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.

Figure 1.

(a) The log2 ratio of control versus treatment for one example from the 20-plex Bar-seq data. The x-axis is the log2 ratio derived from the Bar-seq data, while the y-axis is the log2 ratio derived from barcode microarray data. The profiles are from a 100 µM cisplatin treatment. The black dots represent genes above our threshold; greater than 200 signal intensity in the barcode microarray and greater than 40 barcode sequence counts in the Bar-seq. (b) The log2 ratio of control over treatment for two examples from the 96-plex Bar-seq data. The x-axis is the log2 ratio derived from the Bar-seq data, the y-axis is the log2 ratio derived from barcode microarray data. The left panel shows the result from 91-mM MMS treatment, the right panel shows the profile obtained with an uncharacterized compound 1561-0023 screened at 0.372 mM. The black dots represent genes which were above our threshold; greater than 200 signal intensity in the barcode microarray and greater than 40 barcode sequence counts in Bar-seq.

Figure 2.

Scatter plots of the Log2 ratio of the Pool-constant versus the Pool-variable. The data were generated on three different platforms, barcode microarray, Illumina/Solexa, and SOLiD. The SOLiD data is derived from 20- and 96-plex samples. The r-value is indicated for each comparison and the line of best fit is plotted. Data presented were filtered for barcodes that were present in both Illumina/Solexa and SOLiD sequencing counts with counts greater than zero in both platforms.