Abstract
With the advent of next-generation sequencing (NGS) technology, methods previously developed for microarrays have been adapted for use by NGS. Here we describe in detail a protocol for Barcode analysis by sequencing (Bar-seq) to assess pooled competitive growth of individually barcoded yeast deletion mutants. This protocol has been optimized on two sequencing platforms: Illumina’s Genome Analyzer IIx/HiSeq2000 and Life Technologies SOLiD3/5500. In addition, we provide guidelines for assessment of human knockdown cells using short-hairpin RNAs (shRNA) and an Illumina sequencing readout.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
DiMasi JA, Hansen RW, Grabowski HG (2003) The price of innovation: new estimates of drug development costs. J Health Econ 22(2):151–185
Higgins MJ, Graham SJ (2009) Intellectual property. Balancing innovation and access: patent challenges tip the scales. Science 326(5951):370–371
Waller CL, Shah A, Nolte M (2007) Strategies to support drug discovery through integration of systems and data. Drug Discov Today 12(15–16):634–639
Hopkins AL (2008) Network pharmacology: the next paradigm in drug discovery. Nat Chem Biol 4(11):682–690
Hopkins AL (2009) Drug discovery: predicting promiscuity. Nature 462(7270):167–168
Munos B (2009) Lessons from 60 years of pharmaceutical innovation. Nat Rev Drug Discov 8(12):959–968
Szarenings K et al (2004) Fishing for targets: novel approaches using small molecule baits. Drug Discov Today 1(1):9–15
Roth BL, Sheffler DJ, Kroeze WK (2004) Magic shotguns versus magic bullets: selectively non-selective drugs for mood disorders and schizophrenia. Nature Rev Drug Discov 3(4):353–359
Metz JT, Hajduk PJ (2010) Rational approaches to targeted polypharmacology: creating and navigating protein-ligand interaction networks. Curr Opin Chem Biol 14(4): 498–504
Hillenmeyer ME et al (2008) The chemical genomic potrait of yeast: uncovering a phenotype for all genes. Science 320(5874):362–365
Parsons AB et al (2006) Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast. Cell 126(3): 611–625
Costanzo M et al (2010) The genetic landscape of a cell. Science 327(5964):425–431
Hughes TR et al (2000) Functional discovery via a compendium of expression profiles. Cell 102(1):109–126
Marton MJ et al (1998) Drug target validation and identification of secondary drug target effects using DNA microarrays. Nat Med 4(11):1293–1301
Parsons AB et al (2004) Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways. Nat Biotechnol 22(1):62–69
Giaever G et al (2004) Chemogenomic profiling: identifying the functional interactions of small molecules in yeast. Proc Natl Acad Sci USA 101(3):793–798
Giaever G et al (1999) Genomic profiling of drug sensitivities via induced haploinsufficiency. Nat Genet 21(3):278–283
Giaever G et al (2002) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418(6896):387–391
Winzeler EA et al (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901–906
Ho CH et al (2009) A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds. Nat Biotechnol 27(4):369–377
Davierwala AP et al (2005) The synthetic genetic interaction spectrum of essential genes. Nat Genet 37:1147–1152
Mnaimneh S et al (2004) Exploration of essential gene functions via titratable promoter alleles. Cell 118(1):31–44
Sopko R et al (2006) Mapping pathways and phenotypes by systematic gene overexpression. Mol Cell 21(3):319–330
Tong AH et al (2001) Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294(5550):2364–2368
Tong AH et al (2004) Global mapping of the yeast genetic interaction network. Science 303(5659):808–813
Pierce SE et al (2007) Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures. Nat Protoc 2(11):2958–2974
Pierce SE et al (2006) A unique and universal molecular barcode array. Nat Methods 3(8): 601–603
Lum PY et al (2004) Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes. Cell 116(1):121–137
Hoon S et al (2008) An integrated platform of genomic assays reveals small-molecule bioactivities. Nat Chem Biol 4(8):498–506
Lee W et al (2005) Genome-wide requirements for resistance to functionally distinct DNA-damaging agents. PLoS Genet 1(2):e24
Oh J et al (2010) Gene annotation and drug target discovery in Candida albicans with a tagged transposon mutant collection. PLoS Pathog 6(10):e1001140
Xu D et al (2007) Genome-wide fitness test and mechanism-of-action studies of inhibitory compounds in Candida albicans. PLoS Pathog 3(6):e92
Xu D et al (2009) Chemical genetic profiling and characterization of small-molecule compounds that affect the biosynthesis of unsaturated fatty acids in Candida albicans. J Biol Chem 284(29):19754–19764
Dorer RK et al (2005) A small-molecule inhibitor of Mps1 blocks the spindle-checkpoint response to a lack of tension on mitotic chromosomes. Curr Biol 15(11):1070–1076
Smith AM et al (2009) Quantitative phenotyping via deep barcode sequencing. Genome Res 19(10):1836–1842
St Onge RP et al (2007) Systematic pathway analysis using high-resolution fitness profiling of combinatorial gene deletions. Nat Genet 39(2):199–206
Yan Z et al (2008) Yeast Barcoders: a chemogenomic application of a universal donor-strain collection carrying bar-code identifiers. Nat Methods 5(8):719–725
Ericson E et al (2008) Off-target effects of psychoactive drugs revealed by genome-wide assays in yeast. PLoS Genet 4(8):e1000151
Rock FL et al (2007) An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site. Science 316(5832):1759–1761
Yu H et al (2008) High-quality binary protein interaction map of the yeast interactome network. Science 322(5898):104–110
Goh KI et al (2007) The human disease network. Proc Natl Acad Sci USA 104(21): 8685–8690
Moffat J et al (2006) A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124(6): 1283–1298
Silva JM et al (2005) Second-generation shRNA libraries covering the mouse and human genomes. Nat Genet 37(11): 1281–1288
Schlabach MR et al (2008) Cancer proliferation gene discovery through functional genomics. Science 319(5863):620–624
Silva JM et al (2008) Profiling essential genes in human mammary cells by multiplex RNAi screening. Science 319(5863):617–620
Luo J et al (2009) A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell 137(5): 835–848
Scholl C et al (2009) Synthetic lethal interaction between oncogenic KRAS dependency and STK33 suppression in human cancer cells. Cell 137(5):821–834
Bentley DR et al (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456(7218): 53–59
Mardis ER (2009) New strategies and emerging technologies for massively parallel sequencing: applications in medical research. Genome Med 1(4):40
Mardis ER et al (2009) Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med 361(11): 1058–1066
Miller W et al (2008) Sequencing the nuclear genome of the extinct woolly mammoth. Nature 456(7220):387–390
Green RE et al (2010) A draft sequence of the Neandertal genome. Science 328(5979): 710–722
Nagalakshmi U et al (2008) The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320(5881): 1344–1349
Robertson G et al (2007) Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods 4(8):651–657
Ozsolak F et al (2009) Direct RNA sequencing. Nature 461(7265):814–818
Ozsolak F et al (2007) High-throughput mapping of the chromatin structure of human promoters. Nat Biotechnol 25(2):244–248
Cloonan N et al (2008) Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat Methods 5(7):613–619
Hillier LW et al (2008) Whole-genome sequencing and variant discovery in C. elegans. Nat Methods 5(2):183–188
Lefrancois P et al (2009) Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing. BMC Genomics 10(1):37
Turner EH et al (2009) Massively parallel exon capture and library-free resequencing across 16 genomes. Nat Methods 6(5):315–316
van Opijnen T, Bodi KL, Camilli A (2009) Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6(10): 767–772
Durbin RM et al (2010) A map of human genome variation from population-scale sequencing. Nature 467(7319):1061–1073
Gnirke A et al (2009) Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol 27(2):182–189
Smith AM et al (2010) Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples. Nucleic Acids Res 38:e142
Sambrook J, Russell DW, and Cold Spring Harbor Laboratory (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Root DE et al (2006) Genome-scale loss-of-function screening with a lentiviral RNAi library. Nat Methods 3(9):715–719
Luo B et al (2008) Highly parallel identification of essential genes in cancer cells. Proc Natl Acad Sci USA 105(51):20380–20385
Cummings N et al (2010) Combining target enrichment with barcode multiplexing for high throughput SNP discovery. BMC Genomics 11:641
Daines B et al (2009) High-throughput multiplex sequencing to discover copy number variants in Drosophila. Genetics 182(4):935–941
Han TX et al (2010) Global fitness profiling of fission yeast deletion strains by barcode sequencing. Genome Biol 11(6):R60
Hamady M et al (2008) Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nat Methods 5(3): 235–237
Acknowledgements
A.M.S. is supported by a University of Toronto Open Fellowship. Research in the Giaever and Nislow laboratories is supported by the NHGRI and CIHR.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media New York
About this protocol
Cite this protocol
Smith, A.M., Durbic, T., Kittanakom, S., Giaever, G., Nislow, C. (2012). Barcode Sequencing for Understanding Drug–Gene Interactions. In: Larson, R. (eds) Bioinformatics and Drug Discovery. Methods in Molecular Biology, vol 910. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-965-5_4
Download citation
DOI: https://doi.org/10.1007/978-1-61779-965-5_4
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-964-8
Online ISBN: 978-1-61779-965-5
eBook Packages: Springer Protocols