Spectrum of variations in dog-1/FANCJ and mdf-1/MAD1 defective Caenorhabditis elegans strains after long-term propagation

Whole and partial chromosome losses or gains and structural chromosome changes are hallmarks of human tumors. Guanine-rich DNA, which has a potential to form a G-quadruplex (G4) structure, is particularly vulnerable to changes. In Caenorhabditis elegans, faithful transmission of G-rich DNA is ensured by the DOG-1/FANCJ deadbox helicase. To identify a spectrum of mutations, after long-term propagation, we combined whole genome sequencing (WGS) and oligonucleotide array Comparative Genomic Hybridization (oaCGH) analysis of a C. elegans strain that was propagated, in the absence of DOG-1 and MDF-1/MAD1, for a total of 470 generations, with samples taken for long term storage (by freezing) in generations 170 and 270. We compared the genomes of F170 and F470 strains and identified 94 substitutions, 17 InDels, 3 duplications, and 139 deletions larger than 20 bp. These homozygous variants were predicted to impact 101 protein-coding genes. Phenotypic analysis of this strain revealed remarkable fitness recovery indicating that mutations, which have accumulated in the strain, are not only tolerated but also cooperate to achieve long-term population survival in the absence of DOG-1 and MDF-1. Furthermore, deletions larger than 20 bp were the only variants that frequently occurred in G-rich DNA. We showed that 126 of the possible 954 predicted monoG/C tracts, larger than 14 bp, were deleted in unc-46 mdf-1 such-4; dog-1 F470 (JNC170). Here, we identified variants that accumulated in C. elegans’ genome after long-term propagation in the absence of DOG-1 and MDF-1. We showed that DNA sequences, with G4-forming potential, are vulnerable to deletion-formation in this genetic background.


Background
Genome integrity is crucial for survival of all living organisms. Chromosomal instability (CIN), marked by whole or segmental aneuploidy is hallmark of human tumors, and can drive abnormal proliferation of cancer cells [1]. In Caenorhabditis elegans, mdf-1(gk2) has an essential mdf-1/ MAD-1 component of the spindle assembly checkpoint (SAC) missing and this leads to accumulation of genetic errors and ultimately death by the third generation [2]. The checkpoint prevents CIN by inhibiting anaphasepromoting complex/cyclosome (APC/C), and delaying anaphase onset until all the chromosomes have achieved proper attachment to the spindle [3].
While MDF-1 prevents both loss and gain of whole chromosomes during mitosis, DOG-1 prevents segmental aneuploidies by ensuring proper replication of guanine(G)-rich DNA [4][5][6]. G-rich DNA can adopt a four-stranded helical G-quadruplex (G 4 ) DNA structure [7][8][9], which can pose a barrier to replication fork progression if left unresolved. The ability to form G 4 structures makes the corresponding G-rich DNA sequences particularly vulnerable to chromosomal rearrangements. Studies using C. elegans, as a model organism, were the first to show the striking genomic instability of G-rich DNA sequences when DOG-1, a functional ortholog of the deadbox helicase FANCJ [10], was non-functional [4]. When DOG-1 is functional, G-rich DNA sequences are stable and deletions affecting these regions are not observed [4,5]. Genomewide bioinformatic analysis of the human genome had identified more than 300,000 DNA sites with G 4 -forming potential [11,12]. In humans, mutations in FANCJ/dog-1 have been identified in Fanconi anemia (FA) complementation group J patients [13][14][15], which is a severe, autosomal recessive, disorder with increased spontaneous and DNA crosslink-induced CIN showing a wide range of clinical manifestations [13], and also in early onset breast cancer patients [16,17].
Knowledge of the mutational spectra is crucial for deciphering the cause of heritable genetic disorders as well as the progression of events relevant to cancers. Traditionally, analyses of mutation spectra and rates have been based on a small portion of phenotypically and molecularly detectable loci. In C. elegans, the mutational spectrum of dog-1(gk10) (knockout allele of dog-1) strains, was analyzed using either PCR-based assays [4,10,18] or oligonucleotide array Comparative Genomic Hybridization (oaCGH) [5,6,19]. The rapid advances in "next-generation" DNA sequencing technologies now allows us to perform comprehensive genome-wide analyses of mutational spectra by sequencing whole genomes [20][21][22][23].
Here we undertook a whole genome approach in order to analyze mutational events in a C. elegans strain that is defective for both mdf-1/MAD1 and dog-1/ FANCJ. This strain was propagated for 470 generations and samples were stored frozen at generations 170 and 270. Phenotypic analysis of the strain revealed striking fitness recovery, indicating that accumulated mutations cooperate to bypass the MDF-1 checkpoint requirement and thus achieve long-term population survival. We performed whole-genome sequencing (WGS) and oaCGH analyses of the strain at three different generations (170, 270 and 470). We identified substitutions, InDels, and copy number variants (CNVs) larger than 20 bp, and compared their accumulation over the generations. We showed that only deletions, which are larger than 20 bp, frequently initiated in G-rich DNA (88% of all of the deletions). Consistent with the fitness recovery observed in this strain, rather than a decline in fitness, the mutation spectrum reported here reflects variants that are either advantageous or neutral in this specific genetic background.

Whole genome analysis
In order to propagate mdf-1; dog-1 homozygous worms, it is first necessary to isolate a suppressor of mdf-1(gk2) sterility and lethality that occurs in the double mutant [24]. Previously, such-4(h2168) was isolated, which allowed propagation of mdf-1; dog-1 beyond the third generation ( Figure 1) [24]. The such-4 suppressor allows for an approximately five-fold increase in fertile hermaphrodite progeny of mdf-1; dog-1 [24]. This increase in fertility occurs in the generations immediately after isolation of the suppressor. We outcrossed one worm to obtain KR4233 [unc-46(e177) mdf-1(gk2) such-4(h2168)] [24,25] (Figure 1). A second line was isolated and maintained in the dog-1(gk10) background for 470 generations, with storage by freezing at generations 170 and 270 ( Figure 1). To mark the presence of mdf-1(gk2), unc-46(e177) (a visible marker) was used, which is present in all of our strains ( Figure 1). While propagating unc-46 mdf-1 such-4; dog-1 homozygotes, we observed a further increase in reproductive fitness. This increase was significant, 59% of F 470 unc-46 mdf-1 such-4; dog-1 progeny develop into fertile hermaphrodites, compared to only 10% of the unc-46 mdf-1 such-4 progeny and 2% of unc-46 mdf-1 mutants. Detailed phenotypic analysis of these strains as well as genetic dissection of suppressors has recently been reported [26]. To identify the genomic variations (GVs) that had accumulated in mdf-1 such-4; dog-1 F 470 worms after long-term propagation, the genome was sequenced to a depth of 70× genome equivalents and aligned to the C. elegans reference genome WS235 available at WormBase [27]. We also sequenced the unc-46 mdf-1 such-4; dog-1 strains that were frozen at generations 170 and 270 and compared the progress of mutation accumulation (Additional file 1: Figure S1).

Single Base Substitutions (SBSs) do not occur within G 4 -DNA
We used the variant caller VarScan2; version 2.3.2 [28] to identify 525 homozygous SBSs that occurred with a variant frequency (VF) of 90% or higher in the mdf-1 such-4; dog-1 F470 genome (Additional file 2: Table S1A). All 42 tested substitutions were confirmed by Sanger resequencing (30 were randomly selected; 12 additional substitutions were confirmed by re-sequencing as a result of being adjacent to randomly selected SBSs or as being non-randomly selected as candidates in later analyses), indicating a false positive rate of less than 5%. WGS analysis of the unc-46 mdf-1 such-4; dog-1 strain, which had been frozen at generation 170 ( Figure 1), revealed that the majority (431/525) of the SBSs present in generation 470 are also present in generation 170 (Additional file 1: Figure S1A; Table 1). However, we did observe that 32 additional substitutions had accumulated between F 170 and F 270 , and 62 more between F 270 and F 470 (Table 1). Large number of substitutions observed in F 170 indicate the possibility that the original unc-46 mdf-1 such-4; dog-1 strain ( Figure 1) had a large number of single nucleotide differences from the reference genome (WS235). One way to test this possibility is to estimate the mutation rates based on available data. If we consider the 94 substitutions that had accumulated in 300 generations, between F 170 and F 470 , we estimate the rate of μ bs = 3.1 × 10 -9 /base/ generation. This estimate is similar to the previously reported spontaneous rate of base substitution in C. elegans, 2.7 ± 0.4 × 10 −9 /base/generation [21] and other model organisms, 3.5 × 10 −9 /base/generation in Drosophila melanogaster [23] and 7.1 ± 0.7 × 10 −9 /base/generation in Arabidopsis thaliana [22]. Furthermore, analysis of the 94 substitutions on mutation bias revealed very similar mutation spectrum to the spontaneous mutation spectra in N2 (C. elegans wild-type) [21] (Figure 2), and our analysis of transition bias (transition/transversion -Ts/Tvbase substitution ratios) revealed the Ts/Tv ratio = 0.54, We isolated a suppressor (such-4) in F 4 from the only plate that we set up that propagated in the absence of MDF-1 and DOG-1 for longer than three generations. We generated a strain (KR4233) by crossing away dog-1(gk10). A second line was propagated for the total of 470 generations. The worms were frozen at generations 170 (green), 270 (purple) and 470 (blue). Note that unc-46 (used as a visible marker) is tightly linked to mdf-1(gk2) in all of our strains. which is within the range observed for spontaneous mutations in multiple mutation accumulation lines (average 0.45; range 0.19 -0.79) [21]. Therefore, the similarity of the substitution rate over the last 300 generations to the previously reported μ bs in N2 strongly implies that the majority of the 431 substitutions identified in the F 170 generation are variants originally present in the starting strain; therefore, we focused our analysis on GVs that had occurred between F 170 and F 470 (Additional file 2: Table S1B). Previous studies showed that G 4 DNA secondary structure is mutagenic in the absence of DOG-1 [4][5][6]19]. Using the G 4 DNA signature (G 3+ N 1-7 G 3+ N 1-7 G 3+ N 1-7 G 3+ ), we identified 2,372 such sites in C. elegans' genome (Additional file 2: Table S2). Next, we tested to see if any of the 94 substitutions occurred within the G 4 DNA signature sequence and observed that none of them did (Additional file 2: Table S1B), indicating that the identified substitutions most likely arose spontaneously and were not due to lack of functional DOG-1.

Small InDels do not occur within G 4 -DNA
We applied VarScan2 [28] to identify 150 homozygous InDels that were 20 bp or less in mdf-1 such-4; dog-1 F470 (Additional file 2: Table S3A). We randomly selected 25 of the InDels and confirmed all 25 by Sanger re-sequencing, indicating a false positive rate of less than 5% (Additional file 2: Table S3A). Analysis of InDels in the unc-46 mdf-1 such-4; dog-1 strains propagated for 170 and 270 generations (Additional file 1: Figure S1B) revealed that 88% of the F 470 InDels (133/150) existed in the F 170 generation (Table 1), which again indicates that the majority of InDels were already present in our starting strain. In fact, we only observe eight additional InDels in F 270 (Table 1) and another nine InDels accumulated between F 270 and F 470 (Table 1). Based on the last 300 generations of propagation, we estimate a mutation rate for InDels to be 17/(1 × 10 8 bases × 300 generations ) = 5.7 × 10 -10 /base/generation. The ratio of InDels to SBSs observed was 0.18 or one InDel per 5.5 substitutions, which is much lower than the 1.31 ratio reported previously in C. elegans [29]. However, the lower number of InDels to SBSs that we observe in the unc-46 mdf-1 such-4; dog-1 background is comparable to analyses in yeast [30], A. thaliana [22] and human [31]. Namely, WGS analysis in yeast had revealed ratio of InDels to SBSs of ∼ 0.03 [30], which was consistent with previous findings of one InDel per 33 SBSs [20]. Furthermore, analysis on spontaneous occurrence of InDels in A. thaliana revealed the ratio of 0.13 of InDels to SBSs [22]. It may be possible that the small number of InDels, over the last 300 generations, in our strain may be a result of a mutation that was acquired by propagation; however, it may also be that the spontaneous mutation rate of InDels in C. elegans is comparable to that of other organisms. Next, we tested to see if any of the 17 InDels occurred within the G 4 DNA signature sequence and observed that none of them did (Additional file 2: Table S3B). Thus, we believe that these InDels arose spontaneously and are not due to a lack of functional DOG-1.

Duplications do not occur within G 4 -DNA
Analysis of gene copy-number variant accumulation after long-term propagation in C. elegans using the oaCGH has provided evidence for a high rate of spontaneous gene duplications in this multi-cellular eukaryote [32]. Previously, using the oaCGH we showed that the such-4 suppressor genome contains a large tandem duplication on Chromosome V (LGV) [6]. Here, we used both Pindel [33] and oaCGH [34], and identified four sites with copy number increases in the mdf-1 such-4; dog-1 F470 genome (Additional file 2: Table S4A), including a previously identified large tandem duplication [6]. One of the duplications involves a two-copy addition, making the final count of five duplication events and four different duplication sites. Analysis of the CNVs in the F 170 and F 270 genomes captured a dynamic property of duplications (Additional file 1: Figure S1C). In F 170 , we observed three duplications (Additional file 2: Table S4A). One is the large tandem duplication located on LGV that amplifies 62 protein-coding genes, which we have described previously [6]. In generation F 270 , we detected duplications of two new loci, as well as further amplification of the LGV region to three copies (triplication) (Additional file 1: Figure S1C and Additional file 2: Table S4A). In F 470 , we did not find any new duplications (Additional file 2: Table  S4A), but did observe that the duplication on LGI was lost, resulting in a wild-type copy number for this region (Additional file 2: Table S4A). Thus, the LGV duplication exemplifies the property of duplications to further amplify; while LGI duplication shows that a duplicated region can revert back to a normal copy number.
The gene duplication rate for C. elegans was recently estimated to be 3.4 × 10 -7 /gene/generation [32]. Our data, based on the last 300 generations (from F 170 and F 470 ), also revealed a comparably high rate of gene duplication (6.5 × 10 -7 /gene/generation), specific to the unc-46 mdf-1 such-4; dog-1 background, when a large duplication on LGV is excluded from the analysis. The LGV duplicated region contains 62 protein-coding genes, including cyb-3 [6]. We observed a correlation between increased dosage of CYB-3 and a striking fitness increase in our strains; thus, our experimental protocol selected for LGV amplification (detailed experimental analysis on these findings has been recently published [26]). Importantly, like SBSs and InDels, none of the duplications occurred in the vicinity of the G 4 DNA signature sequences (Additional file 2: Table S4B), also indicating that these CNVs arose spontaneously and were not due to a lack of functional DOG-1.

Deletions frequently initiate at G 4 DNA sites
The major type of mutation observed, in the absence of DOG-1, is a deletion of 300 bp or smaller, initiating at either the 5′-end of C-or the 3′-end of G-tracts [4,5,10,19]. Using the unique alignments generated by Novoalign and Pindel [33], we identified 183 homozygous deletions larger than 20 bp in the mdf-1 such-4; dog-1 F470 genome (Additional file 2: Table S5A), including the known mdf-1(gk2) and dog-1 (gk10) deletions. We randomly selected 28 of the deletions, and confirmed all of them using PCR (Additional file 2: Table S5A). To identify deletions, which may have been missed, we also used oaCGH [34]. We confirmed all of the deletions, predicted by Pindel, that were covered by oaCGH probes and identified an additional 13 deletions not detected by Pindel (Additional file 2: Table S5A), making the final count 196 homozygous deletions in the mdf-1 such-4; dog-1 F470 genome (Additional file 2: Table S5A).
We observed 57 deletions in F 170 , indicating that 139 deletions had accumulated in 300 generations (between F 170 and F 470 ) propagated in the absence of MDF-1 and DOG-1 (Additional file 1: Figure S1D and Table 1). We found that the majority of the deletions (123 of 139) initiated in G 4 DNA (Additional file 2: Table S5A). Previous analysis in C. elegans [32] estimated the spontaneous rate of deletions to be 2.2 × 10 -7 /gene/generation respectively. The 139 deletions accumulating between the generations F 170 and F 470 affect 19 protein-coding genes which allowed us to calculate the mutation rate of deletions over the 300 generations to be 19/(20,400 protein-coding genes × 300 generations ) = 3.1 × 10 -6 /gene/generation in the unc-46 mdf-1 such-4; dog-1 strain, which is approximately 10-fold higher than the estimated rate in N2 [32]. This is similar to the estimated forward mutation frequency of eT1-balanced lethal mutations in dog-1(gk10) background [6]. To determine if the elevated mutation rate of deletions is due to DOG-1 deficiency, we compared mutation rates in non-G 4 sites versus mutation rate in the G 4 sites. While the mutation rate of deletions affecting the non-G 4 sites, 4.9 × 10 -7 /gene/generation or 5.0 × 10 -10 /base/generation, is comparable to the previously reported spontaneous rate of deletions [32]; the mutation rate based on G 4 sites, 1.7 × 10 -4 /G 4 site/generation, illustrates the striking vulnerability of these DNA regions when DOG-1 is absent. Therefore, deletions larger than 20 bp are the only variants in the unc-46 mdf-1 such-4; dog-1 strain that frequently occurred in the G 4 -DNA sites and had significantly higher mutation rate than the spontaneous rate reported previously for the strains with normal DOG-1 function.
monoG/C tracts larger than 14 bp are frequently deleted when DOG-1 is not functional In recent years, the G 4 DNA has been implicated in diverse biological processes, such as gene expression [35] and DNA replication initiation [35]. Consistent with the established role of DOG-1, we found that the majority (114) of the homozygous deletions that we identified between F 170 and F 470 (139) initiated in the previously proposed G 4 DNA signature G 3+ N 1-7 G 3+ N 1-7 G 3+ N 1-7 G 3+ [5] (Additional file 2: Table S5B) where the G-tract was almost completely removed together with 5′ flanking DNA sequence ( Figure 3A and B). In agreement with a previous study [5], we found that the majority of deletions initiate at monoG tracts larger than 14 bp (93 deletions) ( Figure 3A), while 11 deletions initiate at monoG-like structures with no more than two nucleotides that interrupt the homopolymer (Figure 3B), and 10 deletions initiate at sequences that interrupt the homopolymers by three or more nucleotides (e.g., GGGtGGGGaagttatGG GaGGG) (Additional file 2: Table S5B). MonoG/C tracts larger than 14 bp have the highest potential of forming the G-quartet structure. In fact, we find here that the unc-46 mdf-1 such-4; dog-1 F 470 genome has 13.2% of all the predicted monoG/C tracts larger than 14 bp deleted. An interesting question to be addressed with future research would be to determine how mutation rate changes with decreasing numbers of available targets. Furthermore, it would be also important to see how many of the G 4 -DNA sites could be deleted in a strain and still maintain viability of the animals.
To investigate whether there may be additional sequences, which are vulnerable in the absence of DOG-1, we analyzed the 25 deletions that do not initiate in G 4 DNA signature sequences to see if there are common patterns. We found that eight of these deletions initiate at G-rich sequences that correspond to the G 2+ N 1-7 G 2 + N 1-7 G 2+ N 1-7 G 2+ signature (four stretches of two or more guanines, alternated with one to seven nucleotides of any type), while one had a G 2+ N 1-7 G 2+ N 1-7 G 2+ signature at the breakpoint ( Figure 3C and Table 2). Although, it may be possible that our strain had gained a mutation in an unknown gene important for genome stability, it is also possible that additional DNA sequences may be vulnerable to rearrangements in the absence of DOG-1.
Previously, it was found that the deletion sizes in viable lines detected by PCR [4,18] or oaCGH methods [5,6] were predominately smaller than 300 bp. In this study, the 123 deletions that occurred at G-rich sequences ranged between 49 and 10,228 base-pairs with the majority of the deletions (86%) removing less than 300 bp ( Figure 3E). These findings are in agreement with the deletion distribution sizes revealed by the study of a 69 G-tract deletion set [5]. However, larger deletions, initiating at G-tracts, Figure 3 The majority of large deletions initiates at G-rich DNA. Figures (A) through (D) are Genome Browser snapshots of single deletions; x-axis represents genome location, while y-axis represents number of reads that cover the region; blue reads depict coverage of more than 10, while orange depicts 10 or less; the gaps depict no coverage, which is indicative of deletions. The reference sequence is depicted with deleted bases underlined. (A) A 99 bp deletion that initiates at G 20 homopolymer. (B) A 96 bp deletion that initiates at G 14 TG 6 AGAAG 3 sequence. (C) A 112 bp deletion that initiates at a G-rich sequence, G 2 N 3 G 2 N 5 G 2 N 2 G 2 . (D) A 55 bp deletion that initiates at a non G-rich sequence. (E) Size distribution of homozygous deletions that occur at G-rich sequences (n = 123). *Analysis of the sequences revealed that nine match the G 2+ N 1-7 G 2+ N 1-7 G 2+ N 1-have been recovered as lethal mutations [6], consistent with these regions containing essential genes [6]. We also found that 16 deletions, which occurred at non-G-rich sites, removed small regions of less than 300 bp in size (Additional file 2: Table S5B).

protein-coding genes are affected by the 253 GVs
To determine an effect of all the identified variants on protein-coding genes, after long-term propagation in the absence of MDF-1 and DOG-1, we used CooVar [36], a tool developed by our group for annotating variants. Many of the G-rich DNA sites are located in close proximity to protein-coding genes in C. elegans [19,37]. Using CooVar [36], we predicted that 19 genes would be affected by 18 of the deletions that had accumulated between generations F 170 and F 470 (Additional file 2: Table S6). The majority of the deletions (14) represent the first knockout alleles for those genes and thus provide a genetic resource for studying their functions (Additional file 2: Table S6). We found that the majority of the InDels are not located in proteincoding regions, and the one that affects a protein-coding gene is an in-frame deletion (Additional file 2: Table S6). We found that the majority of the detected SBSs are located within non-protein coding regions. However, there are 15 SBSs that fall within protein-coding regions that are predicted to result in missense mutations (Additional file 2: Table S6). In total, 66 protein-coding genes were affected by the duplications (Additional file 2: Table S6). The majority of the genes (62) are affected by the large tandem duplication on LGV (Additional file 2: Table S6). In addition to the 253 GVs that had accumulated between generations F 170 and F 470 , we had also performed analysis of the effect on protein-coding genes of all the variants observed in the mdf-1such-4; dog-1 F470 genome (Additional file 2: Table S7).
Mutations are a source of genetic variation that confers to an organism either advantage due to a beneficial change, disadvantage due to a deleterious alteration, or neither due to a neutral change. Considering the~30-fold fitness recovery in this strain [26], the identified GVs are expected to be either advantageous or neutral in this genetic background. In fact we showed, in a parallel study, that three mutational events discovered in this strain cooperate to increase fitness when MDF-1 absent [26].

Conclusion
In this study we undertook a genomics approach to identify variants that accumulate in the C. elegans genome after long-term propagation in the absence of two genome-guardians, DOG-1/FANCJ and MDF-1/MAD1. Combining WGS analysis with oaCGH analysis allowed us to comprehensively analyze both the small-scale variants (SBSs and InDels of 20 bp or smaller) and largescale variants (CNVs larger than 20 bp). Freezing the strain, for long term storage, at three different time points, F 170 , F 270 and F 470 , allowed us to compare and visualize mutation accumulation over the generations. We were able to estimate the mutation rates in this strain for different types of mutations over the 300 generations (F 170 to F 470 ). We observed a significantly elevated rate of deletions, larger than 20 bp, that initiated at G/C-rich DNA sequence. Our approach had allowed us to show that, in the absence of DOG-1, DNA sequences with G 4 -forming potential are vulnerable to deletion-formations. Previous studies were dependent only on PCR or oaCGH analysis; the two approaches are limited in their ability to detect small-scale variants like SBSs and InDels. Furthermore, genome analysis of the strains at F 170 , F 270 and F 470 allowed us to capture and visualize an intriguing property of CNVs (Additional file 1: Figure S1). We did not find any reversions of deletions, substitutions, or InDels. As expected, once fixed, these types of mutations are propagated indefinitely (Additional file 1: Figure S1). However, we captured a dynamic property of duplications: amplification of a region on LGV from one-to-two-to-three copies and reversion of the LGI duplication back to a normal copy number (Additional file 1: Figure S1C). This is the first extensive analysis of a strain that had been propagated in the absence of DOG-1 helicase for hundreds of generations. We identified 954 monoG/C tracts larger than 14 bp in the C. elegans genome WS325; the polyG/C DNA sequence is the sequence with the highest potential of forming G-quartet. We showed that 13% of these 954 sites are deleted in the mdf-1 such-4; dog-1 F470 genome. This finding raises an important question on the changes in mutation rate when number of mutagenic targets is decreased. Another important question is regarding to the role of G 4 DNA in normal development. Recently, G 4 DNA has been implicated in a variety of biological processes including telomere maintenance, gene expression, epigenetic regulation, and DNA replication [38]. One question to consider is how many G 4 DNA sites could be removed from a genome yet still maintain viability of the animals.
Whole genome sequencing and computational analysis of the unc-46 mdf-1 such-4; dog-1 F470 genome Genomic DNA was prepared from JNC170 following a standard protocol (http://genetics.wustl.edu/tslab/ protocols/genomic-stuff/worm-genomic-dna-prep/) originally set up by the Andy Fire Lab. The library was prepared with average insert size of 300 bp and the genome was then sequenced using Illumina Solexa sequencing (at Canada's Michael Smith Genome Sciences Centre) and 92,282,948 reads of 101 bp in length were obtained. The reads were then aligned to the C. elegans reference genome WS235 (hosted at WormBase) [27] in paired end manner (46,141,474 pairs) using the Novoalign alignment tool. 73,482,133 (79.63%) of the total reads were of base quality 30 or more and were uniquely mapped, generating 70-fold coverage of the genome.
SBSs were detected using the uniquely mapped reads and pileup2snp function of the variant caller VarScan2; version 2.3.2 [28]. We used SAMtools [40] to generate the mpileup file necessary as input for VarScan. We filtered out the SBSs that did not meet the following criteria: depth of coverage > 5 and ≤ 200, variant frequency ≥ 0.9 and base quality ≥ 30. After these filtering steps, we identified 776 substitutions in the mdf-1 such-4; dog-1 F470 genome. However, comparison with the sequenced N2 strains from CGC and Horvitz lab (sequencing reads were kind gift from Dr. Bob Waterston) revealed 525 homozygous substitutions that are unique to mdf-1 such-4; dog-1 F470 genome. The 251 SBSs were not included in our analysis as they did not accumulate during the course of our experiment. To determine rate of false positives in our set, we randomly selected 30 SBSs (Additional file 2: Table S1A), designed primers flanking the predicted substitution sites, amplified the fragments and re-sequenced using Sanger sequencing method at Genewiz, Inc. All 30 substitutions were confirmed by Sanger re-sequencing, suggesting a false positive rate of less than 5% (Additional file 2: Table S1A). Also, 12 additional substitutions were confirmed by re-sequencing as a result of being adjacent to randomly selected SBSs or as being nonrandomly selected as candidates in later analyses.
InDels were detected using the uniquely mapped reads and pileup2indel function of the variant caller VarScan2; version 2.3.2 [28]. We used SAMtools [40] to generate the mpileup file necessary as input for VarS-can2. We filtered out the InDels that did not meet the following criteria: depth of coverage > 5 and ≤ 200, variant frequency ≥ 0.9 and base quality ≥ 30. Additionally, we re-evaluated and filtered the output so that each coordinate corresponds to one variant. After these filtering steps, we identified 556 InDels in the mdf-1 such-4; dog-1 F470 genome. r, comparison with the sequenced N2 strains from CGC and Horvitz lab (sequencing reads were kind gift from Dr. Bob Waterston) revealed 150 homozygous InDels that are unique to mdf-1 such-4; dog-1 F470 genome. The 406 InDels were not included in our analysis as they did not accumulate during the course of our experiment. To determine rate of false positives in our set, we have randomly selected 25 InDels (Additional file 2: Table S3A), designed primers flanking the predicted InDel sites, amplified the fragments and re-sequenced using Sanger sequencing method at Genewiz, Inc. All of the 25 InDels were confirmed by Sanger re-sequencing, suggesting a false positive rate of less than 5% (Additional file 2: Table S3A).
These unique-mapping reads were then used, together with Pindel [33], to identify deletions. The final set of fixed/homozygous deletions was selected based on the following criteria: a = the number of unique reads supporting the breakpoints of the deletion; b = the number of reads within the deleted region; select the predicted deletion if a aþb is larger than 0.5 and the size of the deletion is larger than 20 bp. From the candidate deletions, 28 were randomly selected and the regions were PCRamplified using the same genomic DNA that was used for the WGS and primers designed in the flanking regions of the computationally identified deletions. All of the 28 randomly selected predicted deletion sizes were confirmed using DNA electrophoresis gels, suggesting the rate of false positives of less than 5%.
Whole genome sequencing and computational analysis of the unc-46 mdf-1 such-4; dog-1 F170 and mdf-1 such-4; dog-1 F270 strains The genomic DNA was prepared from JNC168 and JNC169 following a standard protocol (http://genetics.wustl.edu/ tslab/protocols/genomic-stuff/worm-genomic-dna-prep/). The genomic DNA was sheared to generate 500 bp fragment and the library was prepared using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina®. The library was then sequenced using Illumina Solexa sequencing (at Simon Fraser University) and 9,950,748 (F 170 ) and 6,225,488 (F 270 ) reads of 250 bp length were obtained. The reads were then aligned to the C. elegans reference genome WS235 (hosted at WormBase) [27] in paired end manner using the Novoalign alignment tool as described above to achieve 20-fold (F 170 ) and 14-fold (F 270 ) coverage. For both strains, we used Pindel [33] to identify deletions and duplications, and VarScan2; version 2.3.2 [28] to identify small InDels and substitutions, as described above. We also used sequenced N2 strains from CGC and Horvitz lab to remove variants existing in the N2 strain. Then, we compared the variants present in the F 170 and F 270 genomes with the ones identified in the F 470 genome.
oaCGH analysis of the unc-46 mdf-1 such-4; dog-1 strains To perform oaCGH analysis, we used the same genomic DNA from the mdf-1 such-4; dog-1 lines (F 170 , F 270 and F 470 ) that were used for the WGS and the reference N2 DNA that was prepared following a standard protocol. oaCGH analysis was performed as described by Maydan and colleagues [41] with a newly designed microarray. The 3-plex microarray with design name 120618_Cel-e_WS230_JK_CGH was manufactured by Roche Nim-bleGen Inc. with each individual sub-array comprising 720 k 50-mer oligonucleotide probes. The filters used to select the probes primarily followed Maydan and colleagues [41] without focusing on coding regions in order to provide a more uniform coverage of the genome (WormBase release WS230). In regions where unique probes could not be designed selection filters were slightly relaxed in order to allow the inclusion of probes with possible cross-hybridization to at most one other location in the genome.