Seattle SNPs Protocols

Big Dye Sequencing Protocol PCR Protocol
DNA Panel Protocol Ethanol Precipitation Protocol
Big Dye Terminator Precipitation Protocol 96- to 384-Well Plate Transfer
3700 Loading Procedures

Quality Control Statement for Sequence-Based SNP Discovery

1. Sample handling is performed from start to finish in microtiter plates using robotic pipetting devices; each sample is sequenced in an individual capillary.

2. All generated sequences are checked for internal consistency in terms of their location and orientation (forward or reverse reads) with the reference sequence.

3.Sequence quality, which provides an estimate of the error probabilites for each base-call, is used to guide SNP analysis to ensure that only the most accurate regions of the sequence (sequence quality > 30, or < 1 error per 1,000) are used for polymorphism detection.

4. Approximately 30 to 40% of SNPs are confirmed on another strand, due to redundancy from overlapping sequences produced from the forward and reverse strands and overlap between adjacent PCR products.

5. All singletons (single heterozygotes among homozygotes) are re-amplified and re-sequenced for confirmation.

6. Approximately 10% of the samples are resequenced from each gene to check the accuracy of genotypes and SNPs.