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

Published online 11 May 2010 Nucleic Acids Research, 2010, Vol. 38, No. 13 e142 doi:10.1093/nar/gkq368 Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples Andrew M. Smith1,2,3, Lawrence E. Heisler3,4, Robert P. St.Onge5,6, Eveline Farias-Hesson7, Iain M. Wallace2,3, John Bodeau8, Adam N. Harris9, Kathleen M. Perry8, Guri Giaever1,3,4, Nader Pourmand6,7,* and Corey Nislow1,2,3,* 1 Department of Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto, Ontario M5S 1A8, Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario M5G 1L6, 3Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, 4Department of Pharmaceutical Sciences, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada, 5Department of Biochemistry, Stanford University, Stanford, CA 94305, 6 Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, 7Biomolecular Engineering, University of California at Santa Cruz, Santa Cruz, CA 95064, 8Life Technologies Corporation, 850 Lincoln Centre Drive, Foster City, CA 94404 and 9Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92009, USA 2 ABSTRACT INTRODUCTION 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. Next-generation sequencing (NGS) technologies can generate up to several hundred million reads of DNA sequence per lane or slide, and this capacity continues to increase at a rapid pace. This massive capacity has allowed exploration of diverse biological questions (1–4). Although pooled chemogenomic screens of compound– gene interactions in yeast (5–16) and mammalian cells (17,18) are typically assessed using barcode microarrays, counting of individual strains could also be assessed by barcode sequencing. We recently developed such an assay (Bar-seq) to monitor thousands of gene–chemical interactions (19). We now expand upon this proofof-principle to interrogate 96 samples in parallel, developing the methodology and analytical tools to use NGS to simultaneously monitor several hundred thousand gene–environment interactions using a method that should be readily adaptable to an automated workflow. Here, we demonstrate successful multiplexing of samples obtained from 96 distinct pooled yeast growth assays, with each sample comprising 6200 uniquely barcoded yeast mutants. This 96-plex experiment represents a 150-fold increase in unique observations over our proof-of-principle assessment, and provides substantial cost reduction/experiment over microarrays. Furthermore, while many aspects of microarray assay costs are fixed, the cost of multiplex barcode sequencing continues to decline as the number of reads per experiment *To whom correspondence should be addressed. Tel: +1 416 946 8351; Fax: +1 416 978 8287; Email: Correspondence may also be addressed to Nader Pourmand. Tel: +1 831 502 7315; Fax: +1 831 459 2891; Email: ß The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received February 1, 2010; Revised April 5, 2010; Accepted April 24, 2010 PAGE 2 OF 7 e142 Nucleic Acids Research, 2010, Vol. 38, No. 13 METHODS AND MATERIALS Yeast deletion collection The yeast deletion collection was obtained from Angela Chu at the Stanford Genome Technology Centre, and stored in YPD-7% dimethyl sulfoxide (DMSO) in 80 C as individual strains in 96-well plates. The plates were thawed and robotically pinned onto YPD agar plates. Cells are grown in 30 C for 2–3 days until colonies form. Slow growing strains were grown separately for 2–3 additional days. All plates were then flooded with 5–7 ml of media, scraped and pooled in YPD-7% DMSO to a final concentration of OD600 = 50, and frozen at 80 C until use, as described by Pierce et al. (11). Construction of pools with fixed numbers of barcoded strains A pool of 953 different heterozygous mutants was selected to contain two well-known drug targets as heterozygous deletions. ‘Pool-constant’ was constructed by growing each strain in 100 ml of YPD to saturation in 96-well plates then pooling 20 ml from each well, so that all 953 strains in this pool are at approximately the same abundance. ‘Pool-variable’ consisted of the same 953 strains but in this pool, the number of cells of each strain was varied systematically with one-quarter of the 953 strains added at one of the following ratios (2 : 1 : 0.5 : 0.25) with respect to Pool-constant. Pooled growth assays Two deletion pools, a homozygous deletion pool of 5054 strains representing non-essential genes and a heterozygous pool of 1194 strains representing genes essential for viability, were thawed and diluted in YPD to an OD600 of 0.0625. Seven hundred microliters of cultures were grown at 30 C with a chemical inhibitor applied at a dose that produced 10–20% growth inhibition of wild-type. An automated liquid handler was used to maintain logarithmic growth of pools (by dilution), and to collect 0.7 OD600s of heterozygous pool following 20 generations of growth, and 1.4 OD600s of homozygous pool following five generations of growth. Assessing fitness of barcoded yeast strains by barcode microarray Except where indicated, pooled assays were performed as described by Pierce et al. (11). Genomic DNA was isolated from cells and barcodes amplified and hybridized to barcode microarrays, where each barcode deletion mutant is represented by 10 hybridization signals (an uptag and downtag for each strain, each p (...truncated)