Genomic sequence of a mutant strain of Caenorhabditis elegans with an altered recombination pattern

Abstract

Background: 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-1, a strain that alters the distribution of meiotic crossovers without changing the overall frequency. Rec-1 was derived from ethylmethane sulfonate (EMS)-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.

Results: 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 1124 base pair differences between Rec-1 and the reference genome in Wormbase (WS190), and 441 between the mutagenized Rec-1 (BC313) and the wild-type N2 strain (VC2010). The most frequent base-substitution was G:C to A:T, 141 for the entire genome most of which were on chromosomes I or X, 55 and 31 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 11 extra copies of Tc1, and 8 of Tc2 in the Rec-1 genome, most likely the remains of past high-hopper activity in a progenitor strain.

Conclusion: 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-1 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-1 mutation.

Figure 1

Comparision of the Genetic and Physical Maps of chromosome I. The top line shows the wild-type (N2) genetic map of autosome I of C. elegans using genetic distances measured by Zetka and Rose, 1995. Line 2 is the position of the gene markers on the physical map as annotated in WormBase http://www.wormbase.org. The bottom line is the position of markers in the Rec-1 mutant (data taken from Zetka and Rose, 1995).

Figure 2

The number of each of the nonstrand- specific types of base pair differences by chromosome. The chromosomes are identified on the horizontal axis. The number of changes per million base pairs of aligned sequences are plotted on the vertical axis. Data from Table 1.

Figure 3

Distribution of base pair differences between BC313 and VC2010 along the chromosomes. Red crosses (upper) indicate the physical location of G:C to A:Ts in BC313 but not in VC2010. Blue crosses (lower) indicate the physical location of the remaining nonstrand- specific base differences. The chromosome number is shown on the X axis and the distance in Mbp along the Y axis.

Figure 4

Insertions of DNA can result in paired end tags (PETs) shorter than expected. PETs that fall outside the normal size range can be an indication of DNA insertions. Top: In the case of a small insertion, paired end reads can cover a region in the alignment to the reference (Ref) that does not include the inserted sequence in the sample. Lower Right: Insertions in the sample are characterized by multiple PETs with a shorter than average fragment size based on the alignment.

Figure 5

Imperfect direct repeats can result in paired end tags (PETs) longer than expected. PETS for bases 10,941,077-10,960,123 of chromosome I are shown. Below the line in green are the normal sized PETs. Above the line longer, potentially aberrant, PETs are shown. The longer PETs are in regions lacking normal sequence coverage. Typically, the left end of the longer tags detects unique sequence and the right end is aligned with one of a group of imperfect direct repeats producing longer than normal PETs of differing sizes.

Figure 6

Locations of Tc1s and Tc2s specific to Rec-1. A. The red (Tc1) and blue (Tc2) crosses show the position of the elements in WormBase. Triangles show the positions of new full-length insertion sites for Tc1 (Red) and Tc2 (Blue). Three insertions of Tc2 on chromosome I are close together, at 3.2 Mb, 6.9 Mb, and 13.8 Mb (Table 2), and each is shown as a single triangle in the Figure. B. The aCGH data for an insertion of Tc1 into a coding region on the X-chromosome is shown.

Figure 7

A Tc1 insertion with the TATA at position 6992277 on chromosome I. On the left there are four reads adjacent to the 5' end of the Tc1 sequence and on the right are four reads adjacent to 3' end. The ends of the Tc1 sequence are shown in blue. The TA is inserted at position 6,992,279. Lower case indicates read sequences that are partial Tc1's that are unmapped in the genome and shown as mismatches in the alignment. Upper case indicates read sequences that are mapped to the genome. All the reads shown are paired with another read that maps to Tc1 internal sequence.

Figure 8

High density aCGH. An example of aCGH hybridization data identifying a C to T change in Rec-1. The Y-axis is the normalized log2 ratio of fluorescent intensities (Rec-1 versus reference). Each bar represents one 50 mer oligo probe on the oligo array chip. Below the plot is a schematic of the exon structure of predicted gene R05D11.9 and the position of the C-T base pair change identified at position 8,595,578 bp on chromosome I in the Rec-1 strain.