3). The FISH-basedapproach can yield noisy results, especially when used ontissue slides (as opposed to single cell suspensions) [9].Our results are more in agreement with other recent stud-ies that sequenced single neurons [6–8] and reported lowrates (2–5 %) of aneuploid cells in normal brain. Similarto our analysis, these studies all analyzed human frontalcortical cells: McConnell et al. found one chromosomeloss and two gains in 110 neurons (2.7 %) [6], Cai et al.reported four out of the 91 analyzed neurons to beaneuploid (4.4 %) [7], and Knouse et al. found twoaneuploidies 89 cells (2.2 %) [8]. In summary, while ourpre-amplification-free single-cell sequencing methodfaithfully detects aneuploidies such as trisomy 21 in a DSindividual (Fig. 2) or X-chromosome monosomy in malecells (Fig. 3 and Additional file 3: Figure S2), it detects verylow levels of aneuploidy in human adult neurons, indicat-ing that previous FISH approaches may have overesti-mated aneuploidy levels in the human brain.Low level of neuronal aneuploidy in ADWhile several groups have reported an increased level ofaneuploidy in brains of AD patients compared to normalhealthy brains, these observations were also based onFISH studies. Importantly, while our and other’s single-van den Bos et al. Genome Biology  (2016) 17:116 Page 3 of 9cell sequencing experiments [6–8] support that aneu-ploidy in a healthy brain has been overestimated in FISHstudies, no single-cell sequencing data were available forAD patients’ neurons. Therefore, we examined 893neurons from ten individuals with AD to investigate apotential role of neuronal aneuploidy in AD. In contrastto previous studies, we did not find evidence for in-creased aneuploidy in brains of AD patients (Fig. 3 andAdditional file 6: Figure S3, Table 1, Additional file 4:Table S2, and Additional file 5: Table S3). In sevenpatients, no aneuploid cells were found, while in the otherthree patients, out of 261 cells a total of five aneuploidcells were found. Of the neurons from AD2 one cell hadan extra copy of chromosome 6, of AD9 two cells losteither chromosome 3 or 21, and in AD10 one cell lostchromosome 12 and another gained chromosome 22. Noevidence for increased rates of trisomy 21 in the assessedAD samples was found (Table 1 and Additional file 5:Table S3). The total neuronal aneuploidy rates in AD werecomparably low as in control samples (0.6 %, 95 % CI:0.2–1.3 %, n = 893). Again, these aneuploidy rates aresignificantly lower than reported previously (Mann–Whitney–Wilcoxon rank test, p <0.001 for chromosomes1, 7, 11, 14, 17, 18, 21, and X in Iourov et al. [12] and forchr1, 7, 11, 16, 17, 18, and X in Yurov et al. [14]). Im-portantly, we can exclude detection issues, as we observedtrisomy 21 in all neurons sampled from a DS control indi-vidual. Furthermore, we failed to detect selective gains ofthe other recurring AD chromosome gains reported inAD (e.g. trisomy 11 and 17). In fact, the few aneuploidieswe did detect appeared to be random, as no particularchromosome loss or gain was found in more than two cells.Interestingly, a recent study using single-cell quantita-tive PCR reported the presence of local copy numbergains, up to 12 copies, of the APP locus in AD neurons[20]. Even though the goal of our scWGS study was toexamine whole chromosome copy number variation, weinvestigated this region more closely in AD neurons. Nocopy number gains of the APP locus were observed(Additional file 7: Figure S4).Although we do not observe a selective gain ofchromosome 21 in neurons from AD patients, there isstill a very compelling observation that individuals withDS develop early onset dementia with brain lesions simi-lar as observed in AD patients [16]. As we focused oursequencing efforts on neurons only, we cannot rule outthe possibility that aneuploidy in other cell lineages inthe brain is involved in the pathogenesis of AD. Increasingevidence suggests an important contribution of the im-mune system to AD pathogenesis (reviewed in [21, 22]).Both microglia and astrocytes, the CNS-resident innateimmune cells, have been shown to be involved in the on-set and progression of AD. So far, no single-cell sequen-cing data are available of these types of cells from ADbrains. Therefore, we also analyzed some non-neuronal(NeuN-negative) nuclei from a control (n = 63) and an ADFig. 2 Trisomy of chromosome 21 is detected in DS cells. a Genome wide copy number plot of a single DS cell. Arrow denotes gain as identifiedby AneuFinder. b Genome wide copy number profile of a population of DS cells (n = 36). Each row represents a single cell with chromosomesplotted as columns. Cells are clustered based on the similarity of their copy number profile. Copy number states are depicted in different colors(see legend)van den Bos et al. Genome Biology  (2016) 17:116 Page 4 of 9(n = 51) sample by scWGS. We found no aneuploid cellsin either of these non-neuronal controls (Fig. 4 andAdditional file 5: Table S3). However, no clear distinctionwas made between the non-neuronal cells and furtherstudies are needed to exclude a potential role of aneu-ploidy in cell types such as microglia or astrocytes in ADneurodegeneration.Taken together, our analysis using scWGS reveals thatthe prevalence of aneuploid cells in the frontal cortex ofcontrol individuals and AD patients is very low.ConclusionsMany recent studies have reported a high prevalence ofaneuploid neurons in AD brains, which led to thehypothesis that neuronal aneuploidy could be involvedin the pathogenesis of AD. However, using a single-cellsequencing approach, we report low levels of aneuploidyboth in neurons from AD patients as well as in neuronsfrom non-diseased individuals. The level of neuronalaneuploidy in our study is much lower than was previ-ously reported [1, 5, 11–15]. Nevertheless several linesFig. 3 scWGS reveals no common aneuploidy in AD neurons. A representative genome wide copy number profile of a population of cells fromcontrol 6 (male, n = 120) (a) and two AD patients AD 2 (male, n = 37) and AD 4 (female, n = 72) (b) sample. Each row represents a single cell withchromosomes plotted as columns. Cells are clustered based on the similarity of their copy number profile. Copy number states are depicted indifferent colors (see legend)van den Bos et al. Genome Biology  (2016) 17:116 Page 5 of 9of evidence strongly support our results. First, ourmethod clearly detected trisomy of chromosome 21 in aDS sample and monosomy of chromosome X in all malesamples showing the accuracy of our approach. Import-antly, the validity of our scWGS method to study CNVsin leukemic and solid tumor samples was validated witharray CGH in separate studies ([19], Paranita et al.,personal communication). The study by Bakker et al.[19] also provides evidence that our technique candetect complex and partial aneuploidies. Second, the an-euploidy rates that we find in normal healthy neuronsare more in line with recent findings from other single-cell sequencing studies [6–8]. Third, we analyzed over1500 neuronal nuclei, which is to our knowledge thelargest single cell sequencing dataset so far. Therefore,although more AD-affected brains should be assessed toexclude rare cases, our results do not support an im-portant role for neuronal aneuploidy in the pathogenesisof AD.Materials and methodsTissue sourcesFresh-frozen postmortem brain samples from the frontalcortex were obtained from the Dutch Brain Bank andfrom the department of Pathology & Medical Biology ofthe University Medical Center Groningen (UMCG). Inthis study, samples from six non-demented controls(Braak stage 0–I) and ten AD patients (Braak stage III–VI)were used. Patient details are listed in Table 1. A fresh-frozen postmortem brain sample from an individual withDS served as a positive control for the detection of trisomyof chromosome 21.Amyloid plaque stainingAmyloid staining was performed to confirm the presenceof amyloid plaques in the brain samples with Braak stageIII and VI. Immunohistochemical staining with anti-bodies directed at Aβ (4G8, 1:500, Biolegend, 800702)was done on 10-μm frozen brain sections. The sectionswere pre-incubated in 0.3 % H2O2 for 30 min andblocked with 10 % normal horse serum in PBS with0.3 % Triton-X100 (Sigma, 9002-93-1) for 30 min. Here-after, sections were incubated overnight at 4 °C with theAβ primary antibody in PBS containing 0.3 % Triton-X100 and 1 % normal goat serum. Unbound antibodieswere washed away with PBS and sections were incubatedfor 1 h at room temperature with horse anti-mouse bio-tinylated secondary antibody (1:400, Vector, BA-2000).Finally, the sections were incubated in avidin-biotin-peroxidase complex (Vectastain ABC kit, VectorLaboratories, PK-6100) for 30 min and visualized withdiaminobenzidine (Sigma, D-5637). Counterstaining wasperformed with cresyl violet for 2 min.Isolation of neuronal and non-neuronal nucleiFrom each sample, ten sections of 50 μm or a smalltissue block (~0.5–1 cm2), cut into pieces, was used fornuclei isolation. Neuronal nuclei isolation was per-formed as described previously [18] with minor modifi-cations. Samples were kept on ice throughout the nucleiTable 1 Brain samples used and aneuploidy levels found per sampleSample ID Age Sex Braak stage Libraries passing QC Aneuploid cellsControl 1 69 M 0 81 0 (0 %)Control 2 74 M 0 80 0 (0 %)Control 3 79 F I 108 0 (0 %)Control 4 82 F 0 72 4 (5.56 %)Control 5 84 F I 128 0 (0 %)Control 6 93 M I 120 0 (0 %)AD 1 64 F VI 32 0 (0 %)AD 2 66 M IV 37 1 (2.70 %)AD 3 73 F V 63 0 (0 %)AD 4 74 F V 72 0 (0 %)AD 5 76 F III 118 0 (0 %)AD 6 80 F VI 125 0 (0 %)AD 7 85 F III 115 0 (0 %)AD 8 85 F VI 107 0 (0 %)AD 9 91 F III 109 2 (1.83 %)AD 10 92 F VI 116 2 (1.72 %)Non-neuron control 84 F I 63 0 (0 %)Non-neuron AD 92 F VI 51 0 (0 %)van den Bos et al. Genome Biology  (2016) 17:116 Page 6 of 9isolation procedure. In short, tissue sections were incu-bated in nuclear isolation buffer (10 mM Tris-HCl[pH 8], 320 mM sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2,0.1 mM EDTA, 1 mM dithiothreitol [DTT], and 0.1 %Triton X-100) for 5 min and filtered through a 70-μmfilter using a plunger. Hereafter, nuclei were purified byultracentrifugation (107,000 g for 2.5 h at 4 °C) througha dense sucrose buffer (10 mM Tris-HCl (pH 8), 1.8 Msucrose, 3 mM Mg(Ac)2, 0.1 mM EDTA, and 1 mMDTT). Supernatant was removed from the pelletednuclei that were washed and resuspended in PBS con-taining 2 % bovine serum albumin (BSA) (PBS/2%BSA).Isolated nuclei were stored in nuclei storage buffer(50 mM Tris-HCl [pH 8], 5 mM Mg(Ac)2, 0.1 mMEDTA, 5 mM DTT, and 40 % glycerol) at –80 °C. Onthe day of sorting, nuclei were washed with PBS/2%BSAand resuspended in PBS/2%BSA containing an antibodydirected against the nuclear neuronal marker NeuN(1:100.000, Millipore) and 4′,6-diamidino-2-phenylindole(DAPI; 10 μg/mL) and incubated for 45–60 min on ice.Single NeuN-positive or NeuN-negative and DAPI lownuclei were sorted into 5 μL freezing buffer (50 % PBS,7.5 % DMSO, and 42.5 % 2X ProFreeze-CDM [Lonza])in individual wells of a 96-well plate using MoFlo-Astrios(Beckman Coulter). Ninety-two single nuclei were sortedper plate. In two wells of each plate, ten nuclei were sortedas positive control and two wells without nuclei served asnegative control. Plates were subsequently centrifuged at500 g for 5 min at 4 °C before being gradually frozen to –80 °C in styrofoam boxes. Plates were stored at –80 °Cuntil library preparation.Pre-amplification-free scWGS library preparationPre-amplification-free scWGS library preparation wasperformed using a modified version of a protocol de-scribed before [23]. All pipetting steps are performedusing a Bravo Automated Liquid Handling Platform(Agilent Technologies, Santa Clara, CA, USA). All DNApurification steps between enzymatic reactions wereperformed using AMPure XP magnetic beads (AgencourtAMPure, Beckman Coulter, Brea, CA, USA). All enzymesused in the library preparation are obtained from NewEngland Biolabs. After DNA fragmentation by micrococ-cal nuclease, end repair, and A-tailing of the DNAfragments was performed in one reaction mix includingT4 DNA polymerase, T4 polynucleotide kinase, and Bst2.0 warm start polymerase. End repair was performed at25 °C for 30 min followed by the A-tailing reaction at68 °C for 30 min. Subsequently without DNA puri-fication, ligation reaction mixture containing T4 DNAligase was added and Illumina PE forked adapters wereligated to either side of the DNA fragments. After cleanup, the adapter containing DNA fragments weredirectly subjected to 17 cycles of PCR using PhusionHigh Fidelity DNA polymerase and custom barcodedprimers. After PCR amplification, a final AMPure beadclean-up was performed and DNA was eluted in 6 μLelution buffer.Fig. 4 scWGS reveals no common aneuploidy in AD non-neuronal cells. Whole genome copy number profiles from non-neuronal cells fromcontrol 5 (female, n = 63) (a) and AD 10 (female, n = 51) (b). Each row represents a single cell with chromosomes plotted as columns. Cells areclustered based on the similarity of their copy number profile. Copy number states are depicted in different colors (see legend)van den Bos et al. Genome Biology  (2016) 17:116 Page 7 of 9Illumina sequencingSince each single-cell library received a unique barcode,libraries can be pooled (multiplexed) and sequenced to-gether. Per 96-well plate, the full volume (6 μL) of thesingle nuclei and negative controls were pooled togetherwith 1 μL of the ten nuclei controls. Size selection wasperformed on a 2 % E-gel EX (Invitrogen) to isolate themononucleosome fragments of approximately 280 bp(range of 200–400 bp). The DNA was eluted from thegel slices using Zymoclean gel DNA recovery kit (Zymo)according to the manufacturer’s protocol. The DNAquantity and quality were assessed using Qubit fluo-rometer (Invitrogen) and Bioanalyzer with High sensitiv-ity chips (Agilent), respectively. For sequencing, clusterswere generated on the cBot and single-end 50 nt readswere generated using the HiSeq2500 sequencing plat-form (Illumina, San Diego, CA, USA). In all runs, a poolof 192 libraries was sequenced on one lane of a flow cell.Data analysisAfter demultiplexing, all reads were aligned to the hu-man reference genome (GRCh37) using short readaligner Bowtie2 (version 2.2.4) [24] with default settings.The resulting BAM files were sorted using Samtools(version 0.1.18) [25] and duplicate reads were markedusing BamUtil (version 1.0.3). Duplicate reads and am-biguous alignments (MAPQ >10) were filtered out usingAneufinder. Estimated complexity was calculated bydownsampling the reads several times and determiningthe fraction of unique reads each time. Then the numberof reads sequenced (seq_reads) was plotted against thenumber of unique reads (uni_reads) and a curve wasfitted through the data points using the formula:uni reads ¼ Cmax  seq readsð Þ= K þ seq readsð Þ;where Cmax was used as an estimation of the complexityof the library: the theoretical maximum unique reads inthat library. K is the number of sequenced reads atwhich the number of unique reads is half of the librarycomplexity. For subsequent CNV assessment, a custompipeline was developed called AneuFinder [19]. Briefly,uniquely mapped reads are counted in, non-overlappingbins of variable size based on mappability with a meanof 1 Mb in size (for details: see Bakker et al. [19]). GCcorrected uniquely mapped read counts, were used asobservables in a Hidden Markov Model (HMM) withseveral possible hidden copy number states from nullis-omy up to decasomy (ten copies). The emission distribu-tions were modeled with a delta distribution for thenullsomy state and with negative binomial distributionsfor all other states, with means and variances that werefixed to multiples of the monosomy-state ones. Param-eter estimates were obtained using the Baum–Welchalgorithm. The final CNV calls were determined as thestate with the highest posterior probability for each bin.Quality controlThe quality of each library was assessed with severalcriteria: genomic coverage, bin-to-bin variation in readdensity (spikiness), entropy, number of ploidy state seg-ments, and Bhattacharyya distance. Using the AneuFinderfunction “ClusterByQuality,” the libraries were clusteredbased on similarity of the quality control aspects(described in detail in Bakker et al. [19]). From eachsample, the highest quality cluster, each of which had spiki-ness <0.21 and Bhattacharyya distance >1.0, were consid-ered good quality libraries and used for aneuploidy calling.StatisticsWilcoxon rank sum test was used to compare groupsusing wilcox.test in R. P values <0.05 were consideredsignificant.Additional filesAdditional file 1: Figure S1. Single-cell, pre-amplification-free librarypreparation protocol overview. Schematic representation of the librarypreparation protocol used: after sorting in 96-well plates, the DNA isfragmented using MNase. End repair and A-tailing are combined in onereaction and directly followed with ligation of the adapters. Barcodes areintroduced during the PCR and after pooling and size selection thelibraries are ready to be sequenced. (PDF 203 kb)Additional file 2: Table S1. Statistics of all single-cell libraries passingquality control. (XLSX 220 kb)Additional file 3: Figure S2. scWGS reveals no common aneuploidy incontrols. Genome wide copy number profiles from single-cell libraries of fivecontrol individuals. Each row represents a single cell with chromosomesplotted as columns. Cells are clustered based on the similarity of theircopy number profile. Copy number states are depicted in different colors(see legend). (PPTX 327 kb)Additional file 4: Table S2. Copy number states from all chromosomesof all libraries. (XLSX 141 kb)Additional file 5: Table S3. Aneuploidy rates per chromosome of allsamples. (XLSX 13 kb)Additional file 6: Figure S3. scWGS reveals no common aneuploidy inAD. Genome wide copy number profiles from single-cell libraries of eightAD patients. Each row represents a single cell with chromosomes plottedas columns. Cells are clustered based on the similarity of their copynumber profile. Copy number states are depicted in different colors(see legend). (PDF 1238 kb)Additional file 7: Figure S4. No local copy number variations at APPlocus in AD neurons. UCSC genome browser view of the APP locus onchromosome 21 for all AD libraries (n = 893; left) and zoomed in for somelibraries (right). (PDF 600 kb)AbbreviationsAD: Alzheimer’s disease; APP: amyloid precursor protein; Aβ: amyloid β;BSA: bovine serum albumin; CGH: comparative genomic hybridization;CI: confidence interval; CNV: copy number variation; DAPI: 4′,6-diamidino-2-phenylindole; DS: Down’s syndrome; FISH: fluorescence in situ hybridization;HMM: Hidden Markov Model; scWGS: single-cell whole genome sequencing.van den Bos et al. Genome Biology  (2016) 17:116 Page 8 of 9AcknowledgementsWe thank members of the Lansdorp lab for their helpful discussions and theoperators at the Central Flow Cytometry Unit of the University MedicalCenter Groningen, Geert Mesander, Henk Moes, and Roelof Jan van der Lei,for their assistance with single-cell sorting. We also thank Nieske Brouwerand Zhuoran Yin from the Department of Neuroscience for their help withsample collection, sectioning, and staining.FundingWork in the Lansdorp laboratory is supported by a European Research CouncilAdvanced grant (ROOTS-Grant Agreement no. 294740). FF is supported by thePediatric Oncology Foundation Groningen (SKOG) and the Dutch CancerSociety (grant 2012-RUG-5549). MCT is supported by a MEERVOUD grant fromthe Netherlands Organization for Scientific Research (NWO) and RosalindFranklin Fellowship from the University of Groningen.Availability of supporting dataThe dataset(s) supporting the results of this article are available in theArrayExpress repository, under accession numbers E-MTAB-4184 andE-MTAB-4185.Authors’ contributionsHvdB performed single-cell experiments, data analysis, and wrote the manuscript.NH, HGK, and KH-W provided assistance in experiments. EF, CN, NH, and DCJSdeveloped and optimized the scWGS library preparation protocol. WFAdDprovided tissue samples and sections and offered technical advice. AST, BB,MCT, and DP designed and trained the AneuFinder pipeline. AST, DP, MCT,and VG provided bioinformatics support. HWGM provided intellectual input.DCJS supervised single-cell experiments, interpreted results, and wrote themanuscript. FF contributed to scientific discussions and helped with writingthe manuscript. PML conceived and supervised the study and wrote themanuscript. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.EthicsAll participants who donated tissue to the Dutch Brain bank gave writteninformed consent. The remaining tissue samples were treated in accordancewith the national guideline “Code Goed Gebruik van Patienten Materiaal” (Codeof Good Use of Patient Material). All experimental methods comply with theHelsinki Declaration.Author details1European Research Institute for the Biology of Ageing (ERIBA), University ofGroningen, University Medical Center Groningen, 9713 AV Groningen, TheNetherlands. 2Institute for Computational Biology, Helmholtz ZentrumMünchen, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany. 3Terry FoxLaboratory, BC Cancer Agency, Vancouver, BC V5Z 1 L3, Canada. 4Section ofPathology, Department of Pathology and Medical Biology, University ofGroningen, University Medical Center Groningen, 9713 AV Groningen, TheNetherlands. 5Section Medical Physiology, Department of Neuroscience,University of Groningen, University Medical Center Groningen, 9713 AVGroningen, The Netherlands. 6Division of Hematology, Department ofMedicine, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.Received: 14 March 2016 Accepted: 4 May 2016References1. Pack SD, Weil RJ, Vortmeyer AO, Zeng W, Li J, Okamoto H, et al. 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