BACKGROUND
caenorhabditis elegans is an animal model widely used in biomedical and biological research.
c. elegans was the first animal to have its genome completely sequenced  <cit>  and the compiled and annotated sequence is available at wormbase http://www.wormbase.org.
the ready availability of genomic sequence information along with an extensive body of knowledge about gene function in this species provides an exceptional opportunity to examine the consequences of mutational change on the composition of the genome.
high-throughput sequencing of wild type  <cit>  and mutant strains  <cit>  has demonstrated the diverse benefits of examining genomic sequence.
not only is genome-wide sequencing valuable for finding the mutational basis of phenotypic change, but also for understanding evolutionary processes.
denver et al.
<cit>  identified and characterized base-substitution mutations that arose spontaneously in  <dig> lines of c. elegans, providing us with a fuller understanding of the nature of genome-wide base-substitution events.
a question that has long been debated is the relationship of mutational patterns to biological processes such as meiotic recombination  <cit> .
in c. elegans, the central portions of the five autosomes are relatively gene dense compared to the arms  <cit> .
furthermore, traditional genetic approaches using forward mutational screens to recover lethal alleles of essential genes have shown genes in the central clusters of chromosomes i and v to be more mutable to lethality than the arms  <cit> .
however, the most striking feature of the c. elegans autosomes is the recombination suppression associated with the central gene clusters,  <cit>  reviewed in  <cit> .
in wild-type, the frequency of crossing over per length of dna varies as much as ten-fold between the cluster and an arm of chromosome i  <cit> , making this species an excellent model for studying the relationship between sequence variation and recombination rate.
the recombinational suppression of the gene clusters is eliminated in the mutant rec- <dig>  <cit> , resulting in increased crossing over in the autosomal central regions and a compensatory decrease in the arms  <cit> .
the consequence is an altered distribution of meiotic exchange events while retaining the same overall number.
in rec- <dig>  the genetic recombination map resembles more closely the physical length of the chromosome than it does the wild-type pattern of crossovers.
the phenotype was originally identified as a recessive mutation in a strain heterozygous for morphological markers in the central cluster of chromosome i, dpy- <dig> unc- <dig> +/+ +unc- <dig>  a three-fold increase in crossing over was observed in the central region of the autosomes  <cit> .
the visible markers were eventually eliminated by recombination resulting in a wild-type appearing strain, bc <dig>  for which the major phenotype is an altered distribution of recombination, affecting both exchange of flanking markers and apparent intragenic gene conversion  <cit> .
there are no detrimental effects on growth, progeny number or spontaneous mutation rate.
nondisjunction of the x-chromosome is elevated somewhat, but not dramatically.
the rec- <dig> mutation is inherited as a mendelian recessive, and crossover distribution is altered for the entire genome  <cit> , including the x-chromosome, despite the fact that it has a more uniform distribution of recombination events .
the consequence of the mutation is that the recombination map in rec- <dig> more closely reflects the physical map than the genetic map in wild type  <cit>  .
in this paper, we used the high-throughput solexa platform  to sequence the genome of the rec- <dig> strain.
this study provides the first opportunity to examine the consequences on a genome of altering the distribution of meiotic recombination events.
RESULTS
base pair composition of rec- <dig> compared to wormbase and vc2010
there were a total of  <dig> , <dig> forty-two base pair sequence reads, of which  <dig> , <dig>  were aligned to the wormbase reference genome ws <dig> using maq software  <cit>  with a maximum of two mismatches per read resulting in approximately 21-fold redundant sequence coverage.
base pair differences were called using both maq  <cit>  and slider  <cit>  software.
we observed  <dig> base pair differences between rec- <dig>  and wormbase ws <dig>  and  <dig> between rec- <dig> and the wild-type strain vc <dig>  fourteen of the observed differences were tested by either pcr or direct sequencing and all fourteen confirmed.
in this paper, we analyzed those differences that were identified by both maq and slider compared to vc <dig>  the canonical sequence of c. elegans, archived in wormbase, is valuable because it is compiled, annotated and readily accessible.
the wormbase reference sequence  <cit>  was obtained from cloned cosmids and yacs, which for technical reasons came from different strains, and is not the genome sequence of any one existing strain.
thus, we could not experimentally determine the allelic status of rec- <dig> for the wormbase reference sequence.
since many strains of c. elegans carry the s <dig> allelic variant of rec- <dig>  we could not simply assume the reference sequence was wild type.
thus, in this paper we examine the differences between rec- <dig> and vc <dig>  a strain that we confirmed by measuring meiotic crossing over to be wild-type for rec- <dig>
the c. elegans genome is approximately  <dig> million base pairs  in size.
we observed a base pair difference approximately every  <dig>  bps on average.
the number of base pair differences for each of the six nonstrand-specific base substitution mutation types  per mbp of aligned sequence was plotted for each chromosome .
the most frequent change is g:c to a:t on chromosomes i and x,  <dig> and  <dig> respectively.
chromosome i has as many g:c to a:t substitutions as chromosomes ii, iii, iv and v together.
rec- <dig> was originally observed in strains mutagenized with ems, a mutagen known to generate g to a changes.
the gene is linked to chromosome i markers, and due to difficulty in scoring the recombination phenotype, the mutation has not been outcrossed extensively.
most likely many of the a:t differences are attributable to mutational changes retained in the rec- <dig> strain.
the predominance of g:c to a:t substitutions on chromosomes i and x is also reflected in the ratio of transition to transversions  .
ignoring those two chromosomes, the ts/tv ratio is very close to random expectation of  <dig> .
although our study is not designed to follow mutational accumulation over generations, we have analyzed the distribution of base changes along the chromosomes.
table  <dig> shows that the number and distribution of base pair differences on chromosome i  and to a lesser extent the x-chromosome  are predominantly g:c to a:t. in an attempt to separate these changes from what may be due to de novo mutation in rec- <dig>  we have plotted them separately along the chromosomes .
examination of the distribution of change along the chromosomes does not reveal any dramatic pattern, either for the distribution of g:c to a:t changes  or for the distribution of the other types .
in denver et al.
<cit> , after several generations of accumulated mutation in wild-type strains under relaxed selection no distinctive pattern of base substitutions was seen along the chromosomes.
neither do we see any dramatic difference in distribution that might correlate with the absence of a recombinational pattern.
in an attempt to investigate the distribution numerically, we calculated the number base pair differences in the autosomal arms and in the central clusters as defined in  <cit>  per megabasepair.
in the arms there are approximately  <dig>  differences per mbp  compared to  <dig>   in the cluster.
a histogram of the number base changes per mbp along chromosome i is shown in additional file  <dig>  figure s <dig>  when plotted this way, none of the chromosomes show any distinctive pattern .
in the absence of any detectable pattern of mutational distribution, it seems most likely that rec- <dig> has had no significant affect on mutation rate.
we plotted the composition of base pair differences both for the entire genome and for the genome minus chromosome i and the x .
when chromosomes i and x are removed from the analysis, the relative frequency of the different types of base pair differences is similar to that observed by denver et al.
<cit> , with the exception that we see considerably fewer g:c to t:a differences.
the  <dig> base pair differences are shown in additional file  <dig>  table s <dig>  ninety-five of these are in exons .
approximately half of these  were non-synonymous changes.
fifteen of the changes are in untranslated regions ,  <dig> in introns and the rest in intergenic regions.
rec- <dig> is neither caused by nor causes detectable chromosomal rearrangements
the genome of rec- <dig> was sequenced by whole genome shotgun sequencing  using paired end tags  and aligned to the reference genome available in wormbase.
using the alignment to the ws <dig> reference genome, small insertions can be characterized by clusters of pets that are shorter than the average size , whereas deletions in the sample can be detected by pets that are longer.
although this is counter-intuitive to geneticists familiar with interpreting genetic maps, it is true because the sequence reads from the ends  of the genomic fragments are further away in the sequenced dna if there is an insertion of unannotated material than they appear on the wormbase map , which lacks that insertion.
in the example shown in figure  <dig>  the top of the figure shows the size of sequence reads aligned to the wormbase sequence.
although the inserted fragments are actually longer, they appear shorter on the reference genome.
in an analogous way, deletions appear longer.
translocations will have links connecting clusters on different chromosomes with a loss of read coverage at the breakpoints.
in this way, the rec- <dig> sequence was analyzed for chromosomal rearrangements, insertions, deletions, inversions and translocations.
no large chromosomal rearrangements were observed.
confirmational data was obtained using oligo array comparative genome hybridization .
an exon-centric array design that covered the entire genome revealed no major sequence copy number changes relative to the reference dna.
direct repeat sequences can appear as longer paired end reads
examination of fragment sizes in the rec- <dig> strain revealed a number of paired end tags  longer than average, an indication of potential deletions.
the sequence and position of these pets were examined in detail.
an example from the right arm of chromosome i is shown in figure  <dig>  in this case, the observed long sizes resulted from one of the paired ends aligning with an imperfect direct repeat sequence in the sample dna.
the observed longer tag length is the consequence of one of the paired ends aligning to different components of a repeat sequence.
in rec- <dig> as well as in other genomes analyzed, regions like this one that contained imperfect direct repeats gave poor coverage of sequence reads as is illustrated by the absence of normal size pets.
the analysis illustrates how long paired-end tags can be used to identify direct repeat sequences.
analysis of the long pets and alignment to the reference genome revealed the presence of two deletions in the rec- <dig> strain that were not in the vc <dig> wild type.
one of these affected an exonic region on the x-chromosome near  <dig> , <dig> bp and was confirmed by acgh.
the other removed approximately  <dig> base pairs  of intergenic sequence in a region of chromosome i around  <dig> , <dig> bp, a region of dna not on the exon array.
additional transposable elements exist in rec-1
in a progenitor strain of bc <dig>  tc <dig> was observed to actively transpose  <cit> , although in the original cb <dig> strain tc <dig> was apparently inactive.
blot hybridization patterns of the high-hopper strains have been published previously  <cit> .
in this paper, we examined the number and position of the transposable elements, tc <dig>  tc <dig>  tc <dig>  tc <dig>  tc <dig>  tc <dig> and cemar <dig> compared to the positions reported in wormbase and reviewed by  <cit> .
in the wild-type strain of c. elegans there are  <dig> copies of the transposable element tc <dig> and four of tc <dig>  <cit> .
in the rec- <dig> strain, there are  <dig> additional copies of tc <dig>  and  <dig> novel locations for tc <dig> .
an example of how tc <dig> insertion was analyzed is shown in figure  <dig>  reads from within unique sequence paired with a read from the terminus of tc <dig> identified the insertion.
all the full length tc <dig> and tc2's had ta termini.
we were not able to uniquely identify the progenitor tc <dig> since none of the tc1's analyzed had a unique base pair change in the portion of the element sequenced.
as might be expected, most of the new insertions were in either introns or intergenic regions.
one tc <dig> that inserted into a coding region of a gene on the x was detectable also by acgh.
no empty sites, that is, sites vacated by tc <dig>  were found by searching unmapped reads for the dna sequence ta or tata.
there were no new locations for tc <dig>  tc <dig>  tc <dig>  tc <dig>  these elements had positions identical to those reported in wormbase.
there is an additional copy of cemar <dig> reported in wormbase that correlates with a duplication of a portion of chromosome v, which is present in several wild-type strains, but not in vc <dig>  <cit>  or rec- <dig>
acgh high density chip analysis agrees with the genomic sequencing
in addition to the genome-wide acgh, a specially designed high density array was used to examine the central portion of the gene cluster of chromosome i. the array identified five base pair differences relative to the reference dna, an example of one is shown in figure  <dig>  all five of these differences were also identified in the sequence analysis  of the rec- <dig> genomic dna and confirmed by either restriction enzyme analysis followed by pcr or direct sequencing across the site using primers.
discussion
the sequence of the rec- <dig> genome was obtained by whole genome shotgun sequencing  with the illumina genome analyzer and compared to both the reference genome available at wormbase http://www.wormbase.org and a laboratory wild type  using maq  <cit>  and slider  <cit>  software.
the base pair composition of rec- <dig> was more similar to the wild strain vc <dig> than to the reference genome, ws <dig>  vc <dig> is a line of n <dig>  separated from the original brenner strain at some time in the past.
there are actually two major n <dig> derivative lines distinguishable by the presence or absence of a duplication of a portion of the left arm of chromosome v  <cit> .
although the rec- <dig> strain, bc <dig>  is not directly derived from vc <dig>  both strains lack the chromosome v duplication.
rec- <dig> was originally detected in strains that along with a wild-type male strain from brenner's original collection were transported to the bc laboratory and maintained on plates for approximately two years before being preserved by freezing in liquid nitrogen.
the major detectable phenotype of rec- <dig> is its alteration in crossover frequency between markers, a phenotype that is difficult and time consuming to follow through genetic crosses.
for this reason, once the strain was constructed and confirmed, it was maintained without additional out-crossing.
the number, type and location of the base pair differences detected by both maq  <cit>  and slider  <cit>  software have been analyzed for the genome.
the slider software was developed to enhance the quality of alignment possible from a low read number and improve the accuracy in base pair change prediction  <cit>  and indeed the number of differences detected was considerably higher with the slider software .
we confirmed the existence of a subset of the differences detected by restriction enzyme analysis of base pair differences that created new cut sites.
in addition, in one region of  <dig> million bp chromosome i, five differences that were observed by slider were confirmed by acgh.
acgh has been proposed as a method for detection of single nucleotide mutations in homozygous c. elegans strains  <cit> .
a large fraction of the base pair differences in rec- <dig> were potentially g to a changes  and may represent remnants of ems mutagenesis.
thus, we attempted to separate these from other types of changes, which may be more representative of spontaneous changes having occurred in the rec- <dig> background.
when plotted and compared to the mutations accumulated under relaxed selection in ten wild-type lines observed by denver et al  <cit> , we see a similar pattern of nonstrand-specific base substitution mutation types, with the exception that there were fewer g:c to t:a changes in rec- <dig> than in their ma-lines.
the number and location of transposable elements was examined.
tc <dig>  tc <dig>  tc <dig>  tc <dig> and cemar <dig> were unchanged.
however, in addition to the ones reported in the reference genome,  <dig> tc <dig> and  <dig> tc <dig> were observed.
these are most likely the remnants of transposition events that occurred in a progenitor strain  <cit> .
the results emphasize the advantage over previous technologies of having the genomic sequence.
we identified not only the location of the new tc <dig> insertions but also the number and location of a large number of tc <dig> insertions.
tc <dig> is  <dig>  bp long and contains two 54-bp terminal inverted repeats and transposes by excision and reinsertion into target dna containing a ta dinucleotide, leaving behind a double-strand dna break which is repaired by the cellular machinery.
tc <dig> is a  <dig>  base pair element that has perfect terminal inverted repeats of  <dig> bp and like tc <dig>  insertions are flanked by a ta dinucleotide at either end.
it may not be obvious why tc <dig> and tc <dig>  which are members of different transposable element superfamilies  <cit> , would have been the two elements to have transposed in the high hopper strain  <cit> , although both elements have been observed to transpose in bristol bergerac hybrids and proposed to move together possibly by a mechanism involving mut- <dig>  <cit> .
the original strain in which mobility was first observed  <cit>  is maintained as a frozen archive and available for further characterization with regard to aspects of tc <dig> and tc <dig> mobilization.
the rec- <dig> strain is unique in that it alters the pattern of meiotic exchange events without affecting the total number of crossovers  <cit>  and has little other phenotypic effects  <cit> .
rattray and rose  <cit>  investigated fitness of rec- <dig> relative to wild type in a short-term experiment performed in a laboratory setting.
no difference was observed under those conditions.
in addition, mutational damage as measured by capture of lethal events using a genetic balancer did not differ from wild type  <cit> .
in the present study, no major chromosomal rearrangements, which might reduce the fitness of the strain, were observed.
there were however, a large number of base pair differences from wild type, and many of these were in coding regions.
these changes are presumably non-detrimental based on their benign effect on the phenotype of the strain.
the genetic maps of many sexually reproducing species reveal that relative to physical distance recombination occurs more frequently in some regions than in others.
furthermore, the position of the crossover event can be influenced by a number of factors, including treatment with ionizing radiation.
in drosophila, ionizing radiation increases crossing over  <cit> , primarily in regions of centric heterochromatin  <cit> , a region known to have low recombination relative to the amount of dna  <cit> .
similarly in c. elegans gamma radiation has been shown to increase crossing over across the recombination-poor central region of autosome i  <cit> .
in yeast, dna damaging agents have been shown to stimulate homologous recombination between ectopic repeats resulting in translocations  <cit> .
in addition, induced double-strand breaks within dispersed small repeats can generate rearrangements resulting in genome reshaping and are a potential source for evolutionary change  <cit> .
c. elegans, which is a self-fertilizing hermaphrodite with out-crossing, a relatively rare situation in nature, provides a useful model for the study of genomic characteristics as they relate to recombination and short term evolution in a self-fertilizing hermaphrodite  <cit> .
in both the denver et al.
<cit>  analysis of ten mutation-accumulating wild-type strains and in our study of rec- <dig>  there is no dramatic pattern of mutational alterations along the chromosome.
these results may be interesting in the context of whether or not recombination is mutagenic.
both the wild-type ma-lines, which presumably have regions of both high and low recombination, and rec- <dig>  in which the variation in recombination is randomized, have very similar patterns of base pair differences.
the data are compatible with a model in which mutational events are independent of the meiotic recombination processes.
CONCLUSIONS
our analysis of high-throughput sequencing was able to detect regions of direct repeat sequences, deletions, insertions of transposable elements, and base pair differences.
a subset of sequence alterations affecting coding regions were confirmed by an independent approach using oligo array comparative genome hybridization.
the major phenotype of the rec- <dig> strain is an alteration in the preferred position of the meiotic recombination event with no other significant phenotypic consequences.
in this study, we observed no evidence of a mutator effect at the nucleotide level attributable to the rec- <dig> mutation.

 caenorhabditis elegans is an animal model widely used in biomedical and biological research. c. elegans was the first animal to have its genome completely sequenced and the compiled and annotated sequence is available at wormbase http://www.wormbase.org. the ready availability of genomic sequence information along with an extensive body of knowledge about gene function in this species provides an exceptional opportunity to examine the consequences of mutational change on the composition of the genome. high-throughput sequencing of wild type and mutant strains has demonstrated the diverse benefits of examining genomic sequence. not only is genome-wide sequencing valuable for finding the mutational basis of phenotypic change, but also for understanding evolutionary processes. denver et al. identified and characterized base-substitution mutations that arose spontaneously in lines of c. elegans, providing us with a fuller understanding of the nature of genome-wide base-substitution events. a question that has long been debated is the relationship of mutational patterns to biological processes such as meiotic recombination. in c. elegans, the central portions of the five autosomes are relatively gene dense compared to the arms. furthermore, traditional genetic approaches using forward mutational screens to recover lethal alleles of essential genes have shown genes in the central clusters of chromosomes i and v to be more mutable to lethality than the arms. however, the most striking feature of the c. elegans autosomes is the recombination suppression associated with the central gene clusters, reviewed in. in wild-type, the frequency of crossing over per length of dna varies as much as ten-fold between the cluster and an arm of chromosome i, making this species an excellent model for studying the relationship between sequence variation and recombination rate. the recombinational suppression of the gene clusters is eliminated in the mutant rec-, resulting in increased crossing over in the autosomal central regions and a compensatory decrease in the arms. the consequence is an altered distribution of meiotic exchange events while retaining the same overall number. in rec- the genetic recombination map resembles more closely the physical length of the chromosome than it does the wild-type pattern of crossovers. the phenotype was originally identified as a recessive mutation in a strain heterozygous for morphological markers in the central cluster of chromosome i, dpy- unc- +/+ +unc- a three-fold increase in crossing over was observed in the central region of the autosomes. the visible markers were eventually eliminated by recombination resulting in a wild-type appearing strain, bc for which the major phenotype is an altered distribution of recombination, affecting both exchange of flanking markers and apparent intragenic gene conversion. there are no detrimental effects on growth, progeny number or spontaneous mutation rate. nondisjunction of the x-chromosome is elevated somewhat, but not dramatically. the rec- mutation is inherited as a mendelian recessive, and crossover distribution is altered for the entire genome, including the x-chromosome, despite the fact that it has a more uniform distribution of recombination events. the consequence of the mutation is that the recombination map in rec- more closely reflects the physical map than the genetic map in wild type. in this paper, we used the high-throughput solexa platform to sequence the genome of the rec- strain. this study provides the first opportunity to examine the consequences on a genome of altering the distribution of meiotic recombination events.  base pair composition of rec- compared to wormbase and vc2010 there were a total of, forty-two base pair sequence reads, of which, were aligned to the wormbase reference genome ws using maq software with a maximum of two mismatches per read resulting in approximately 21-fold redundant sequence coverage. base pair differences were called using both maq and slider software. we observed base pair differences between rec- and wormbase ws and between rec- and the wild-type strain vc fourteen of the observed differences were tested by either pcr or direct sequencing and all fourteen confirmed. in this paper, we analyzed those differences that were identified by both maq and slider compared to vc the canonical sequence of c. elegans, archived in wormbase, is valuable because it is compiled, annotated and readily accessible. the wormbase reference sequence was obtained from cloned cosmids and yacs, which for technical reasons came from different strains, and is not the genome sequence of any one existing strain. thus, we could not experimentally determine the allelic status of rec- for the wormbase reference sequence. since many strains of c. elegans carry the s allelic variant of rec- we could not simply assume the reference sequence was wild type. thus, in this paper we examine the differences between rec- and vc a strain that we confirmed by measuring meiotic crossing over to be wild-type for rec- the c. elegans genome is approximately million base pairs in size. we observed a base pair difference approximately every bps on average. the number of base pair differences for each of the six nonstrand-specific base substitution mutation types per mbp of aligned sequence was plotted for each chromosome. the most frequent change is g:c to a:t on chromosomes i and x, and respectively. chromosome i has as many g:c to a:t substitutions as chromosomes ii, iii, iv and v together. rec- was originally observed in strains mutagenized with ems, a mutagen known to generate g to a changes. the gene is linked to chromosome i markers, and due to difficulty in scoring the recombination phenotype, the mutation has not been outcrossed extensively. most likely many of the a:t differences are attributable to mutational changes retained in the rec- strain. the predominance of g:c to a:t substitutions on chromosomes i and x is also reflected in the ratio of transition to transversions. ignoring those two chromosomes, the ts/tv ratio is very close to random expectation of. although our study is not designed to follow mutational accumulation over generations, we have analyzed the distribution of base changes along the chromosomes. table shows that the number and distribution of base pair differences on chromosome i and to a lesser extent the x-chromosome are predominantly g:c to a:t. in an attempt to separate these changes from what may be due to de novo mutation in rec- we have plotted them separately along the chromosomes. examination of the distribution of change along the chromosomes does not reveal any dramatic pattern, either for the distribution of g:c to a:t changes or for the distribution of the other types. in denver et al., after several generations of accumulated mutation in wild-type strains under relaxed selection no distinctive pattern of base substitutions was seen along the chromosomes. neither do we see any dramatic difference in distribution that might correlate with the absence of a recombinational pattern. in an attempt to investigate the distribution numerically, we calculated the number base pair differences in the autosomal arms and in the central clusters as defined in per megabasepair. in the arms there are approximately differences per mbp compared to in the cluster. a histogram of the number base changes per mbp along chromosome i is shown in additional file figure s when plotted this way, none of the chromosomes show any distinctive pattern. in the absence of any detectable pattern of mutational distribution, it seems most likely that rec- has had no significant affect on mutation rate. we plotted the composition of base pair differences both for the entire genome and for the genome minus chromosome i and the x. when chromosomes i and x are removed from the analysis, the relative frequency of the different types of base pair differences is similar to that observed by denver et al., with the exception that we see considerably fewer g:c to t:a differences. the base pair differences are shown in additional file table s ninety-five of these are in exons. approximately half of these were non-synonymous changes. fifteen of the changes are in untranslated regions, in introns and the rest in intergenic regions. rec- is neither caused by nor causes detectable chromosomal rearrangements the genome of rec- was sequenced by whole genome shotgun sequencing using paired end tags and aligned to the reference genome available in wormbase. using the alignment to the ws reference genome, small insertions can be characterized by clusters of pets that are shorter than the average size, whereas deletions in the sample can be detected by pets that are longer. although this is counter-intuitive to geneticists familiar with interpreting genetic maps, it is true because the sequence reads from the ends of the genomic fragments are further away in the sequenced dna if there is an insertion of unannotated material than they appear on the wormbase map, which lacks that insertion. in the example shown in figure the top of the figure shows the size of sequence reads aligned to the wormbase sequence. although the inserted fragments are actually longer, they appear shorter on the reference genome. in an analogous way, deletions appear longer. translocations will have links connecting clusters on different chromosomes with a loss of read coverage at the breakpoints. in this way, the rec- sequence was analyzed for chromosomal rearrangements, insertions, deletions, inversions and translocations. no large chromosomal rearrangements were observed. confirmational data was obtained using oligo array comparative genome hybridization. an exon-centric array design that covered the entire genome revealed no major sequence copy number changes relative to the reference dna. direct repeat sequences can appear as longer paired end reads examination of fragment sizes in the rec- strain revealed a number of paired end tags longer than average, an indication of potential deletions. the sequence and position of these pets were examined in detail. an example from the right arm of chromosome i is shown in figure in this case, the observed long sizes resulted from one of the paired ends aligning with an imperfect direct repeat sequence in the sample dna. the observed longer tag length is the consequence of one of the paired ends aligning to different components of a repeat sequence. in rec- as well as in other genomes analyzed, regions like this one that contained imperfect direct repeats gave poor coverage of sequence reads as is illustrated by the absence of normal size pets. the analysis illustrates how long paired-end tags can be used to identify direct repeat sequences. analysis of the long pets and alignment to the reference genome revealed the presence of two deletions in the rec- strain that were not in the vc wild type. one of these affected an exonic region on the x-chromosome near, bp and was confirmed by acgh. the other removed approximately base pairs of intergenic sequence in a region of chromosome i around, bp, a region of dna not on the exon array. additional transposable elements exist in rec-1 in a progenitor strain of bc tc was observed to actively transpose, although in the original cb strain tc was apparently inactive. blot hybridization patterns of the high-hopper strains have been published previously. in this paper, we examined the number and position of the transposable elements, tc tc tc tc tc tc and cemar compared to the positions reported in wormbase and reviewed by. in the wild-type strain of c. elegans there are copies of the transposable element tc and four of tc. in the rec- strain, there are additional copies of tc and novel locations for tc. an example of how tc insertion was analyzed is shown in figure reads from within unique sequence paired with a read from the terminus of tc identified the insertion. all the full length tc and tc2's had ta termini. we were not able to uniquely identify the progenitor tc since none of the tc1's analyzed had a unique base pair change in the portion of the element sequenced. as might be expected, most of the new insertions were in either introns or intergenic regions. one tc that inserted into a coding region of a gene on the x was detectable also by acgh. no empty sites, that is, sites vacated by tc were found by searching unmapped reads for the dna sequence ta or tata. there were no new locations for tc tc tc tc these elements had positions identical to those reported in wormbase. there is an additional copy of cemar reported in wormbase that correlates with a duplication of a portion of chromosome v, which is present in several wild-type strains, but not in vc or rec- acgh high density chip analysis agrees with the genomic sequencing in addition to the genome-wide acgh, a specially designed high density array was used to examine the central portion of the gene cluster of chromosome i. the array identified five base pair differences relative to the reference dna, an example of one is shown in figure all five of these differences were also identified in the sequence analysis of the rec- genomic dna and confirmed by either restriction enzyme analysis followed by pcr or direct sequencing across the site using primers. discussion the sequence of the rec- genome was obtained by whole genome shotgun sequencing with the illumina genome analyzer and compared to both the reference genome available at wormbase http://www.wormbase.org and a laboratory wild type using maq and slider software. the base pair composition of rec- was more similar to the wild strain vc than to the reference genome, ws vc is a line of n separated from the original brenner strain at some time in the past. there are actually two major n derivative lines distinguishable by the presence or absence of a duplication of a portion of the left arm of chromosome v. although the rec- strain, bc is not directly derived from vc both strains lack the chromosome v duplication. rec- was originally detected in strains that along with a wild-type male strain from brenner's original collection were transported to the bc laboratory and maintained on plates for approximately two years before being preserved by freezing in liquid nitrogen. the major detectable phenotype of rec- is its alteration in crossover frequency between markers, a phenotype that is difficult and time consuming to follow through genetic crosses. for this reason, once the strain was constructed and confirmed, it was maintained without additional out-crossing. the number, type and location of the base pair differences detected by both maq and slider software have been analyzed for the genome. the slider software was developed to enhance the quality of alignment possible from a low read number and improve the accuracy in base pair change prediction and indeed the number of differences detected was considerably higher with the slider software. we confirmed the existence of a subset of the differences detected by restriction enzyme analysis of base pair differences that created new cut sites. in addition, in one region of million bp chromosome i, five differences that were observed by slider were confirmed by acgh. acgh has been proposed as a method for detection of single nucleotide mutations in homozygous c. elegans strains. a large fraction of the base pair differences in rec- were potentially g to a changes and may represent remnants of ems mutagenesis. thus, we attempted to separate these from other types of changes, which may be more representative of spontaneous changes having occurred in the rec- background. when plotted and compared to the mutations accumulated under relaxed selection in ten wild-type lines observed by denver et al, we see a similar pattern of nonstrand-specific base substitution mutation types, with the exception that there were fewer g:c to t:a changes in rec- than in their ma-lines. the number and location of transposable elements was examined. tc tc tc tc and cemar were unchanged. however, in addition to the ones reported in the reference genome, tc and tc were observed. these are most likely the remnants of transposition events that occurred in a progenitor strain. the results emphasize the advantage over previous technologies of having the genomic sequence. we identified not only the location of the new tc insertions but also the number and location of a large number of tc insertions. tc is bp long and contains two 54-bp terminal inverted repeats and transposes by excision and reinsertion into target dna containing a ta dinucleotide, leaving behind a double-strand dna break which is repaired by the cellular machinery. tc is a base pair element that has perfect terminal inverted repeats of bp and like tc insertions are flanked by a ta dinucleotide at either end. it may not be obvious why tc and tc which are members of different transposable element superfamilies, would have been the two elements to have transposed in the high hopper strain, although both elements have been observed to transpose in bristol bergerac hybrids and proposed to move together possibly by a mechanism involving mut-. the original strain in which mobility was first observed is maintained as a frozen archive and available for further characterization with regard to aspects of tc and tc mobilization. the rec- strain is unique in that it alters the pattern of meiotic exchange events without affecting the total number of crossovers and has little other phenotypic effects. rattray and rose investigated fitness of rec- relative to wild type in a short-term experiment performed in a laboratory setting. no difference was observed under those conditions. in addition, mutational damage as measured by capture of lethal events using a genetic balancer did not differ from wild type. in the present study, no major chromosomal rearrangements, which might reduce the fitness of the strain, were observed. there were however, a large number of base pair differences from wild type, and many of these were in coding regions. these changes are presumably non-detrimental based on their benign effect on the phenotype of the strain. the genetic maps of many sexually reproducing species reveal that relative to physical distance recombination occurs more frequently in some regions than in others. furthermore, the position of the crossover event can be influenced by a number of factors, including treatment with ionizing radiation. in drosophila, ionizing radiation increases crossing over, primarily in regions of centric heterochromatin, a region known to have low recombination relative to the amount of dna. similarly in c. elegans gamma radiation has been shown to increase crossing over across the recombination-poor central region of autosome i. in yeast, dna damaging agents have been shown to stimulate homologous recombination between ectopic repeats resulting in translocations. in addition, induced double-strand breaks within dispersed small repeats can generate rearrangements resulting in genome reshaping and are a potential source for evolutionary change. c. elegans, which is a self-fertilizing hermaphrodite with out-crossing, a relatively rare situation in nature, provides a useful model for the study of genomic characteristics as they relate to recombination and short term evolution in a self-fertilizing hermaphrodite. in both the denver et al. analysis of ten mutation-accumulating wild-type strains and in our study of rec- there is no dramatic pattern of mutational alterations along the chromosome. these results may be interesting in the context of whether or not recombination is mutagenic. both the wild-type ma-lines, which presumably have regions of both high and low recombination, and rec- in which the variation in recombination is randomized, have very similar patterns of base pair differences. the data are compatible with a model in which mutational events are independent of the meiotic recombination processes.  our analysis of high-throughput sequencing was able to detect regions of direct repeat sequences, deletions, insertions of transposable elements, and base pair differences. a subset of sequence alterations affecting coding regions were confirmed by an independent approach using oligo array comparative genome hybridization. the major phenotype of the rec- strain is an alteration in the preferred position of the meiotic recombination event with no other significant phenotypic consequences. in this study, we observed no evidence of a mutator effect at the nucleotide level attributable to the rec- mutation. 
the most frequent base-substitution was g:c to a:t, for the entire genome most of which were on chromosomes i or x, and respectively. the original sequencing and annotation of the caenorhabditis elegans genome along with recent advances in sequencing technology provide an exceptional opportunity for the genomic analysis of wild-type and mutant strains. a subset of sequence alterations affecting coding regions were confirmed by an independent approach using oligo array comparative genome hybridization. the major phenotype of the rec- strain is an alteration in the preferred position of the meiotic recombination event with no other significant phenotypic consequences. no major chromosomal rearrangements were observed, but additional insertions of transposable elements were detected. our analysis of high-throughput sequencing was able to detect regions of direct repeat sequences, deletions, insertions of transposable elements, and base pair differences. in this study, we observed no evidence of a mutator effect at the nucleotide level attributable to the rec- mutation. sequencing of this strain provides an opportunity to examine the consequences on the genome of altering the distribution of meiotic recombination events. using the software programs maq and slider, we observed base pair differences between rec- and the reference genome in wormbase, and between the mutagenized rec- and the wild-type n strain. using illumina sequencing and maq software, 83% of the base pair sequence reads were aligned to the reference genome available at wormbase, providing a 21-fold coverage of the genome. there are extra copies of tc and of tc in the rec- genome, most likely the remains of past high-hopper activity in a progenitor strain. using the illumina genome analyzer, we sequenced the entire genome of rec- a strain that alters the distribution of meiotic crossovers without changing the overall frequency.
 the original sequencing and annotation of the caenorhabditis elegans genome along with recent advances in sequencing technology provide an exceptional opportunity for the genomic analysis of wild-type and mutant strains. using the illumina genome analyzer, we sequenced the entire genome of rec- a strain that alters the distribution of meiotic crossovers without changing the overall frequency. rec- was derived from ethylmethane sulfonate -treated strains, one of which had a high level of transposable element mobility. sequencing of this strain provides an opportunity to examine the consequences on the genome of altering the distribution of meiotic recombination events.  using illumina sequencing and maq software, 83% of the base pair sequence reads were aligned to the reference genome available at wormbase, providing a 21-fold coverage of the genome. using the software programs maq and slider, we observed base pair differences between rec- and the reference genome in wormbase, and between the mutagenized rec- and the wild-type n strain. the most frequent base-substitution was g:c to a:t, for the entire genome most of which were on chromosomes i or x, and respectively. with this data removed, no obvious pattern in the distribution of the base differences along the chromosomes was apparent. no major chromosomal rearrangements were observed, but additional insertions of transposable elements were detected. there are extra copies of tc and of tc in the rec- genome, most likely the remains of past high-hopper activity in a progenitor strain.  our analysis of high-throughput sequencing was able to detect regions of direct repeat sequences, deletions, insertions of transposable elements, and base pair differences. a subset of sequence alterations affecting coding regions were confirmed by an independent approach using oligo array comparative genome hybridization. the major phenotype of the rec- strain is an alteration in the preferred position of the meiotic recombination event with no other significant phenotypic consequences. in this study, we observed no evidence of a mutator effect at the nucleotide level attributable to the rec- mutation. 