Adam N. Harris

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

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.

Abstract 4851: A single-tube protocol for next gen library construction increases complexity and simplifies parallel sample handling

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL With the advent of second-generation sequencing technologies, exome- and transcriptome- and whole genome sequencing are tools of choice in studying cancer to detect alterations in the genome correlating with tumorigenesis. It is essential to compare genomic alterations in cancer with matched normal DNA from the same individual largely due to incomplete knowledge of the “normal” variations. There have been concerted efforts for targeted sequencing of cancer-related exomes in a high throughput manner. However, the needs to process multiple samples simultaneously while maintaining the complexity of libraries have not been addressed adequately. Fragment library generation for single- and paired-end reads is typically performed by a series of enzymatic steps following gDNA shearing by ultrasonication. These may include end-repair, A-tailing, and ligation, each followed by bead- or column-based purification. The clean-ups are necessary to prevent enzyme activity carryover from one step to another. We have developed a single-tube method to take sheared, bead size-selected DNA through end-repair, A-tailing, and ligation without intermittent purifications. The A-tailing is done by a thermophilic polymerase at a temperature sufficient to denature the end-repair enzymes. Ligation is performed by adding ligase and adaptors after cooling the reaction. A bead- or column-based purification is then performed after the ligation reaction to produce template for amplification. With the removal of the clean-up steps, the workflow is simplified, allowing faster processing of samples with less hands-on time. We also reduce loss of sample associated with the purifications, increasing the complexity of the output library or enabling a lower sample input concentration. Our method is well suited for labs which are producing multiple libraries each week but have not scaled up to full automation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4851. doi:10.1158/1538-7445.AM2011-4851