Dataset Information

Comparison of the Illumina Genome Analyzer and Roche 454 GS FLX for resequencing of hypertrophic cardiomyopathy-associated genes.

ABSTRACT: Next-generation sequencing (NGS) is widely used in biomedical research, but its adoption has been limited in molecular diagnostics. One application of NGS is the targeted resequencing of genes whose mutations lead to an overlapping clinical phenotype. This study evaluated the comparative performance of the Illumina Genome Analyzer and Roche 454 GS FLX for the resequencing of 16 genes associated with hypertrophic cardiomyopathy (HCM). Using a single human genomic DNA sample enriched by long-range PCR (LR-PCR), 40 GS FLX and 31 Genome Analyzer exon variants were identified using >or=30-fold read-coverage and >or=20% read-percentage selection criteria. Twenty-seven platform concordant variants were Sanger-confirmed. The discordant variants segregated into two categories: variants with read coverages >or=30 on one platform but <30-fold on the alternate platform and variants with read percentages >or=20% on one platform but <20% on the alternate platform. All variants with <30-fold coverage were Sanger-confirmed, suggesting that the coverage criterion of >or=30-fold is too stringent for variant discovery. The variants with <20% read percentage were identified as reference sequence based on Sanger sequencing. These variants were found in homopolymer tracts and short-read misalignments, specifically in genes with high identity. The results of the current study demonstrate the feasibility of combining LR-PCR with the Genome Analyzer or GS FLX for targeted resequencing of HCM-associated genes.

SUBMITTER: Dames S

PROVIDER: S-EPMC2884316 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Comparison of the Illumina Genome Analyzer and Roche 454 GS FLX for resequencing of hypertrophic cardiomyopathy-associated genes.

Dames Shale S Durtschi Jacob J Geiersbach Katherine K Stephens Jack J Voelkerding Karl V KV

Journal of biomolecular techniques : JBT 20100701 2

Next-generation sequencing (NGS) is widely used in biomedical research, but its adoption has been limited in molecular diagnostics. One application of NGS is the targeted resequencing of genes whose mutations lead to an overlapping clinical phenotype. This study evaluated the comparative performance of the Illumina Genome Analyzer and Roche 454 GS FLX for the resequencing of 16 genes associated with hypertrophic cardiomyopathy (HCM). Using a single human genomic DNA sample enriched by long-range ...[more]

PMID: 20592870

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Project description:BackgroundLycoris aurea, also called Golden Magic Lily, is an ornamentally and medicinally important species of the Amaryllidaceae family. To date, the sequencing of its whole genome is unavailable as a non-model organism. Transcriptomic information is also scarce for this species. In this study, we performed de novo transcriptome sequencing to produce the first comprehensive expressed sequence tag (EST) dataset for L. aurea using high-throughput sequencing technology.Methodology and principal findingsTotal RNA was isolated from leaves with sodium nitroprusside (SNP), salicylic acid (SA), or methyl jasmonate (MeJA) treatment, stems, and flowers at the bud, blooming, and wilting stages. Equal quantities of RNA from each tissue and stage were pooled to construct a cDNA library. Using 454 pyrosequencing technology, a total of 937,990 high quality reads (308.63 Mb) with an average read length of 329 bp were generated. Clustering and assembly of these reads produced a non-redundant set of 141,111 unique sequences, comprising 24,604 contigs and 116,507 singletons. All of the unique sequences were involved in the biological process, cellular component and molecular function categories by GO analysis. Potential genes and their functions were predicted by KEGG pathway mapping and COG analysis. Based on our sequence analysis and published literatures, many putative genes involved in Amaryllidaceae alkaloids synthesis, including PAL, TYDC OMT, NMT, P450, and other potentially important candidate genes, were identified for the first time in this Lycoris. Furthermore, 6,386 SSRs and 18,107 high-confidence SNPs were identified in this EST dataset.ConclusionsThe transcriptome provides an invaluable new data for a functional genomics resource and future biological research in L. aurea. The molecular markers identified in this study will provide a material basis for future genetic linkage and quantitative trait loci analyses, and will provide useful information for functional genomic research in future.

Dataset Information

Comparison of the Illumina Genome Analyzer and Roche 454 GS FLX for resequencing of hypertrophic cardiomyopathy-associated genes.

Publications

Comparison of the Illumina Genome Analyzer and Roche 454 GS FLX for resequencing of hypertrophic cardiomyopathy-associated genes.

Similar Datasets

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