Characterization of synthetic DNA bar codes in Saccharomyces cerevisiae gene-deletion strains

Abstract

Incorporation of strain-specific synthetic DNA tags into yeast Saccharomyces cerevisiae gene-deletion strains has enabled identification of gene functions by massively parallel growth rate analysis. However, it is important to confirm the sequences of these tags, because mutations introduced during construction could lead to significant errors in hybridization performance. To validate this experimental system, we sequenced 11,812 synthetic 20-mer molecular bar codes and adjacent sequences (>1.8 megabases synthetic DNA) by pyrosequencing and Sanger methods. At least 31% of the genome-integrated 20-mer tags contain differences from those originally synthesized. However, these mutations result in anomalous hybridization in only a small subset of strains, and the sequence information enables redesign of hybridization probes for arrays. The robust performance of the yeast genedeletion dual oligonucleotide bar-code design in array hybridization validates the use of molecular bar codes in living cells for tracking their growth phenotype.

Large‐scale Pyrosequencing of synthetic DNA: A comparison with results from Sanger dideoxy sequencing

“…In the present study, we analyzed in detail the sequence data of a large number of amplicons (barcode targets) from our earlier study [5] using two techniques (Pyrosequencing and Sanger dideoxy sequencing). These data were originally generated to validate barcodes in a yeast deletion project.…”

“…However, in the Sanger sequencing of the barcodes reported in [5], a large number (~500) of sequences were redone independently two, or sometimes three times. In about 20% of these cases, results were obtained with a q of <20 for a given point mutation, whereas in the independent sequencing of the same barcode, the same mutation was found to have q of >40 in subsequent runs.…”

“…Synthetic oligonucleotides inserted in the yeast gene-deletion strains were those originally acquired from Research Genetics Corp. (Huntsville, AL, USA) for our earlier study [5]. Culture conditions and DNA extraction were performed as described in that report.…”

“…Thus in the present work, we compare Pyrosequencing technology and the Sanger dideoxy method for base-calling accuracy, sequence resolution, and convenience using 4747 synthetic DNA fragments [5]. We demonstrate here that Pyrosequencing is optimal for short stretches of DNA.…”

dSLAM analysis of genome-wide genetic interactions in Saccharomyces cerevisiae

“…Many freshly converted haploid YKO mutants are lethal or grow poorly and therefore are severely underrepresented in the pool. In addition, direct sequencing of the TAGs and their associated primer sites have revealed many mutations that may hinder their PCR amplification or hybridization to the array (20). Here we describe a protocol we use to select from a dSLAM screen the candidates meriting followup confirmation.…”