Journal
ELECTROPHORESIS
Volume 27, Issue 15, Pages 3042-3047Publisher
WILEY
DOI: 10.1002/elps.200500834
Keywords
DNA sequencing; pyrosequencing technology; Sanger dideoxy sequencing; synthetic oligonucleotides; yeast deletion strains
Funding
- NHGRI NIH HHS [P01 HG000205, P01 HG 000205, P01 HG000205-190004] Funding Source: Medline
- NIAID NIH HHS [1R21 AI 059499-01] Funding Source: Medline
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Pyrosequencing is a relatively recent method for sequencing short stretches of DNA. Because both Pyrosequencing and Sanger dideoxy sequencing were recently used to characterize and validate DNA molecular barcodes in a large yeast gene-deletion project, a meta-analysis of those data allow an excellent and timely opportunity for evaluating Pyrosequencing against the current gold standard, Sanger dideoxy sequencing. Starting with yeast genomic DNA, parallel PGR amplification methods were used to prepared 4747 short barcode-containing constructs from 6000 Saccharomyces cerevisiae gene-deletion strains. Pyrosequencing was optimized for average read lengths of 25-30 bases, which included in each case a 20-mer barcode sequence. Results were compared with sequence data obtained by the standard Sanger dideoxy chain termination method. In most cases, sequences obtained by Pyrosequencing and Sanger dideoxy sequencing were of comparable accuracy, and the overall rate of failure was similar. The DNA in the barcodes is derived from synthetic oligonucleotide sequences that were inserted into yeast-deletion-strain genomic DNA by homologous recombination and represents the most significant amount of DNA from a synthetic source that has been sequenced to date. Although more automation and quality control measures are needed, Pyrosequencing was shown to be a fast and convenient method for determining short stretches of DNA sequence.
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