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.

Transposase mediated construction of RNA-seq libraries

“…Because every enzymatic step is followed by magnetic bead purification (Hawkins et al 1994), the full library construction protocol can easily be applied to a 96-well plate format, where steps can be completed with robotics. The protocol also allows for multiplex sequencing of samples (Smith et al 2010). Molecular barcodes can be added during the final PCR step by using different primers, which can result in a significant cost savings.…”

Adaptive mechanisms in pathogens: universal aneuploidy in Leishmania

“…As a result of increased sequencing, this removes the need for the time-consuming task of sample cloning for genetic tests. Moreover, the increasing scope to pool many barcoded samples together on next-generation platforms means that chromosome copy number estimation is an inexpensive and effective method for sample karyotyping [74].…”

Genomic breeding for food, environment and livelihoods

“…Two factors, however, have substantially improved the usefulness of genomic breeding in recent years: next-generation sequencing (Smith et al 2010) and high-throughput phenomics (Yang et al 2013). The cost, in time and money, of genetic sequencing has dropped precipitously in the last few years through combined innovations in chemistry, robotics and computing (Smith et al 2010;Harakalova et al 2011).…”

“…The cost, in time and money, of genetic sequencing has dropped precipitously in the last few years through combined innovations in chemistry, robotics and computing (Smith et al 2010;Harakalova et al 2011). Genomic sequencing is faster, cheaper and more reliable than ever before.…”

“…A paucity of genomic resources has, for example, limited lentil breeding; a recent transcriptome sequencing effort will reverse this by providing molecular markers to assist breeding efforts (Sharpe et al 2013). Traditional and/or regional crops that have been overlooked by past, commercial, breeding efforts will thus benefit from greater use of genomic breeding.…”