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Comments on Daniel C. Koboldt et al. (2007) International Worm Meeting "High-throughput identification of structural variations from C. briggsae sequence traces." (0)
Overview
Daniel C. Koboldt, Julia E. Staisch, & Raymond D. Miller (2007). High-throughput identification of structural variations from C. briggsae sequence traces presented in International Worm Meeting. Unpublished information; cite only with author permission.
Recent reports of widespread copy number differences including large insertion/deletions (indels) have demonstrated that this variation affects such substantial portions of the human genome that it is likely to play a significant role in human disease. C. elegans strains also harbor large indels (Maydan, J.S. et al., 2007, Genome Res 17:337). While comparative genome hybridization and SNP genotyping technologies have been adapted to detect structural variants, doing so at the nucleotide level remains a challenge. We have developed a method that allows for the high-throughput identification of structural variation from sequence trace data. We tested the method using sequence traces from C. briggsae strain HK104, which has a SNP on average every 115 bp when compared to the canonical strain (AF16). Each set of sequence traces was used to create a BLAST database against which the reference genome was aligned using WU-BLAST. The BLAST results were parsed and filtered to identify reads that appear to span an indel. Alignment positions and orientations were analyzed to characterize the size and precise molecular boundaries of the apparent indels. From a database of 13,632 sequencing traces from strain HK104 we identified 399 candidate indel events of 50 - 1,000 bp, 62 indels of 1-10 kbp, and a number of larger indels. We chose an initial test set of two indels for each of the six chromosomes, and designed PCR assays for them. Nine of the 12 PCR assays were successful under uniform conditions and each of the nine reflected the predicted indel. We also designed and successfully used three FP-TDI genotyping assays for the indels. Additional candidates are being tested. Our method is both efficient and sensitive. It has shown that large indels are common in C. briggsae. The large indels are very useful as tools for mapping studies and they raise the question about their effects on gene expression.
