Dataset Information

Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples.

ABSTRACT: Next-generation sequencing has proven an extremely effective technology for molecular counting applications where the number of sequence reads provides a digital readout for RNA-seq, ChIP-seq, Tn-seq and other applications. The extremely large number of sequence reads that can be obtained per run permits the analysis of increasingly complex samples. For lower complexity samples, however, a point of diminishing returns is reached when the number of counts per sequence results in oversampling with no increase in data quality. A solution to making next-generation sequencing as efficient and affordable as possible involves assaying multiple samples in a single run. Here, we report the successful 96-plexing of complex pools of DNA barcoded yeast mutants and show that such 'Bar-seq' assessment of these samples is comparable with data provided by barcode microarrays, the current benchmark for this application. The cost reduction and increased throughput permitted by highly multiplexed sequencing will greatly expand the scope of chemogenomics assays and, equally importantly, the approach is suitable for other sequence counting applications that could benefit from massive parallelization.

SUBMITTER: Smith AM

PROVIDER: S-EPMC2910071 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples.

Smith Andrew M AM Heisler Lawrence E LE St Onge Robert P RP Farias-Hesson Eveline E Wallace Iain M IM Bodeau John J Harris Adam N AN Perry Kathleen M KM Giaever Guri G Pourmand Nader N Nislow Corey C

Nucleic acids research 20100511 13

Next-generation sequencing has proven an extremely effective technology for molecular counting applications where the number of sequence reads provides a digital readout for RNA-seq, ChIP-seq, Tn-seq and other applications. The extremely large number of sequence reads that can be obtained per run permits the analysis of increasingly complex samples. For lower complexity samples, however, a point of diminishing returns is reached when the number of counts per sequence results in oversampling with ...[more]

PMID: 20460461

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Project description:BackgroundAimMassively parallel sequencing (MPS) holds promise for expanding cancer translational research and diagnostics. As yet, it has been applied on paraffin DNA (FFPE) with commercially available highly multiplexed gene panels (100s of DNA targets), while custom panels of low multiplexing are used for re-sequencing. Here, we evaluated the performance of two highly multiplexed custom panels on FFPE DNA.MethodsTwo custom multiplex amplification panels (B, 373 amplicons; T, 286 amplicons) were coupled with semiconductor sequencing on DNA samples from FFPE breast tumors and matched peripheral blood samples (n samples: 316; n libraries: 332). The two panels shared 37% DNA targets (common or shifted amplicons). Panel performance was evaluated in paired sample groups and quartets of libraries, where possible.ResultsAmplicon read ratios yielded similar patterns per gene with the same panel in FFPE and blood samples; however, performance of common amplicons differed between panels (p<0.001). FFPE genotypes were compared for 1267 coding and non-coding variant replicates, 999 out of which (78.8%) were concordant in different paired sample combinations. Variant frequency was highly reproducible (Spearman's rho 0.959). Repeatedly discordant variants were of high coverage / low frequency (p<0.001). Genotype concordance was (a) high, for intra-run duplicates with the same panel (mean±SD: 97.2±4.7, 95%CI: 94.8-99.7, p<0.001); (b) modest, when the same DNA was analyzed with different panels (mean±SD: 81.1±20.3, 95%CI: 66.1-95.1, p = 0.004); and (c) low, when different DNA samples from the same tumor were compared with the same panel (mean±SD: 59.9±24.0; 95%CI: 43.3-76.5; p = 0.282). Low coverage / low frequency variants were validated with Sanger sequencing even in samples with unfavourable DNA quality.ConclusionsCustom MPS may yield novel information on genomic alterations, provided that data evaluation is adjusted to tumor tissue FFPE DNA. To this scope, eligibility of all amplicons along with variant coverage and frequency need to be assessed.

Dataset Information

Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples.

Publications

Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples.

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