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

Rose AM, O'Neil NJ, Bilenky M, Butterfield YS, Malhis N, Flibotte S, Jones MR, Marra M, Baillie DL, Jones SJ - BMC Genomics (2010)

Bottom Line: With this data removed, no obvious pattern in the distribution of the base differences along the chromosomes was apparent.A subset of sequence alterations affecting coding regions were confirmed by an independent approach using oligo array comparative genome hybridization.In this study, we observed no evidence of a mutator effect at the nucleotide level attributable to the Rec-1 mutation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada. ann.rose@ubc.ca

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.

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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.
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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.

Mentions: The genome of Rec-1 was sequenced by whole genome shotgun sequencing (WGSS) using paired end tags (PETs) and aligned to the reference genome available in WormBase. Using the alignment to the WS190 reference genome, small insertions can be characterized by clusters of PETs that are shorter than the average size (Figure 4), 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 (paired end tags) 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 (reference DNA), which lacks that insertion. In the example shown in Figure 4, 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-1 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 (aCGH). An exon-centric array design that covered the entire genome revealed no major sequence copy number changes relative to the reference DNA.


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

Rose AM, O'Neil NJ, Bilenky M, Butterfield YS, Malhis N, Flibotte S, Jones MR, Marra M, Baillie DL, Jones SJ - BMC Genomics (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2837035&req=5

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.
Mentions: The genome of Rec-1 was sequenced by whole genome shotgun sequencing (WGSS) using paired end tags (PETs) and aligned to the reference genome available in WormBase. Using the alignment to the WS190 reference genome, small insertions can be characterized by clusters of PETs that are shorter than the average size (Figure 4), 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 (paired end tags) 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 (reference DNA), which lacks that insertion. In the example shown in Figure 4, 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-1 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 (aCGH). An exon-centric array design that covered the entire genome revealed no major sequence copy number changes relative to the reference DNA.

Bottom Line: With this data removed, no obvious pattern in the distribution of the base differences along the chromosomes was apparent.A subset of sequence alterations affecting coding regions were confirmed by an independent approach using oligo array comparative genome hybridization.In this study, we observed no evidence of a mutator effect at the nucleotide level attributable to the Rec-1 mutation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada. ann.rose@ubc.ca

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.

Show MeSH
Related in: MedlinePlus