Multi-sample pooling and illumina genome analyzer sequencing methods to determine gene sequence variation for database development.

ABSTRACT: Determination of sequence variation within a genetic locus to develop clinically relevant databases is critical for molecular assay design and clinical test interpretation, so multisample pooling for Illumina genome analyzer (GA) sequencing was investigated using the RET proto-oncogene as a model. Samples were Sanger-sequenced for RET exons 10, 11, and 13-16. Ten samples with 13 known unique variants ("singleton variants" within the pool) and seven common changes were amplified and then equimolar-pooled before sequencing on a single flow cell lane, generating 36 base reads. For comparison, a single "control" sample was run in a different lane. After alignment, a 24-base quality score-screening threshold and 3; read end trimming of three bases yielded low background error rates with a 27% d

SUBMITTER: Margraf RL 

PROVIDER: S-EPMC2922832 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature