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SNP discovery via 454 transcriptome sequencing.

Barbazuk WB, Emrich SJ, Chen HD, Li L, Schnable PS - Plant J. (2007)

Bottom Line: 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.These results demonstrate that 454-based transcriptome sequencing is an excellent method for the high-throughput acquisition of gene-associated SNPs.

View Article: PubMed Central - PubMed

Affiliation: Donald Danforth Plant Science Center, St Louis, MO 63132, USA.

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.

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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.
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fig01: 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.

Mentions: POLYBAYES is designed to use template-driven MSAs, in which sequences are scaffolded across a high-quality template sequence that serves as an anchor. In addition to being highly accurate (Marth et al., 1999), this approach eliminates the need to perform de novo assemblies of 454 ESTs, which are complicated by the short lengths of 454 reads. Furthermore, gaps and insertions in this template-driven multiple sequence alignment approach are propagated throughout all members, so 454 semi-random indels can be easily identified and ignored (Figure 1). Finally, the ability of POLYBAYES to use quality scores during SNP detection provides the option of utilizing 454 sequence calls once they are better accepted by the research community, or if Sanger sequences are also used, or if the base accuracy of the template is suspect. In all of these cases, the availability of accurate base quality data could improve the accuracy of SNP detection.


SNP discovery via 454 transcriptome sequencing.

Barbazuk WB, Emrich SJ, Chen HD, Li L, Schnable PS - Plant J. (2007)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169515&req=5

fig01: 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.
Mentions: POLYBAYES is designed to use template-driven MSAs, in which sequences are scaffolded across a high-quality template sequence that serves as an anchor. In addition to being highly accurate (Marth et al., 1999), this approach eliminates the need to perform de novo assemblies of 454 ESTs, which are complicated by the short lengths of 454 reads. Furthermore, gaps and insertions in this template-driven multiple sequence alignment approach are propagated throughout all members, so 454 semi-random indels can be easily identified and ignored (Figure 1). Finally, the ability of POLYBAYES to use quality scores during SNP detection provides the option of utilizing 454 sequence calls once they are better accepted by the research community, or if Sanger sequences are also used, or if the base accuracy of the template is suspect. In all of these cases, the availability of accurate base quality data could improve the accuracy of SNP detection.

Bottom Line: 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.These results demonstrate that 454-based transcriptome sequencing is an excellent method for the high-throughput acquisition of gene-associated SNPs.

View Article: PubMed Central - PubMed

Affiliation: Donald Danforth Plant Science Center, St Louis, MO 63132, USA.

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.

Show MeSH