SNP discovery via 454 transcriptome sequencing

Abstract

A massively parallel pyro-sequencing technology commercialized by 454 Life Sciences Corporation was used to sequence the transcriptomes of shoot apical meristems isolated from two inbred lines of maize using laser capture microdissection (LCM). A computational pipeline that uses the POLYBAYES polymorphism detection system was adapted for 454 ESTs and used to detect SNPs (single nucleotide polymorphisms) between the two inbred lines. Putative SNPs were computationally identified using 260,000 and 280,000 454 ESTs from the B73 and Mo17 inbred lines, respectively. Over 36,000 putative SNPs were detected within 9980 unique B73 genomic anchor sequences (MAGIs). Stringent post-processing reduced this number to > 7000 putative SNPs. Over 85% (94/110) of a sample of these putative SNPs were successfully validated by Sanger sequencing. Based on this validation rate, this pilot experiment conservatively identified > 4900 valid SNPs within > 2400 maize genes. These results demonstrate that 454-based transcriptome sequencing is an excellent method for the high-throughput acquisition of gene-associated SNPs.

Figure 1

A portion of the CROSS_MATCH-produced, template-driven, padded alignment between B73 and Mo17 454 EST sequences and the high-quality MAGI_105195 sequence assembly constructed from the B73 maize genomic survey sequence that serves as an alignment template. A G/A polymorphism occurs at position 2846 of the template (green highlight), with the Mo17 allele (A) in red and the B73 allele (G) in blue. Two insertions have occurred (yellow), one within a Mo17 454 EST and the second within a B73 454 EST. Because these insertions are not supported by other sequences, they are easily identified as errors by the POLYBAYSE pipeline and are not called as polymorphisms.