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Oligonucleotide Array Comparative Genomic Hybridization (oaCGH) based characterization of genetic deficiencies… Jones, Martin R; Maydan, Jason S; Flibotte, Stephane; Moerman, Donald G; Baillie, David L Nov 7, 2007

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ralssBioMed CentBMC GenomicsOpen AcceMethodology articleOligonucleotide Array Comparative Genomic Hybridization (oaCGH) based characterization of genetic deficiencies as an aid to gene mapping in Caenorhabditis elegansMartin R Jones*1, Jason S Maydan3, Stephane Flibotte2, Donald G Moerman3 and David L Baillie1Address: 1Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby BC V35 1S6 Canada, 2Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia V5Z 4S6 Canada and 3Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, CanadaEmail: Martin R Jones* - mrjones@sfu.ca; Jason S Maydan - jmaydan@interchange.ubc.ca; Stephane Flibotte - sflibotte@bcgsc.ca; Donald G Moerman - moerman@zoology.ubc.ca; David L Baillie - baillie@sfu.ca* Corresponding author    AbstractBackground: A collection of genetic deficiencies covering over 70% of the Caenorhabditis elegansgenome exists, however the application of these valuable biological tools has been limited due tothe incomplete correlation between their genetic and physical characterization.Results: We have applied oligonucleotide array Comparative Genomic Hybridization (oaCGH) tothe high resolution, molecular characterization of several genetic deficiency and duplication strainsin a 5 Mb region of Chromosome III. We incorporate this data into a physical deficiency map whichis subsequently used to direct the positional cloning of essential genes within the region. From thisanalysis we are able to quickly determine the molecular identity of several previously unidentifiedmutations.Conclusion: We have applied accurate, high resolution molecular analysis to the characterizationof genetic mapping tools in Caenorhabditis elegans. Consequently we have generated a valuablephysical mapping resource, which we have demonstrated can aid in the rapid molecularidentification of mutations of interest.BackgroundA large resource of deletion strains (also known as geneticdeficiencies) accounting for over 70% of the Caenorhabdi-tis elegans genome has been generated by various researchgroups over the past three decades [1]. These genetic defi-ciencies have proven advantageous for a variety of pur-poses including; characterization of mutant alleles [2],bility [4,5] and, most significantly, as tools for positionalcloning of unmapped mutations to discrete regions of thegenome [1,6-8].The full potential of these biological tools has howeverbeen limited due to the lack of high resolution character-ization at a genome wide scale. The mapping of physicalPublished: 7 November 2007BMC Genomics 2007, 8:402 doi:10.1186/1471-2164-8-402Received: 11 August 2007Accepted: 7 November 2007This article is available from: http://www.biomedcentral.com/1471-2164/8/402© 2007 Jones et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 10(page number not for citation purposes)identification of specific loci affecting developmentalprocesses [3], investigation of genome replication and sta-breakpoint positions within each deficiency strain isrequired to allow the deleted gene complement for thatBMC Genomics 2007, 8:402 http://www.biomedcentral.com/1471-2164/8/402strain to be precisely defined. Additionally, genetic defi-ciencies may exhibit molecular complexity preventingtheir reliable use in mapping experiments [9].Previously, characterization of genetic deficiencies hasbeen performed by fairly low resolution or labor intensivetechniques such as genetic linkage mapping, PCR analysis[7] and, more recently, by the application of snip-SNP[9,10] and as a consequence many available deficiencystrains remain poorly characterized.Oligonucleotide array Comparative Genomic Analysis(oaCGH) is an emerging technology for high resolutionmapping of chromosomal copy number changes at agenome wide scale through the comparison of the DNAratio between two samples from the same organism[11,12]. The recent development of a C. elegans specificoaCGH platform for identification of novel single genedeletions [13] represents a powerful technology that canbe adapted to the rapid and precise characterization ofdeficiency mapping strains.In this study we demonstrate the successful application ofoaCGH to the physical characterization of deficiencystrains in C. elegans. We use this data to annotate a physi-cal deficiency map within a 5 Mb region of chromosomeIII and demonstrate the application of this map to aid inthe molecular identification of previously generatedmutations known to reside within this region.Results and DiscussionoaCGH mapped deletions and duplications physically define 17 zones around the dpy-17 region of Chromosome III7 deficiencies and 2 duplications lying in the region ofdpy-17 on chromosome III were chosen for oaCGH analy-sis (Nimblegen) as they have been previously character-ized by both genetic linkage and PCR analysis, and usedto roughly position a large number of unidentified EMSgenerated lethal mutants [7]. After oaCGH mapping hadprecisely defined the gene complement for each of thesedeficiencies a refined candidate gene approach was imple-mented to rapidly identify mutations in essential geneswhich map to this region.Available mapping data for deficiency strains positioneddeletion and duplication breakpoints with an average res-olution of 117 kb. With the incorporation of the oaCGHdata however, breakpoints resolved to within an averageof 5.6 kb and subsequent analysis through PCR amplifica-tion and sequencing allowed for the identification of pre-cise physical breakpoints in strains containing nDf20,sDf121, sDf125, sD128 and sDf135 (Table 1). The remain-ing deficiency strains, sDf127 and sDf130, both containTable 1: Summary of mapping resolution for deficiencies using both genetic and molecular mapping techniques.Genetic/PCR Mapping Data oaCGH Mapping Data aSequenced Breakpoint CoordinatesStrain Deficiency Mutagen Position LEFT Cosmid RIGHT Cosmid LEFT (bp) RIGHT (bp) LEFT (bp) RIGHT (bp) Size of deficiency (kb)MT3022 nDf20 GRI Out egl-5 sma-2 7895240 8678216 7896734 8676211 780In lin-36 dpy-19 7900966 8676164BC4698 sDf121 GRI Out F10G11 C05D2 4568979 5637318 4568042 5637312 1069In F35G12 F54E7 4569058 5636068BC4634 sDf125 UVI Out F42A10 K04C2 6178564 6620842 6178994 6620106 441In C23G10 R13F6 6181034 6619737BC4638 sDf127 UVI Out C23G10 R13A5 6200904 7453868 ND ND d1194–1253In C23G10 T20B12 6200951 7395215BC4690 sDf128 GRI Out C29E4 C06G4 7945153 8023540 7945392 8023387 78In C06G4 F44B9 7945526 8019512BC4637 sDf130 UV Out C32A3 C05D2 3640014 5566628 3640101 5565581 1925In 3640119 5565449Out 5635618 5664335 ND ND e16–29In R13G10 ZC155 5645065 5661035BC4677 sDf135 GRI Out B0361 T20B12 7276848 7479897 7277386 7476402 199In T20B12 R13A5 7277471 7467958bNA sDp3 GRI Out daf-7 dpy-19 cComplex 8611252 NA NA NAIn dpy-18 mig-10 8611166BC3737 sDp8 UVI Out let-721 sma-2 6559307 8714234 NA NA NAIn M01G4 dpy-19 6562088 8710120Average resolution (kb) 117 5.6 0 0aBreakpoint coordinates aligned to Wormbase (release WS170).bsDp3 structure is inferred from the background of the oaCGH data.cThe left breakpoint of sDp3 is fragmented over a large region.Page 2 of 10(page number not for citation purposes)dThe right breakpoint of sDf127 falls within a 50 kb region of low complexity and therefore low probe density and could not be accurately defined.eThe second deleted region in sDf130 was not be resolved by PCR analysis.BMC Genomics 2007, 8:402 http://www.biomedcentral.com/1471-2164/8/402breakpoints which reside in relatively large intergenicregions and since the oaCGH array used for these sampleshas low probe density in intergenic regions [13] PCR andsequencing analysis was not feasible in these cases.By using the refined mapping data obtained from theoaCGH analysis we annotated the deleted gene comple-ment within each mapping strain, creating a physical mapof zones defined by the overlap of the deficiencies andduplications within the region. The resulting map extendsacross almost 5 Mb of the genome and contains 17 zoneswith an average size of 323 kb (Figure 1). Zone 13 is thelargest region, covering 929 kb and containing 204 pre-dicted genes, while zone 8 is the smallest at 22 kb andcontains 5 predicted genes (Table 2). Finally, positionalmapping data available for mutations known to fallwithin this region was incorporated into the map, and inthis way we have been able to assign each mutation to anaccurate and precisely defined list of gene candidates (Fig-ure 1. and Additional file 1).The free translocation sDp3 undergoes significant modification within deficiency strainsIn our initial set of experiments we performed oaCGHusing genomic DNA from three representative deficiencystrains balanced by the free duplication sDp3 ; BC4697(sDf121(s2098) [sDp3]), BC4638 (sDf127(s2428) [sDp3])and BC4690 (sDf128(s2786) [sDp3]) (Figure 2), usingA revised genetic and physical map of the dpy-17 region of chromosome III after incorporation of oaCGH deficiency mapping dataFigure 1A revised genetic and physical map of the dpy-17 region of chromosome III after incorporation of oaCGH defi-ciency mapping data. Deficiencies are represented as single lines and duplications as double lines. The outermost genes residing within each deficiency are shown. 17 zones defined by the deficiencies and duplications are represented by grey boxes. Note that the transition between zones 11 and 12 is defined by the position of dpy-17. *8 lethal mutants have not been posi-sDp3sDf135sDf127sDf121sDf130sDp8dpy-19sDf125sDf1281013A14 12 9 7 4 3 2B 1B 1A56118Zonenas-28 Y40D12A.1F44B9.2kle-2B0361.11 H14A12.5T20B12.9rpn-2ckk-1sel-5C32A3.3 mlc-3F27B3.5rha-2Y53G8B.1le t-713 le t-716 le t-721 le t-725 le t-728 le t-747 le t-756 le t-767 le t-774 le t-814 le t-815 le t-816 le t-817 le t-818sma-2mig-10sma-3unc-93dpy-17nDf20ZK688.10 fli -113B2Ale t-706 le t-720 le t-758 le t-759 le t-760 le t-776 le t-791 le t-811 le t-831le t-700 le t-710 le t-715 le t-736 le t-737 le t-738 le t-763 le t-796 le t-812 le t-813 le t-819 le t-820 mel-33le t-707 le t-732 le t-733 le t-734le t-741 le t-761 le t-778 le t-782 le t-822 le t-823 le t-824 le t-830le t-836 le t-840le t-748 le t-775 le t-777 le t-789 le t-826 le t-827 le t-837 mel-31le t-719 le t-727 le t-829le t-753le t-784 le t-825 le t-832le t-718 le t-780 le t-797 le t-798 le t-799 le t-809 le t-810le t-755 le t-765le t-711 le t-714 le t-717 le t-724 le t-764 le t-769 le t-771 le t-783 le t-792 le t-793 le t-794 le t-795 le t-843le t-702le t-768le t-704 le t-743 le t-746 le t-751 le t-752 le t-821 le t-841 le t-842 mup-4le t-709 le t-712 le t-729 le t-740 le t-750 le t-786 le t-834 le t-835 le t-838 le t-839 le t-844 le t-972*let-722 le t-754 le t-766par -3ckk-1B0303.11Page 3 of 10(page number not for citation purposes)tioned left/right with respect to dpy-17. Figure is not to scale.BMC Genomics 2007, 8:402 http://www.biomedcentral.com/1471-2164/8/402wild type Bristol N2 DNA as the reference sample. Theexpected ratio of reference DNA to test using thisapproach is 1:1 over the region of the chromosome notcovered by sDp3, 2:3 over the region covered by sDp3alone and 2:1 in the region of the deficiency balanced bysDp3 (Figure 2A).This initial analysis revealed that the extent of sDp3 variesdramatically between each of the strains tested (Figure2B–D). Since duplications maintained in balancer strainsare known to breakdown both spontaneously and underthe mutagenic conditions used to induce deficiencies [14-16] it is likely that the observed modifications to sDp3 areas a result of these factors.In addition to the variability of sDp3 coverage, discreteduplications internal to sDp3 are observed within each ofthe strains tested (Figure 2). This lack of uniformity makesdetermination of deficiency structure problematic insome cases. For example, the presence of an internalduplication in BC4697 across the region containing theleft breakpoint of sDf121 results in an increase of pre-dicted copy number in this region (Figure 2F), while mul-tiple variations within both sDp3 balanced strainscontaining sDf127 and sDf128 complicates the distinctionbetween where DNA has been deleted or duplicated (Fig-ure 2C–E and data not shown).oaCGH of heterozygous deficiencies resolves copy number ambiguity and results in precise mapping of deletion breakpointsSince heterozygous deletions can be easily resolved withoaCGH [13] we surmised that the creation of single copydeficiency strains would eliminate the ambiguity intro-duced through the use of the sDp3 balancer. By using thisapproach the expected ratio of reference DNA to test is 1:1along the whole length of the chromosome except withinthe deleted region where a ratio of 2:1 will be expected(Figure 2B).To implement this strategy all of the sDp3 balanced defi-ciency strains to be tested were out-crossed to JK2689,which contains the GFP marked heterozygous balancerhT2 (qIs48) known to cover the region under investigationin this study [1]. For this analysis genomic JK2689 DNAwas used as the reference sample to eliminate anygenomic variability that may have been introduced intothis strain through the heavy mutagenesis used for its orig-inal construction [17].With the elimination of sDp3 from the background theability to accurately resolve breakpoint positions in theambiguously mapped deficiency strains improved signifi-cantly. A comparison of oaCGH data for sDf127 balancedby sDp3 and this same deficiency out-crossed to hT2(qIs48) highlights this improvement (Figure 3). As part oftheir construction, deficiency strains have been out-crossed several times and it is therefore unlikely that theTable 2: Physical and genetic structure of zones 1A-14.Zone Start (bp) End (bp) Left Gene Right Gene Size (kb) # genes b# RNAi Lethal # Lethal Mutants1A 8023387 8611252 F44B9.2 rha-2 588 151 39 121B 7945392 8023387 kle-2 F44B9.2 78 23 6 32A 7896734 7945392 ZK688.2 kle-2 49 14 5 02B 7476402 7896734 H14A12.5 ZK688.2 420 81 23 113 7395219 7476402 T20B12.9 H14A12.5 81 8 5 14 7277442 7395219 B0361.11 T20B12.9 118 35 11 15 6620106 7277442 Y40D12A.1 B0361.11 657 154 48 136 6559307 6620106 F27B3.5 Y40D12A.1 61 14 4 27 6200904 6562088 rpn-2 F27B3.5 361 97 25 158 6178994 6200951 nas-28 rpn-2 22 5 3 09 5662800 6178564 par-3 nas-28 516 123 29 7a13 5637312 5664335 ckk-1 par-3 27 5 1 010 5565581 5637312 mlc-3 ckk-1 72 18 2 011 5107800 5565581 dpy-17 mlc-3 458 98 31 7 c(+/- 8)12 4569043 5107800 F35G12.2 dpy-17 539 163 46 14 c(+/- 8)13 3640101 4569043 C32A3.3 F35G21.1 929 204 54 1314 3115819 3640101 Y53G8B.1 C32A3.3 524 85 21 9aZones 11 and 12 are defined by dpy-17.b RNAi Lethal encompasses all genes annotated in Wormbase (release WS170) as having either emb, let, lvl and/or ste phenotypes by RNAi.c 8 lethal mutations have not been position right/left with respect to dpy-17.Page 4 of 10(page number not for citation purposes)process of creating the hT2 balanced strains has removedany features of these deficiencies. The oaCGH dataBMC Genomics 2007, 8:402 http://www.biomedcentral.com/1471-2164/8/402Page 5 of 10(page number not for citation purposes)oaCGH experimental overview and sDp3 balanced deficiency dataFigure 2oaCGH experimental overview and sDp3 balanced deficiency data. (A-B) Schematic representation of the oaCGH experimental approaches; (A) using sDp3 balanced deficiency strains and (B) modified to use hT2 (qIs48) heterozygous bal-ancer. Colored bars beneath schematic indicate expected DNA ratios. (C-E) oaCGH data obtained in GFF file format for three sDp3 balanced deficiency strains visualized using the SignalMap™ browser software [20]. (C) BC4697 (sDf121 [sDp3]), (D) BC4638 (sDf127 [sDp3]) and (E) BC4690 (sDf128 [sDp3]). Regions covered by sDp3 are represented as blue lines above the data; deletions are represented by red lines below the data. (F) Expansion of oaCGH data across the left breakpoint region of sDf121. Region of deletion ambiguity is represented by broken lines. *Apparent internal duplications of sDp3.CDE***N2sDfxAhT2sDfx/hT2B1:1 2:3 2:1 2:3 1:11:1 2:1 1:1sDp3*FBMC Genomics 2007, 8:402 http://www.biomedcentral.com/1471-2164/8/402obtained for each strain using the modified strategy istherefore likely to represent the initial structure of each ofthe deficiencies tested.Combining data from multiple array experiments allows for bias removal resulting in reliable deficiency characterizationoaCGH data obtained from separate strains using thesame chip design performs consistently enough to allowfor the integration and direct comparison of the data gen-erated from multiple experiments. Through this data inte-gration, common inconsistencies that are not specific tothe deletion of interest are identified and eliminated fromthe annotation, resulting in a reliable characterization ofthe whole deficiency genome (Figure 3B and 3C). This isan important step given that small mutagenic eventswhich may have occurred outside the known boundariesof a given deficiency may remain unidentified by tradi-tional mapping methods and could lead to conflictingmapping data.oaCGH analysis reveals deficiency complexity on a genome-wide scaleWe have analysed a number of deficiencies generated withboth GRI and UV irradiation (Table 1 and data notshown), and have been unable to detect any significantmutagen specific deletion characteristics, the majorityhaving a simple contiguous structure (Figure 3B and 3C,and data not shown). Exceptions to this observation areseen in the deficiencies sDf130, which contains two dis-tinct deletions, the smaller of which defines zone 10 (Fig-ure 1), and sDf128 which exhibits associated complexity.In a similar case as to that seen with sDp3 balancedsDf121, several duplications of various sizes extend intothe deficiency region of sDf128 (Figure 2C and 2E).Unlike sDf121 however, these duplications are retainedwhen sDf128 is out-crossed to hT2 (Figure 4), suggestingthat they have been integrated into the deficiencygenome. Attempts to further characterize the complexityof sDf128 through PCR amplification across the predicteddeletion breakpoints failed, suggesting that integration ofa duplicated region has occurred within the deficiencyComparison of oaCGH data in the sDp3 and hT2 (qIs48) balancer backgroundsFigure 3Comparison of oaCGH data in the sDp3 and hT2 (qIs48) balancer backgrounds: (A) BC4638 (sDf127 [sDp3]) (B) BC7163 (sDf127/hT2 [qIs48]) (C) BC7162 (sDf125/hT2 [qIs48]). Unambiguous deficiency mapping for each experiment is depicted as a solid black line while ambiguous regions are represented as a dashed grey line. Genes residing at the confirmed sDf127 breakpoints are shown (grey bars). Non-deficiency specific copy number changes introduced by the background strain can be seen (B and C solid arrows). Note: oaCGH array used in A is the Nimblegen design [20] while the array used in B and C is from the exon-centric array described by Mayden et.al [13] (see Methods).Page 6 of 10(page number not for citation purposes)itself. Subsequent amplification of a PCR product span-ning the left end of the largest duplication and the rightBMC Genomics 2007, 8:402 http://www.biomedcentral.com/1471-2164/8/402end of the deleted region confirms the integration of theduplicated fragment within the deletion (Figure 4). Con-firmation of the structure of the left end of the insertion,or elucidation of the position of the smaller duplicatedregion, could not be achieved leading to the conclusionthat this region is physically complex.Accurate identification of lethal mutations positioned within the deficiency mapA more detailed analysis of Zone 1B highlights the valueof applying the physical deficiency map to the identifica-tion of uncloned mutations. This zone is defined by thedeletion breakpoints of the deficiency sDf128 and con-tains three molecularly unknown lethal mutants; let-722,let-754 and let-766. The region is 78 kb in size, contains 21predicted genes and is spanned by the cosmids; C29E4,F54H12, C06G4 and F44B9 (Figure 5).Since 39 of the 40 molecularly identified lethal mutants inWormbase [18] exhibit a variety of lethal or sterile RNAiphenotypes (Additional file 1), genes within each zonewhich display these RNAi phenotypes make good candi-dates for the lethal mutations mapping to the sameregion. Of the 21 genes within zone 1 B, only 6 exhibitlethal phenotypes by RNAi (Figure 5 and Additional file1) and these 6 genes were consequently taken as initialcandidates of the three lethal mutants in this region.To identify DNA lesions the coding region of each candi-date gene was PCR amplified and sequenced usinghomozygous mutant DNA from a representative lethalstrain as template. In this way both let-722 and let-754,were identified as being alleles of aco-2 and C29E4.8respectively (summarized in Table 3). let-766 howeverwas not identified from this approach and we concludethat this mutation either maps into one of the remaining19 genes within this region, or a gene outside the defi-ciency which may have be disrupted through integrationof the small unmapped duplication present in this strain.Finally, through the incorporation of mapping data gen-erated by our group from ongoing efforts to clone lethalmutations, we have been able to further identify the muta-tions let-716 and let-768 as alleles of the genes C16A3.3and fum-1, respectively (our unpublished data and sum-marized in Table 3).sDf128 associated complexityFigure 4sDf128 associated complexity. (A) BC7164 (sDf128/hT2 [qIs48]) oaCGH data showing retention of duplications. (B) Sche-matic representation of sDf128 PCR analysis and confirmed duplication insertion. (C) Predicted insertion of the duplicated Wildtype DNA   Duplication      Deletion            PCR PrimerAC ?Original deletionRegion replaced by duplicationsDf128 deficiencyBPage 7 of 10(page number not for citation purposes)region into the sDf127 genome. (?) Unconfirmed deficiency structure.BMC Genomics 2007, 8:402 http://www.biomedcentral.com/1471-2164/8/402ConclusionWe have demonstrated that oaCGH can be successfullyapplied to the rapid and precise characterization of exist-ing C. elegans genetic deficiencies. This study highlightshow this type of analysis can transform low resolutiongenetic mapping tools into a precise physical mappingresource with which to accurately position molecularlyunidentified mutations.The implementation of oaCGH technology for this pur-pose is straightforward, requiring only the preparation ofhigh quality genomic DNA from the deficiency strains ofinterest. Data is generated in a short time and can be veri-Table 3: Summary of the mutations identified in essential genes as a result of this study.Gene Description Genomic Position (bp)Chromosome Mutant Allele Mutation Protein Change RNAi PhenotypeMutagenC16A3.3 RRP5 Ribosomal RNA Processing Enzyme6383494..6389343 III let-716 s2457 G-A 3rd Intron Splice Site emb EMSs2626 TGG-TGA W1489 (opal)fum-1 Fumarase 7465428..7467668 III let-768 s2482 TCG-TTG S149L emb EMSs2592 CGT-CAT R151Hs2628 GGA-AGA G88RC29E4.8 Adenylate Kinase 7951844..7949909 III let-754 s2622 AAG-AAA R150C emb EMSaco-2 Aconitase 7973646..7977254 III let-722 s2448 GAA-AAA E285K emb EMSs2450 GGG-GGA G663Ds2588 CGC-CGT A687VSchematic representation of zone 1 B as defined by the deficiency sDf128Figure 5Schematic representation of zone 1 B as defined by the deficiency sDf128. Cosmids as represented as grey bars above predicted gene positions taken from Wormbase (release WS170). Essential Gene candidates as defined by RNAi pheno-type are numbered. Identified mutation positions are shown in gene expansions.Page 8 of 10(page number not for citation purposes)emb = embryonic lethalityEMS = Ethylmethane SulphonateBMC Genomics 2007, 8:402 http://www.biomedcentral.com/1471-2164/8/402fied experimentally by standard molecular techniques.Some caveats to the application of this methodology havebeen highlighted by the initial difficulties we describeresulting from the use of animals balanced by a free dupli-cation, though we have demonstrated that by consideredevaluation of the experimental approach these issues canbe circumvented easily.We envisage that this resource will be instrumental inresolving previously ambiguous genetic mapping data.Additionally, we propose that integration of a physicaldeficiency map into a high-throughput molecular map-ping strategy such as snip-SNP will improve the efficiencyof positional cloning in C. elegans. In such an approachsnip-SNP would be employed to map the mutant of inter-est into a sub-chromosomal region, this region could thenbe further broken up by physically defined deficiencies,and a candidate sequencing pipeline, such as the one wedescribe here, employed to identify the molecular posi-tion of the mutation of interest.MethodsNematode culture, harvest, and DNA preparationNematodes were grown as previously described [19] on150 mm NGM agar plates seeded with Escherichia colistrain OP50. Strains used: Bristol N2, JK2689 – pop-1(q645) dpy-5(e61) I/hT2 [bli-4(e937) let-?(q782) qIs48](I;III), BC4697 – sDf121(s2098) unc-32(e189) III; sDp3(III;f), BC7162 – dpy-17(e164) sDf125(s2424) unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48], BC4638– dpy-17(e164) sDf127(s2428) unc-32(e189) III; sDp3(III;f), BC7163 – dpy-17(e164) sDf127(s2428) unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48], BC4690– dpy-17(e164) sDf128(s2786) unc-32(e189) III; sDp3(III;f), BC7164 – dpy-17(e164) sDf128(s2786) unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48], BC7244– sDf130(s2427) unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48], BC7181 – dpy-17(e164) sDf135(s2767)unc-32(e189) III; hT2 [bli-4(e937) let-?(q782) qIs48],BC3737 – sDp8(III;I); eT1(III;V), BC4158 sDp3(III;f); dpy-17(e164) let-722(s2448) unc-32(e189) III, BC4160 –sDp3(III;f); dpy-17(e164) let-722(s2450) unc-32(e189) III,BC4228 – sDp3(III;f); dpy-17(e164) let-722(s2588) unc-32(e189), BC4262 – sDp3(III;f); dpy-17(e164) let-754(s2622) unc-32(e189) III, BC4173 – dpy-17(e164) let-766(s2463) unc-32(e189) III; sDp3(III;f), BC4167 –sDp3(III;f); dpy-17(e164) let-716(s2457) unc-32(e189) III,BC4266 – sDp3(III;f); dpy-17(e164) let-716(s2626) unc-32(e189) III, BC4192 – sDp3(III;f); dpy-17(e164) let-768(s2482) unc-32(e189) III, BC4232 – sDp3(III;f); dpy-17(e164) let-768(s2592) unc-32(e189) III, BC4268 –sDp3(III;f); dpy-17(e164) let-768(s2628) unc-32(e189) III,BC2842 – dpy-18(e364)/eT1 III; unc-46(e177) let-DNA preparation for oaCGH samples was done as previ-ously described [13]. For mutant sequencing ~50homozygous lethal animals were picked from the progenyof sDp3 balanced strains into 5 ul of lysis buffer (50 mMKCl, 10 mM Tris pH 8.3, 2.5 mM MgCl2, 0.45% Tween-20, 0.01% Gelatin) and freeze cracked in Liquid N2 fol-lowed by digestion (1 hour at 60°C followed by 15 minat 95°C). 1 ul of this DNA preparation was subsequentlyused in a 25 ul PCR reaction.oaCGH Data analysisFor the analysis of sDp3 balanced strains a C. eleganswhole genome array was used, based on Wormbaserelease WS120, and available from NimbleGen SystemsInc. [20]. Analysis of the hT2 (qIs48) balanced strains wasperformed with a whole genome C. elegans array designedwith overlapping 50-mer probes targeting primarily anno-tated exons and micro-RNAs [13]. oaCGH sample prepa-ration, hybridization and analysis was done as previouslydescribed [13]. Copy number aberrations were detectedby visual inspection using the SignalMap™ browser soft-ware [20]. The data discussed in this publication havebeen deposited in NCBIs Gene Expression Omnibus(GEO) [21] and are accessible through GEO Series acces-sion numbers GSE9214 and GPL6047.Molecular identification of deficiency breakpoints and mutationsPCR amplification across the region of the breakpoint wasperformed with nested primers (Primer sequences andamplification conditions available upon request) andsequenced using standard molecular methods.Authors' contributionsMRJ carried out the experiments, data analysis and draftedthe manuscript. JSM assisted in sample preparation. SFdesigned the oaCGH array. DGM helped to draft the man-uscript. DLB conceived of the study, and participated in itsdesign and coordination. All authors read and approvedthe final manuscript.Additional materialAcknowledgementsWe thank Dr Nigel O'Neil for his enthusiasm and advice concerning this project and his comments on the manuscript. This work was supported by grants from the Canadian Space Agency, the Canadian Institute of Health Additional file 1Identified mutants of essential genes and their associated RNAi pheno-type.Page 9 of 10(page number not for citation purposes)444(s1418)/eT1 [let-500(s2165)]V, MT3022 – nDf20/sma-2(e502) unc-32(e189)III.Research and from Genome Canada to DGM.Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central BMC Genomics 2007, 8:402 http://www.biomedcentral.com/1471-2164/8/402References1. 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