title: 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.
authors.affiliation: Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Smith
authors.forename: Lawrence E
authors.initials: LE
authors.lastname: Heisler
authors.forename: Robert P
authors.initials: RP
authors.lastname: St Onge
authors.forename: Eveline
authors.initials: E
authors.lastname: Farias-Hesson
authors.forename: Iain M
authors.initials: IM
authors.lastname: Wallace
authors.forename: John
authors.initials: J
authors.lastname: Bodeau
authors.forename: Adam N
authors.initials: AN
authors.lastname: Harris
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Perry
authors.forename: Guri
authors.initials: G
authors.lastname: Giaever
authors.forename: Nader
authors.initials: N
authors.lastname: Pourmand
authors.forename: Corey
authors.initials: C
authors.lastname: Nislow
mesh_terms: D009154:Mutation; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*
references.citation: Nat Methods. 2008 Feb;5(2):183-8
references.pmid: 18204455
references.citation: Nat Methods. 2007 Nov;4(11):931-6
references.pmid: 17934468
references.citation: Science. 2008 Apr 18;320(5874):362-5
references.pmid: 18420932
references.citation: PLoS Genet. 2008 Aug 08;4(8):e1000151
references.pmid: 18688276
references.citation: Nat Genet. 2007 Feb;39(2):199-206
references.pmid: 17206143
references.citation: Nat Genet. 1999 Mar;21(3):278-83
references.pmid: 10080179
references.citation: Proc Natl Acad Sci U S A. 2004 Jan 20;101(3):793-8
references.pmid: 14718668
references.citation: Cell Host Microbe. 2009 Sep 17;6(3):279-89
references.pmid: 19748469
references.citation: Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W577-81
references.pmid: 15980540
references.citation: BMC Genomics. 2009 Jan 21;10:37
references.pmid: 19159457
references.citation: Nat Protoc. 2007;2(11):2958-74
references.pmid: 18007632
references.citation: Nat Methods. 2009 May;6(5):315-6
references.pmid: 19349981
references.citation: PLoS Genet. 2005 Aug;1(2):e24
references.pmid: 16121259
references.citation: Mol Cell. 2004 Nov 5;16(3):487-96
references.pmid: 15525520
references.citation: Nat Methods. 2008 Oct;5(10):887-93
references.pmid: 18794863
references.citation: Nat Methods. 2006 Aug;3(8):601-3
references.pmid: 16862133
references.citation: Nat Methods. 2008 Mar;5(3):235-7
references.pmid: 18264105
references.citation: Cell. 2006 Mar 24;124(6):1283-98
references.pmid: 16564017
references.citation: Nat Biotechnol. 2009 Apr;27(4):369-77
references.pmid: 19349972
references.citation: Proc Natl Acad Sci U S A. 2009 Feb 17;106(7):2289-94
references.pmid: 19171886
references.citation: Nat Biotechnol. 2009 Dec;27(12):1173-5
references.pmid: 19915551
references.citation: Science. 2008 Jun 6;320(5881):1344-9
references.pmid: 18451266
references.citation: Nature. 2008 Nov 6;456(7218):53-9
references.pmid: 18987734
references.citation: Science. 2008 Feb 1;319(5863):617-20
references.pmid: 18239125
references.citation: Cell. 2009 May 29;137(5):835-48
references.pmid: 19490893
references.citation: BMC Bioinformatics. 2009 Oct 29;10:362
references.pmid: 19874596
references.citation: Genome Res. 2009 Oct;19(10):1836-42
references.pmid: 19622793
references.citation: Nat Methods. 2007 Aug;4(8):651-7
references.pmid: 17558387
references.citation: Nature. 2008 Nov 6;456(7218):66-72
references.pmid: 18987736
references.citation: Nat Methods. 2008 Aug;5(8):719-25
references.pmid: 18622398
references.citation: Nat Methods. 2009 Oct;6(10):767-72
references.pmid: 19767758
references.citation: Nucleic Acids Res. 2008 Nov;36(19):e122
references.pmid: 18753151
references.citation: Nucleic Acids Res. 2007;35(19):e130
references.pmid: 17932070
references.citation: Nat Methods. 2010 Feb;7(2):111-8
references.pmid: 20111037
references.citation: Nat Genet. 2009 Oct;41(10):1061-7
references.pmid: 19718026
references.citation: Cell. 2006 Aug 11;126(3):611-25
references.pmid: 16901791
references.citation: Nat Biotechnol. 2007 Feb;25(2):244-8
references.pmid: 17220878