BACKGROUND
genome integrity is crucial for survival of all living organisms.
chromosomal instability , marked by whole or segmental aneuploidy is hallmark of human tumors, and can drive abnormal proliferation of cancer cells  <cit> .
in caenorhabditis elegans, mdf- <dig> has an essential mdf-1/mad- <dig> component of the spindle assembly checkpoint  missing and this leads to accumulation of genetic errors and ultimately death by the third generation  <cit> .
the checkpoint prevents cin by inhibiting anaphase-promoting complex/cyclosome , and delaying anaphase onset until all the chromosomes have achieved proper attachment to the spindle  <cit> .
while mdf- <dig> prevents both loss and gain of whole chromosomes during mitosis, dog- <dig> prevents segmental aneuploidies by ensuring proper replication of guanine-rich dna  <cit> .
g-rich dna can adopt a four-stranded helical g-quadruplex  dna structure  <cit> , which can pose a barrier to replication fork progression if left unresolved.
the ability to form g <dig> 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- <dig>  a functional ortholog of the deadbox helicase fancj  <cit> , was non-functional  <cit> .
when dog- <dig> is functional, g-rich dna sequences are stable and deletions affecting these regions are not observed  <cit> .
genome-wide bioinformatic analysis of the human genome had identified more than  <dig>  dna sites with g4-forming potential  <cit> .
in humans, mutations in fancj/dog- <dig> have been identified in fanconi anemia  complementation group j patients  <cit> , which is a severe, autosomal recessive, disorder with increased spontaneous and dna crosslink-induced cin showing a wide range of clinical manifestations  <cit> , and also in early onset breast cancer patients  <cit> .
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- <dig>  strains, was analyzed using either pcr-based assays  <cit>  or oligonucleotide array comparative genomic hybridization   <cit> .
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  <cit> .
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/mad <dig> and dog-1/fancj.
this strain was propagated for  <dig> generations and samples were stored frozen at generations  <dig> and  <dig>  phenotypic analysis of the strain revealed striking fitness recovery, indicating that accumulated mutations cooperate to bypass the mdf- <dig> checkpoint requirement and thus achieve long-term population survival.
we performed whole-genome sequencing  and oacgh analyses of the strain at three different generations .
we identified substitutions, indels, and copy number variants  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 .
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.
RESULTS
whole genome analysis
in order to propagate mdf-1; dog- <dig> homozygous worms, it is first necessary to isolate a suppressor of mdf- <dig> sterility and lethality that occurs in the double mutant  <cit> .
previously, such- <dig> was isolated, which allowed propagation of mdf-1; dog- <dig> beyond the third generation   <cit> .
the such- <dig> suppressor allows for an approximately five-fold increase in fertile hermaphrodite progeny of mdf-1; dog- <dig>  <cit> .
this increase in fertility occurs in the generations immediately after isolation of the suppressor.
we outcrossed one worm to obtain kr <dig>   <cit>  .
a second line was isolated and maintained in the dog- <dig> background for  <dig> generations, with storage by freezing at generations  <dig> and  <dig> .
to mark the presence of mdf- <dig>  unc- <dig>  was used, which is present in all of our strains .
while propagating unc- <dig> mdf- <dig> such-4; dog- <dig> homozygotes, we observed a further increase in reproductive fitness.
this increase was significant, 59% of f470unc- <dig> mdf- <dig> such-4; dog- <dig> progeny develop into fertile hermaphrodites, compared to only 10% of the unc- <dig> mdf- <dig> such- <dig> progeny and 2% of unc- <dig> mdf- <dig> mutants.
detailed phenotypic analysis of these strains as well as genetic dissection of suppressors has recently been reported  <cit> .
to identify the genomic variations  that had accumulated in mdf- <dig> such-4; dog-1 f <dig> worms after long-term propagation, the genome was sequenced to a depth of 70× genome equivalents and aligned to the c. elegans reference genome ws <dig> available at wormbase  <cit> .
we also sequenced the unc- <dig> mdf- <dig> such-4; dog- <dig> strains that were frozen at generations  <dig> and  <dig> and compared the progress of mutation accumulation .figure  <dig>
a schematic representation of the long-term propagation of
unc- <dig> mdf- <dig> such-4; dog-1
homozygotes for  <dig> generations.
we isolated a suppressor  in f <dig> from the only plate that we set up that propagated in the absence of mdf- <dig> and dog- <dig> for longer than three generations.
we generated a strain  by crossing away dog- <dig>  a second line was propagated for the total of  <dig> generations.
the worms were frozen at generations  <dig> ,  <dig>  and  <dig> .
note that unc- <dig>  is tightly linked to mdf- <dig> in all of our strains.
single base substitutions  do not occur within g4-dna
we used the variant caller varscan2; version  <dig> .
<dig>  <cit>  to identify  <dig> homozygous sbss that occurred with a variant frequency  of 90% or higher in the mdf- <dig> such-4; dog-1f <dig> genome .
all  <dig> tested substitutions were confirmed by sanger re-sequencing , indicating a false positive rate of less than 5%.
wgs analysis of the unc- <dig> mdf- <dig> such-4; dog- <dig> strain, which had been frozen at generation  <dig> , revealed that the majority  of the sbss present in generation  <dig> are also present in generation  <dig> .
however, we did observe that  <dig> additional substitutions had accumulated between f <dig> and f <dig>  and  <dig> more between f <dig> and f <dig> .
large number of substitutions observed in f <dig> indicate the possibility that the original unc- <dig> mdf- <dig> such-4; dog- <dig> strain  had a large number of single nucleotide differences from the reference genome .
one way to test this possibility is to estimate the mutation rates based on available data.
if we consider the  <dig> substitutions that had accumulated in  <dig> generations, between f <dig> and f <dig>  we estimate the rate of μbs =  <dig>  × 10-9/base/generation.
this estimate is similar to the previously reported spontaneous rate of base substitution in c. elegans,  <dig>  ±  <dig>  × 10− <dig> /base/generation  <cit>  and other model organisms,  <dig>  × 10− <dig> /base/generation in drosophila melanogaster  <cit>  and  <dig>  ±  <dig>  × 10− <dig> /base/generation in arabidopsis thaliana  <cit> .
furthermore, analysis of the  <dig> substitutions on mutation bias revealed very similar mutation spectrum to the spontaneous mutation spectra in n <dig>   <cit>  , and our analysis of transition bias  revealed the ts/tv ratio =  <dig> , which is within the range observed for spontaneous mutations in multiple mutation accumulation lines   <cit> .
therefore, the similarity of the substitution rate over the last  <dig> generations to the previously reported μbs in n <dig> strongly implies that the majority of the  <dig> substitutions identified in the f <dig> generation are variants originally present in the starting strain; therefore, we focused our analysis on gvs that had occurred between f <dig> and f <dig> .table  <dig>
summary of the variants identified in the
unc- <dig> mdf- <dig> such-4; dog-1
strains at generations f
170
, f
270
and f
470
sbss
indels ≤ 20 bp
duplications
deletions >20 bp
*two new duplications and amplification of the cyb- <dig> locus to three copies.
mutation rate estimates.
the variants analyzed are the  <dig> sbss that were identified between generations  <dig> and  <dig>
previous studies showed that g <dig> dna secondary structure is mutagenic in the absence of dog- <dig>  <cit> .
using the g <dig> dna signature , we identified  <dig>  such sites in c. elegans’ genome .
next, we tested to see if any of the  <dig> substitutions occurred within the g <dig> dna signature sequence and observed that none of them did , indicating that the identified substitutions most likely arose spontaneously and were not due to lack of functional dog- <dig>
small indels do not occur within g4-dna
we applied varscan <dig>  <cit>  to identify  <dig> homozygous indels that were 20 bp or less in mdf- <dig> such-4; dog-1f <dig> .
we randomly selected  <dig> of the indels and confirmed all  <dig> by sanger re-sequencing, indicating a false positive rate of less than 5% .
analysis of indels in the unc- <dig> mdf- <dig> such-4; dog- <dig> strains propagated for  <dig> and  <dig> generations  revealed that 88% of the f <dig> indels  existed in the f <dig> generation , 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 <dig>  and another nine indels accumulated between f <dig> and f <dig> .
based on the last  <dig> generations of propagation, we estimate a mutation rate for indels to be 17/ =  <dig>  × 10-10/base/generation.
the ratio of indels to sbss observed was  <dig>  or one indel per  <dig>  substitutions, which is much lower than the  <dig>  ratio reported previously in c. elegans  <cit> .
however, the lower number of indels to sbss that we observe in the unc- <dig> mdf- <dig> such-4; dog- <dig> background is comparable to analyses in yeast  <cit> , a. thaliana  <cit>  and human  <cit> .
namely, wgs analysis in yeast had revealed ratio of indels to sbss of ∼  <dig>   <cit> , which was consistent with previous findings of one indel per  <dig> sbss  <cit> .
furthermore, analysis on spontaneous occurrence of indels in a. thaliana revealed the ratio of  <dig>  of indels to sbss  <cit> .
it may be possible that the small number of indels, over the last  <dig> 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  <dig> indels occurred within the g <dig> dna signature sequence and observed that none of them did .
thus, we believe that these indels arose spontaneously and are not due to a lack of functional dog- <dig>
duplications do not occur within g4-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  <cit> .
previously, using the oacgh we showed that the such- <dig> suppressor genome contains a large tandem duplication on chromosome v   <cit> .
here, we used both pindel  <cit>  and oacgh  <cit> , and identified four sites with copy number increases in the mdf- <dig> such-4; dog-1f <dig> genome , including a previously identified large tandem duplication  <cit> .
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 <dig> and f <dig> genomes captured a dynamic property of duplications .
in f <dig>  we observed three duplications .
one is the large tandem duplication located on lgv that amplifies  <dig> protein-coding genes, which we have described previously  <cit> .
in generation f <dig>  we detected duplications of two new loci, as well as further amplification of the lgv region to three copies  .
in f <dig>  we did not find any new duplications , but did observe that the duplication on lgi was lost, resulting in a wild-type copy number for this region .
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  <dig>  × 10-7/gene/generation  <cit> .
our data, based on the last  <dig> generations , also revealed a comparably high rate of gene duplication , specific to the unc- <dig> mdf- <dig> such-4; dog- <dig> background, when a large duplication on lgv is excluded from the analysis.
the lgv duplicated region contains  <dig> protein-coding genes, including cyb- <dig>  <cit> .
we observed a correlation between increased dosage of cyb- <dig> and a striking fitness increase in our strains; thus, our experimental protocol selected for lgv amplification .
importantly, like sbss and indels, none of the duplications occurred in the vicinity of the g <dig> dna signature sequences , also indicating that these cnvs arose spontaneously and were not due to a lack of functional dog- <dig>
deletions frequently initiate at g <dig> dna sites
the major type of mutation observed, in the absence of dog- <dig>  is a deletion of 300 bp or smaller, initiating at either the 5′-end of c- or the 3′-end of g-tracts  <cit> .
using the unique alignments generated by novoalign and pindel  <cit> , we identified  <dig> homozygous deletions larger than 20 bp in the mdf- <dig> such-4; dog-1f <dig> genome , including the known mdf- <dig> and dog- <dig> deletions.
we randomly selected  <dig> of the deletions, and confirmed all of them using pcr .
to identify deletions, which may have been missed, we also used oacgh  <cit> .
we confirmed all of the deletions, predicted by pindel, that were covered by oacgh probes and identified an additional  <dig> deletions not detected by pindel , making the final count  <dig> homozygous deletions in the mdf- <dig> such-4; dog-1f <dig> genome .
we observed  <dig> deletions in f <dig>  indicating that  <dig> deletions had accumulated in  <dig> generations  propagated in the absence of mdf- <dig> and dog- <dig> .
we found that the majority of the deletions  initiated in g <dig> dna .
previous analysis in c. elegans  <cit>  estimated the spontaneous rate of deletions to be  <dig>  × 10-7/gene/generation respectively.
the  <dig> deletions accumulating between the generations f <dig> and f <dig> affect  <dig> protein-coding genes which allowed us to calculate the mutation rate of deletions over the  <dig> generations to be 19/ =  <dig>  × 10-6/gene/generation in the unc- <dig> mdf- <dig> such-4; dog- <dig> strain, which is approximately 10-fold higher than the estimated rate in n <dig>  <cit> .
this is similar to the estimated forward mutation frequency of et1-balanced lethal mutations in dog- <dig> background  <cit> .
to determine if the elevated mutation rate of deletions is due to dog- <dig> deficiency, we compared mutation rates in non-g <dig> sites versus mutation rate in the g <dig> sites.
while the mutation rate of deletions affecting the non-g <dig> sites,  <dig>  × 10-7/gene/generation or  <dig>  × 10-10/base/generation, is comparable to the previously reported spontaneous rate of deletions  <cit> ; the mutation rate based on g <dig> sites,  <dig>  × 10- <dig> /g4site/generation, illustrates the striking vulnerability of these dna regions when dog- <dig> is absent.
therefore, deletions larger than 20 bp are the only variants in the unc- <dig> mdf- <dig> such-4; dog- <dig> strain that frequently occurred in the g4-dna sites and had significantly higher mutation rate than the spontaneous rate reported previously for the strains with normal dog- <dig> function.
monog/c tracts larger than 14 bp are frequently deleted when dog- <dig> is not functional
in recent years, the g <dig> dna has been implicated in diverse biological processes, such as gene expression  <cit>  and dna replication initiation  <cit> .
consistent with the established role of dog- <dig>  we found that the majority  of the homozygous deletions that we identified between f <dig> and f <dig>  initiated in the previously proposed g <dig> dna signature g3+n1-7g3+n1-7g3+n1-7g3+  <cit>   where the g-tract was almost completely removed together with 5′ flanking dna sequence .
in agreement with a previous study  <cit> , we found that the majority of deletions initiate at monog tracts larger than 14 bp  , while  <dig> deletions initiate at monog-like structures with no more than two nucleotides that interrupt the homopolymer , and  <dig> deletions initiate at sequences that interrupt the homopolymers by three or more nucleotides  .
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- <dig> mdf- <dig> such-4; dog-1 f <dig> genome has  <dig> % 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 g4-dna sites could be deleted in a strain and still maintain viability of the animals.figure  <dig>
the majority of large deletions initiates at g-rich dna.
figures  through  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  <dig>  while orange depicts  <dig> or less; the gaps depict no coverage, which is indicative of deletions.
the reference sequence is depicted with deleted bases underlined.
a 99 bp deletion that initiates at g <dig> homopolymer.
a 96 bp deletion that initiates at g14tg6agaag <dig> sequence.
a 112 bp deletion that initiates at a g-rich sequence, g2n3g2n5g2n2g <dig>   a 55 bp deletion that initiates at a non g-rich sequence.
size distribution of homozygous deletions that occur at g-rich sequences .
to investigate whether there may be additional sequences, which are vulnerable in the absence of dog- <dig>  we analyzed the  <dig> deletions that do not initiate in g <dig> 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 g2+n1-7g2+n1-7g2+n1-7g2+ signature , while one had a g2+n1-7g2+n1-7g2+ signature at the breakpoint .
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- <dig> table  <dig>
schematic representation of the nine deletions that initiate at g-rich regions within sequences that deviate from the g <dig> dna signature, g
3+
n
1-7
g
3+
n
1-7
g
3+
n
1-7
g
3+
lg
locus
signature
*analysis of the sequences revealed that nine match the g2+n1-7g2+n1-7g2+n1-7g2+ signature, while one  matches g2+n1-7g2+n1-7g2+ signature at the breakpoint.
previously, it was found that the deletion sizes in viable lines detected by pcr  <cit>  or oacgh methods  <cit>  were predominately smaller than 300 bp.
in this study, the  <dig> deletions that occurred at g-rich sequences ranged between  <dig> and  <dig>  base-pairs with the majority of the deletions  removing less than 300 bp .
these findings are in agreement with the deletion distribution sizes revealed by the study of a 69 g-tract deletion set  <cit> .
however, larger deletions, initiating at g-tracts, have been recovered as lethal mutations  <cit> , consistent with these regions containing essential genes  <cit> .
we also found that  <dig> deletions, which occurred at non-g-rich sites, removed small regions of less than 300 bp in size .
<dig> protein-coding genes are affected by the  <dig> gvs
to determine an effect of all the identified variants on protein-coding genes, after long-term propagation in the absence of mdf- <dig> and dog- <dig>  we used coovar  <cit> , 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  <cit> .
using coovar  <cit> , we predicted that  <dig> genes would be affected by  <dig> of the deletions that had accumulated between generations f <dig> and f <dig> .
the majority of the deletions  represent the first knockout alleles for those genes and thus provide a genetic resource for studying their functions .
we found that the majority of the indels are not located in protein-coding regions, and the one that affects a protein-coding gene is an in-frame deletion .
we found that the majority of the detected sbss are located within non-protein coding regions.
however, there are  <dig> sbss that fall within protein-coding regions that are predicted to result in missense mutations .
in total,  <dig> protein-coding genes were affected by the duplications .
the majority of the genes  are affected by the large tandem duplication on lgv .
in addition to the  <dig> gvs that had accumulated between generations f <dig> and f <dig>  we had also performed analysis of the effect on protein-coding genes of all the variants observed in the mdf-1such-4; dog-1f <dig> genome .
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  <cit> , 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- <dig> absent  <cit> .
CONCLUSIONS
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/mad <dig>  combining wgs analysis with oacgh analysis allowed us to comprehensively analyze both the small-scale variants  and large-scale variants .
freezing the strain, for long term storage, at three different time points, f <dig>  f <dig> and f <dig>  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  <dig> generations .
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- <dig>  dna sequences with g4-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 <dig>  f <dig> and f <dig> allowed us to capture and visualize an intriguing property of cnvs .
we did not find any reversions of deletions, substitutions, or indels.
as expected, once fixed, these types of mutations are propagated indefinitely .
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 .
this is the first extensive analysis of a strain that had been propagated in the absence of dog- <dig> helicase for hundreds of generations.
we identified  <dig> 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  <dig> sites are deleted in the mdf- <dig> such-4; dog-1f <dig> 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 <dig> dna in normal development.
recently, g <dig> dna has been implicated in a variety of biological processes including telomere maintenance, gene expression, epigenetic regulation, and dna replication  <cit> .
one question to consider is how many g <dig> dna sites could be removed from a genome yet still maintain viability of the animals.

 genome integrity is crucial for survival of all living organisms. chromosomal instability, marked by whole or segmental aneuploidy is hallmark of human tumors, and can drive abnormal proliferation of cancer cells. in caenorhabditis elegans, mdf- has an essential mdf-1/mad- component of the spindle assembly checkpoint missing and this leads to accumulation of genetic errors and ultimately death by the third generation. the checkpoint prevents cin by inhibiting anaphase-promoting complex/cyclosome, and delaying anaphase onset until all the chromosomes have achieved proper attachment to the spindle. while mdf- prevents both loss and gain of whole chromosomes during mitosis, dog- prevents segmental aneuploidies by ensuring proper replication of guanine-rich dna. g-rich dna can adopt a four-stranded helical g-quadruplex dna structure, which can pose a barrier to replication fork progression if left unresolved. the ability to form g 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- a functional ortholog of the deadbox helicase fancj, was non-functional. when dog- is functional, g-rich dna sequences are stable and deletions affecting these regions are not observed. genome-wide bioinformatic analysis of the human genome had identified more than dna sites with g4-forming potential. in humans, mutations in fancj/dog- have been identified in fanconi anemia complementation group j patients, which is a severe, autosomal recessive, disorder with increased spontaneous and dna crosslink-induced cin showing a wide range of clinical manifestations, and also in early onset breast cancer patients. 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- strains, was analyzed using either pcr-based assays or oligonucleotide array comparative genomic hybridization. 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. 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/mad and dog-1/fancj. this strain was propagated for generations and samples were stored frozen at generations and phenotypic analysis of the strain revealed striking fitness recovery, indicating that accumulated mutations cooperate to bypass the mdf- checkpoint requirement and thus achieve long-term population survival. we performed whole-genome sequencing and oacgh analyses of the strain at three different generations. we identified substitutions, indels, and copy number variants 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. 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- homozygous worms, it is first necessary to isolate a suppressor of mdf- sterility and lethality that occurs in the double mutant. previously, such- was isolated, which allowed propagation of mdf-1; dog- beyond the third generation. the such- suppressor allows for an approximately five-fold increase in fertile hermaphrodite progeny of mdf-1; dog-. this increase in fertility occurs in the generations immediately after isolation of the suppressor. we outcrossed one worm to obtain kr. a second line was isolated and maintained in the dog- background for generations, with storage by freezing at generations and. to mark the presence of mdf- unc- was used, which is present in all of our strains. while propagating unc- mdf- such-4; dog- homozygotes, we observed a further increase in reproductive fitness. this increase was significant, 59% of f470unc- mdf- such-4; dog- progeny develop into fertile hermaphrodites, compared to only 10% of the unc- mdf- such- progeny and 2% of unc- mdf- mutants. detailed phenotypic analysis of these strains as well as genetic dissection of suppressors has recently been reported. to identify the genomic variations that had accumulated in mdf- such-4; dog-1 f worms after long-term propagation, the genome was sequenced to a depth of 70× genome equivalents and aligned to the c. elegans reference genome ws available at wormbase. we also sequenced the unc- mdf- such-4; dog- strains that were frozen at generations and and compared the progress of mutation accumulation.figure a schematic representation of the long-term propagation of unc- mdf- such-4; dog-1 homozygotes for generations. we isolated a suppressor in f from the only plate that we set up that propagated in the absence of mdf- and dog- for longer than three generations. we generated a strain by crossing away dog- a second line was propagated for the total of generations. the worms were frozen at generations, and. note that unc- is tightly linked to mdf- in all of our strains. single base substitutions do not occur within g4-dna we used the variant caller varscan2; version. to identify homozygous sbss that occurred with a variant frequency of 90% or higher in the mdf- such-4; dog-1f genome. all tested substitutions were confirmed by sanger re-sequencing, indicating a false positive rate of less than 5%. wgs analysis of the unc- mdf- such-4; dog- strain, which had been frozen at generation, revealed that the majority of the sbss present in generation are also present in generation. however, we did observe that additional substitutions had accumulated between f and f and more between f and f. large number of substitutions observed in f indicate the possibility that the original unc- mdf- such-4; dog- strain had a large number of single nucleotide differences from the reference genome. one way to test this possibility is to estimate the mutation rates based on available data. if we consider the substitutions that had accumulated in generations, between f and f we estimate the rate of μbs =  × 10-9/base/generation. this estimate is similar to the previously reported spontaneous rate of base substitution in c. elegans,  ±   × 10− /base/generation and other model organisms,  × 10− /base/generation in drosophila melanogaster and  ±   × 10− /base/generation in arabidopsis thaliana. furthermore, analysis of the substitutions on mutation bias revealed very similar mutation spectrum to the spontaneous mutation spectra in n, and our analysis of transition bias revealed the ts/tv ratio = , which is within the range observed for spontaneous mutations in multiple mutation accumulation lines. therefore, the similarity of the substitution rate over the last generations to the previously reported μbs in n strongly implies that the majority of the substitutions identified in the f generation are variants originally present in the starting strain; therefore, we focused our analysis on gvs that had occurred between f and f.table summary of the variants identified in the unc- mdf- such-4; dog-1 strains at generations f 170, f 270 and f 470 sbss indels ≤ 20 bp duplications deletions >20 bp *two new duplications and amplification of the cyb- locus to three copies. mutation rate estimates. the variants analyzed are the sbss that were identified between generations and previous studies showed that g dna secondary structure is mutagenic in the absence of dog-. using the g dna signature, we identified such sites in c. elegans’ genome. next, we tested to see if any of the substitutions occurred within the g dna signature sequence and observed that none of them did, indicating that the identified substitutions most likely arose spontaneously and were not due to lack of functional dog- small indels do not occur within g4-dna we applied varscan to identify homozygous indels that were 20 bp or less in mdf- such-4; dog-1f. we randomly selected of the indels and confirmed all by sanger re-sequencing, indicating a false positive rate of less than 5%. analysis of indels in the unc- mdf- such-4; dog- strains propagated for and generations revealed that 88% of the f indels existed in the f generation, 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 and another nine indels accumulated between f and f. based on the last generations of propagation, we estimate a mutation rate for indels to be 17/ =  × 10-10/base/generation. the ratio of indels to sbss observed was or one indel per substitutions, which is much lower than the ratio reported previously in c. elegans. however, the lower number of indels to sbss that we observe in the unc- mdf- such-4; dog- background is comparable to analyses in yeast, a. thaliana and human. namely, wgs analysis in yeast had revealed ratio of indels to sbss of ∼ , which was consistent with previous findings of one indel per sbss. furthermore, analysis on spontaneous occurrence of indels in a. thaliana revealed the ratio of of indels to sbss. it may be possible that the small number of indels, over the last 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 indels occurred within the g dna signature sequence and observed that none of them did. thus, we believe that these indels arose spontaneously and are not due to a lack of functional dog- duplications do not occur within g4-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. previously, using the oacgh we showed that the such- suppressor genome contains a large tandem duplication on chromosome v. here, we used both pindel and oacgh, and identified four sites with copy number increases in the mdf- such-4; dog-1f genome, including a previously identified large tandem duplication. 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 and f genomes captured a dynamic property of duplications. in f we observed three duplications. one is the large tandem duplication located on lgv that amplifies protein-coding genes, which we have described previously. in generation f we detected duplications of two new loci, as well as further amplification of the lgv region to three copies. in f we did not find any new duplications, but did observe that the duplication on lgi was lost, resulting in a wild-type copy number for this region. 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 × 10-7/gene/generation. our data, based on the last generations, also revealed a comparably high rate of gene duplication, specific to the unc- mdf- such-4; dog- background, when a large duplication on lgv is excluded from the analysis. the lgv duplicated region contains protein-coding genes, including cyb-. we observed a correlation between increased dosage of cyb- and a striking fitness increase in our strains; thus, our experimental protocol selected for lgv amplification. importantly, like sbss and indels, none of the duplications occurred in the vicinity of the g dna signature sequences, also indicating that these cnvs arose spontaneously and were not due to a lack of functional dog- deletions frequently initiate at g dna sites the major type of mutation observed, in the absence of dog- is a deletion of 300 bp or smaller, initiating at either the 5′-end of c- or the 3′-end of g-tracts. using the unique alignments generated by novoalign and pindel, we identified homozygous deletions larger than 20 bp in the mdf- such-4; dog-1f genome, including the known mdf- and dog- deletions. we randomly selected of the deletions, and confirmed all of them using pcr. to identify deletions, which may have been missed, we also used oacgh. we confirmed all of the deletions, predicted by pindel, that were covered by oacgh probes and identified an additional deletions not detected by pindel, making the final count homozygous deletions in the mdf- such-4; dog-1f genome. we observed deletions in f indicating that deletions had accumulated in generations propagated in the absence of mdf- and dog-. we found that the majority of the deletions initiated in g dna. previous analysis in c. elegans estimated the spontaneous rate of deletions to be × 10-7/gene/generation respectively. the deletions accumulating between the generations f and f affect protein-coding genes which allowed us to calculate the mutation rate of deletions over the generations to be 19/ =  × 10-6/gene/generation in the unc- mdf- such-4; dog- strain, which is approximately 10-fold higher than the estimated rate in n. this is similar to the estimated forward mutation frequency of et1-balanced lethal mutations in dog- background. to determine if the elevated mutation rate of deletions is due to dog- deficiency, we compared mutation rates in non-g sites versus mutation rate in the g sites. while the mutation rate of deletions affecting the non-g sites, × 10-7/gene/generation or × 10-10/base/generation, is comparable to the previously reported spontaneous rate of deletions; the mutation rate based on g sites, × 10- /g4site/generation, illustrates the striking vulnerability of these dna regions when dog- is absent. therefore, deletions larger than 20 bp are the only variants in the unc- mdf- such-4; dog- strain that frequently occurred in the g4-dna sites and had significantly higher mutation rate than the spontaneous rate reported previously for the strains with normal dog- function. monog/c tracts larger than 14 bp are frequently deleted when dog- is not functional in recent years, the g dna has been implicated in diverse biological processes, such as gene expression and dna replication initiation. consistent with the established role of dog- we found that the majority of the homozygous deletions that we identified between f and f initiated in the previously proposed g dna signature g3+n1-7g3+n1-7g3+n1-7g3+ where the g-tract was almost completely removed together with 5′ flanking dna sequence. in agreement with a previous study, we found that the majority of deletions initiate at monog tracts larger than 14 bp, while deletions initiate at monog-like structures with no more than two nucleotides that interrupt the homopolymer, and deletions initiate at sequences that interrupt the homopolymers by three or more nucleotides. 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- mdf- such-4; dog-1 f genome has % 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 g4-dna sites could be deleted in a strain and still maintain viability of the animals.figure the majority of large deletions initiates at g-rich dna. figures through 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 while orange depicts or less; the gaps depict no coverage, which is indicative of deletions. the reference sequence is depicted with deleted bases underlined. a 99 bp deletion that initiates at g homopolymer. a 96 bp deletion that initiates at g14tg6agaag sequence. a 112 bp deletion that initiates at a g-rich sequence, g2n3g2n5g2n2g a 55 bp deletion that initiates at a non g-rich sequence. size distribution of homozygous deletions that occur at g-rich sequences. to investigate whether there may be additional sequences, which are vulnerable in the absence of dog- we analyzed the deletions that do not initiate in g 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 g2+n1-7g2+n1-7g2+n1-7g2+ signature, while one had a g2+n1-7g2+n1-7g2+ signature at the breakpoint. 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- table schematic representation of the nine deletions that initiate at g-rich regions within sequences that deviate from the g dna signature, g 3+ n 1-7 g 3+ n 1-7 g 3+ n 1-7 g 3+ lg locus signature *analysis of the sequences revealed that nine match the g2+n1-7g2+n1-7g2+n1-7g2+ signature, while one matches g2+n1-7g2+n1-7g2+ signature at the breakpoint. previously, it was found that the deletion sizes in viable lines detected by pcr or oacgh methods were predominately smaller than 300 bp. in this study, the deletions that occurred at g-rich sequences ranged between and base-pairs with the majority of the deletions removing less than 300 bp. these findings are in agreement with the deletion distribution sizes revealed by the study of a 69 g-tract deletion set. however, larger deletions, initiating at g-tracts, have been recovered as lethal mutations, consistent with these regions containing essential genes. we also found that deletions, which occurred at non-g-rich sites, removed small regions of less than 300 bp in size. protein-coding genes are affected by the gvs to determine an effect of all the identified variants on protein-coding genes, after long-term propagation in the absence of mdf- and dog- we used coovar, 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. using coovar, we predicted that genes would be affected by of the deletions that had accumulated between generations f and f. the majority of the deletions represent the first knockout alleles for those genes and thus provide a genetic resource for studying their functions. we found that the majority of the indels are not located in protein-coding regions, and the one that affects a protein-coding gene is an in-frame deletion. we found that the majority of the detected sbss are located within non-protein coding regions. however, there are sbss that fall within protein-coding regions that are predicted to result in missense mutations. in total, protein-coding genes were affected by the duplications. the majority of the genes are affected by the large tandem duplication on lgv. in addition to the gvs that had accumulated between generations f and f we had also performed analysis of the effect on protein-coding genes of all the variants observed in the mdf-1such-4; dog-1f genome. 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, 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- absent.  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/mad combining wgs analysis with oacgh analysis allowed us to comprehensively analyze both the small-scale variants and large-scale variants. freezing the strain, for long term storage, at three different time points, f f and f 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 generations. 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- dna sequences with g4-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 f and f allowed us to capture and visualize an intriguing property of cnvs. we did not find any reversions of deletions, substitutions, or indels. as expected, once fixed, these types of mutations are propagated indefinitely. 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. this is the first extensive analysis of a strain that had been propagated in the absence of dog- helicase for hundreds of generations. we identified 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 sites are deleted in the mdf- such-4; dog-1f 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 dna in normal development. recently, g dna has been implicated in a variety of biological processes including telomere maintenance, gene expression, epigenetic regulation, and dna replication. one question to consider is how many g dna sites could be removed from a genome yet still maintain viability of the animals. 
to identify a spectrum of mutations, after long-term propagation, we combined whole genome sequencing and oligonucleotide array comparative genomic hybridization analysis of a c. elegans strain that was propagated, in the absence of dog- and mdf-1/mad for a total of generations, with samples taken for long term storage in generations and we compared the genomes of f and f strains and identified substitutions, indels, duplications, and deletions larger than 20 bp. whole and partial chromosome losses or gains and structural chromosome changes are hallmarks of human tumors. 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- and mdf- furthermore, deletions larger than 20 bp were the only variants that frequently occurred in g-rich dna. we showed that of the possible predicted monog/c tracts, larger than 14 bp, were deleted in unc- mdf- such-4; dog-1 f. in caenorhabditis elegans, faithful transmission of g-rich dna is ensured by the dog-1/fancj deadbox helicase. here, we identified variants that accumulated in c. elegans’ genome after long-term propagation in the absence of dog- and mdf- we showed that dna sequences, with g4-forming potential, are vulnerable to deletion-formation in this genetic background.
 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 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 and oligonucleotide array comparative genomic hybridization analysis of a c. elegans strain that was propagated, in the absence of dog- and mdf-1/mad for a total of generations, with samples taken for long term storage in generations and we compared the genomes of f and f strains and identified substitutions, indels, duplications, and deletions larger than 20 bp. these homozygous variants were predicted to impact 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- and mdf- furthermore, deletions larger than 20 bp were the only variants that frequently occurred in g-rich dna. we showed that of the possible predicted monog/c tracts, larger than 14 bp, were deleted in unc- mdf- such-4; dog-1 f.  here, we identified variants that accumulated in c. elegans’ genome after long-term propagation in the absence of dog- and mdf- we showed that dna sequences, with g4-forming potential, are vulnerable to deletion-formation in this genetic background. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords c. eleganswhole genome sequencing oligonucleotide array comparative genomic hybridization mutation accumulation genomic variation spindle assembly checkpoint dog-1/fancjg-quadruplex structureissue-copyright-statement© the author 2015 