Abstract
Identification of essential genes is key to understanding the required processes and gene products of organisms under one or more conditions. Transposon sequencing (Tn-seq) has been used to predict essential genes or ones that conditionally impact fitness in a wide variety of organisms. Here, we describe the generation of genome-scale mutant libraries and the analysis of Tn-seq data to identify essential genes from cultures grown in a single condition as well as those that are conditionally important by analyzing the behavior of these mutant libraries in different growth environments. While we illustrate the approach using data derived from Tn-seq analysis of the α-proteobacteria Rhodobacter sphaeroides and Zymomonas mobilis, the protocols and systems we describe should be generally applicable to a variety of organisms.
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References
Loman NJ, Constantinidou C, Chan JZ et al (2012) High-throughput bacterial genome sequencing: an embarrassment of choice, a world of opportunity. Nat Rev Microbiol 10:599–606
Galperin MY, Koonin EV (2010) From complete genome sequence to ‘complete’ understanding? Trends Biotechnol 28:398–406
Baba T, Ara T, Hasegawa M et al (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:2006.0008
Yamamoto N, Nakahigashi K, Nakamichi T et al (2009) Update on the Keio collection of Escherichia coli single-gene deletion mutants. Mol Syst Biol 5:335
Koo B-M, Kritikos G, Farelli JD et al (2017) Construction and analysis of two genome-scale deletion libraries for Bacillus subtilis. Cell Syst 4:291–305.e7
van Opijnen T, Camilli A (2013) Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol 11:435–442
Pettitt SJ, Krastev DB, Pemberton HN et al (2017) Genome-wide barcoded transposon screen for cancer drug sensitivity in haploid mouse embryonic stem cells. Sci Data 4:170020
Friedrich MJ, Rad L, Bronner IF et al (2017) Genome-wide transposon screening and quantitative insertion site sequencing for cancer gene discovery in mice. Nat Protoc 12:289–309
Yum S-Y, Lee S-J, Kim H-M et al (2016) Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing. Sci Rep 6:27185
Bai J, Li K, Tang W et al (2019) A high-throughput screen for genes essential for PRRSV infection using a piggyBac-based system. Virology 531:19–30
Ni TK, Landrette SF, Bjornson RD et al (2013) Low-copy piggyBac transposon mutagenesis in mice identifies genes driving melanoma. Proc National Acad Sci U S A 110:E3640–E3649
Friedel RH, Friedel CC, Bonfert T et al (2013) Clonal expansion analysis of transposon insertions by high-throughput sequencing identifies candidate cancer genes in a PiggyBac mutagenesis screen. PLoS One 8:e72338
Choi J, Landrette SF, Wang T et al (2014) Identification of PLX4032-resistance mechanisms and implications for novel RAF inhibitors. Pigment Cell Melanoma Res 27:253–262
Goodman AL, McNulty NP, Zhao Y et al (2009) Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host Microbe 6:279–289
van Opijnen T, Bodi KL, Camilli A (2009) Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods 6:767–772
Gawronski J, Wong S, Giannoukos G et al (2009) Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung. Proc National Acad Sci U S A 106:16422–16427
Langridge G, Phan DM, Turner D et al (2009) Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Res 19:2308–2316
Burger BT, Imam S, Scarborough MJ et al (2017) Combining genome-scale experimental and computational methods to identify essential genes in Rhodobacter sphaeroides. Msystems 2:e00015–e00017
Chihiro S, Masanosuke Y (1987) A series of Tn5 variants with various drug-resistance markers and suicide vector for transposon mutagenesis. Gene 56:283–288
Sistrom W (1960) A requirement for sodium in the growth of Rhodopseudomonas spheroides. J Gen Microbiol 22:778–785
Bertani G (1951) Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300
Martien JI, Hebert AS, Evenson D et al (2019) Systems-level analysis of oxygen exposure in Zymomonas mobilis: implications for isoprenoid production. Msystems 4:e00284–e00218
Lal P, Wells FM, Lyu Y et al (2019) A Markerless method for genome engineering in Zymomonas mobilis ZM4. Front Microbiol 10:2216
Larsen R, Wilson M, Guss A et al (2002) Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch Microbiol 178:193–201
Dabney J, Meyer M (2012) Length and GC-biases during sequencing library amplification: a comparison of various polymerase-buffer systems with ancient and modern DNA sequencing libraries. BioTechniques 52:87–94
Aird D, Ross MG, Chen W-S et al (2011) Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol 12:R18
Arkin AP, Cottingham RW, Henry CS et al (2018) KBase: the United States Department of Energy Systems Biology Knowledgebase. Nat Biotechnol 36:566–569
Langmead B, Trapnell C, Pop M et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25
Langmead B, Salzberg SL (2012) Fast gapped-read alignment with bowtie 2. Nat Methods 9:357–359
Li H, Durbin R (2009) Fast and accurate short read alignment with burrows–wheeler transform. Bioinformatics 25:1754–1760
Goecks J, Nekruenko A, Talor J (2010) Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol 11:1–38
Hannon GJ (2010) FASTX-Toolkit
Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17:10–12
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120
Homann OR, Johnson AD (2010) MochiView: versatile software for genome browsing and DNA motif analysis. BMC Biol 8:49
Robinson JT, Thorvaldsdóttir H, Winckler W et al (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26
Kent JW, Sugnet CW, Furey TS et al (2002) The human genome browser at UCSC. Genome Res 12:996–1006
Pritchard JR, Chao MC, Abel S et al (2014) ARTIST: high-resolution genome-wide assessment of fitness using transposon-insertion sequencing. PLoS Genet 10:e1004782
Barquist L, Mayho M, Cummins C et al (2016) The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries. Bioinformatics 32:1109–1111
McCoy K, Antonio ML, van Opijnen T (2017) MAGenTA: a galaxy implemented tool for complete Tn-Seq analysis and data visualization. Bioinformatics 33:2781–2783
Blanchard AM, Leigh JA, Egan SA et al (2015) Transposon insertion mapping with PIMMS - pragmatic insertional mutation mapping system. Front Genet 6:139
Solaimanpour S, Sarmiento F, Mrázek J (2015) Tn-seq explorer: a tool for analysis of high-throughput sequencing data of transposon mutant libraries. PLoS One 10:e0126070
Turner KH, Wessel AK, Palmer GC et al (2015) Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum. Proc Natl Acad Sci U S A 112:4110–4115
DeJesus MA, Ambadipudi C, Baker R et al (2015) TRANSIT--A software tool for Himar1 TnSeq analysis. PLoS Comput Biol 11:e1004401
Goodman AL, Wu M, Gordon JI (2011) Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries. Nat Protoc 6:1969–1980
Mandal RK, Jiang T, Kwon Y (2017) Essential genome of Campylobacter jejuni. BMC Genomics 18:616
Nilsson M, Givskov M, Tolker-Nielsen T (2019) Transposon mutagenesis in Streptococcus species. Methods Mol Biol 2016:39–49
Lin T, Gao L (2018) Genome-wide mutagenesis in Borrelia burgdorferi. Methods Mol Biol 1690:201–223
Caruccio N (2011) Preparation of next-generation sequencing libraries using Nextera™ technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition. Methods Mol Biol 733:241–255
Gorbacheva T, Quispe-Tintaya W, Popov VN et al (2015) Improved transposon-based library preparation for the ion torrent platform. BioTechniques 58:200–202
Solonenko SA, Ignacio-Espinoza CJ, Alberti A et al (2013) Sequencing platform and library preparation choices impact viral metagenomes. BMC Genomics 14:320
Gordon A (2008) FASTX-Toolkit
Langmead B (2010) Aligning short sequencing reads with Bowtie. Curr Protoc Bioinformatics. Chapter 11:Unit 11.7
Institute B (2019) Picard toolkit
Li H, Handsaker B, Wysoker A et al (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25:2078–2079
Quinlan AR and Kindlon N (2019) Bedtools
Ponstingl H (2015) SMALT
Li H (2011) Tabix: fast retrieval of sequence features from generic TAB-delimited files. Bioinformatics 27:718–719
Team CR (2013) R: a language and environment for statistical computing
Bates D, Carey V, Dettling M, et al (2002) Bioconductor
Anaconda (2016) Anaconda Software Distribution
Afgan E, Baker D, Batut B et al (2018) The galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res 46:gky379
Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11:R106
Wickham H, François R, Henry L, et al (2018) Dplyr: a grammar of data manipulation
Walt S, Colbert CS, Varoquaux G (2011) The NumPy array: a structure for efficient numerical computation. Comput Sci Eng 13:22–30
Seabold S and Perktold J (2010) Statsmodels: econometric and statistical modeling with python
Virtanen P, Gommers R, Oliphant TE, et al (2019) SciPy 1.0--fundamental algorithms for scientific computing in python
Hunter JD (2007) Matplotlib: a 2D graphics environment. Comput Sci Eng 9:90–95
wiredfool, Clark A, Hugo, et al (2016) Pillow: 3.1.0
Team T (2019) wxPython
Schoenborn O (2016) PyPubSub
Goryshin I, Reznikoff WS (1998) Tn 5 in vitro transposition. J Biol Chem 273:7367–7374
Acknowledgements
This material is based upon work supported by the Great Lakes Bioenergy Research Center, U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number DE-SC0018409.
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Myers, K.S., Behari Lal, P., Noguera, D.R., Donohue, T.J. (2022). Using Genome Scale Mutant Libraries to Identify Essential Genes. In: Zhang, R. (eds) Essential Genes and Genomes. Methods in Molecular Biology, vol 2377. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1720-5_12
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DOI: https://doi.org/10.1007/978-1-0716-1720-5_12
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