Tn-Seq/INSeq
Transposon Sequencing or Insertion Sequencing
Tn-seq (van Opijnen et al., 2009) and INSeq (Goodman et al., 2009) are nearly identical methods that determine quantitative genetic interactions accurately. INSeq includes a polyacrylamide gel purification step following adapter ligation and PCR, whereas Tn_seq requires agarose gel purification (van Opijnen et al., 2013). In this method, a transposon with flanking Mmel digestion sites is transposed into bacteria which, after culturing, can help detect the frequency of mutations within the transposon. After MmeI digestion and subsequent adapter ligation, PCR amplification and sequencing can provide information about the transposon insertion sites.
Advantages:
- Can study mutational frequency of transposons
- Can be used to deduce fitness of genes within microorganisms
- Robust, reproducible, and sensitive
Disadvantages:
- Limited to bacterial studies
- Errors during PCR amplification can lead to inaccurate sequence reads
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Gray A. N., Koo B. M., Shiver A. L., Peters J. M., Osadnik H. and Gross C. A. High-throughput bacterial functional genomics in the sequencing era. Curr Opin Microbiol. 2015;27:86-95
van Opijnen T. and Camilli A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol. 2013;11:435-442
References:
Hooven T. A., Catomeris A. J., Akabas L. H., et al. The essential genome of Streptococcus agalactiae. BMC Genomics. 2016;17:406
Le Breton Y., Belew A. T., Valdes K. M., et al. Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes. Sci Rep. 2015;5:9838
Liu F., Wang C., Wu Z., Zhang Q. and Liu P. A zero-inflated Poisson model for insertion tolerance analysis of genes based on Tn-seq data. Bioinformatics. 2016;32:1701-1708
DeJesus M. A., Ambadipudi C., Baker R., Sassetti C. and Ioerger T. R. TRANSIT–A Software Tool for Himar1 TnSeq Analysis. PLoS Comput Biol. 2015;11:e1004401
Lee S. A., Gallagher L. A., Thongdee M., et al. General and condition-specific essential functions of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 2015;112:5189-5194
Meeske A. J., Sham L. T., Kimsey H., et al. MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis. Proc Natl Acad Sci U S A. 2015;112:6437-6442
Rubin B. E., Wetmore K. M., Price M. N., et al. The essential gene set of a photosynthetic organism. Proc Natl Acad Sci U S A. 2015;112:E6634-6643
Turner K. H., Wessel A. K., Palmer G. C., Murray J. L. and Whiteley M. Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum. Proc Natl Acad Sci U S A. 2015;112:4110-4115
Wu M., McNulty N. P., Rodionov D. A., et al. Genetic determinants of in vivo fitness and diet responsiveness in multiple human gut Bacteroides. Science. 2015;350:aac5992
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History: Tn-Seq/INSeq
Revision by sbrumpton on 2017-06-21 07:50:21 - Show/Hide
Transposon Sequencing or Insertion Sequencing
Tn-seq (van Opijnen et al., 2009) and INSeq (Goodman et al., 2009) are nearly identical methods that determine quantitative genetic interactions accurately. INSeq includes a polyacrylamide gel purification step following adapter ligation and PCR, whereas Tn_seq requires agarose gel purification (van Opijnen et al., 2013). In this method, a transposon with flanking Mmel digestion sites is transposed into bacteria which, after culturing, can help detect the frequency of mutations within the transposon. After MmeI digestion and subsequent adapter ligation, PCR amplification and sequencing can provide information about the transposon insertion sites.
Advantages:- Can study mutational frequency of transposons
- Can be used to deduce fitness of genes within microorganisms
- Robust, reproducible, and sensitive
Disadvantages:- Limited to bacterial studies
- Errors during PCR amplification can lead to inaccurate sequence reads
Reagents:Illumina Library prep and Array Kit SelectorReviews:Gray A. N., Koo B. M., Shiver A. L., Peters J. M., Osadnik H. and Gross C. A. High-throughput bacterial functional genomics in the sequencing era. Curr Opin Microbiol. 2015;27:86-95van Opijnen T. and Camilli A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol. 2013;11:435-442References:Hooven T. A., Catomeris A. J., Akabas L. H., et al. The essential genome of Streptococcus agalactiae. BMC Genomics. 2016;17:406Le Breton Y., Belew A. T., Valdes K. M., et al. Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes. Sci Rep. 2015;5:9838Liu F., Wang C., Wu Z., Zhang Q. and Liu P. A zero-inflated Poisson model for insertion tolerance analysis of genes based on Tn-seq data. Bioinformatics. 2016;32:1701-1708DeJesus M. A., Ambadipudi C., Baker R., Sassetti C. and Ioerger T. R. TRANSIT--A Software Tool for Himar1 TnSeq Analysis. PLoS Comput Biol. 2015;11:e1004401Lee S. A., Gallagher L. A., Thongdee M., et al. General and condition-specific essential functions of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 2015;112:5189-5194Meeske A. J., Sham L. T., Kimsey H., et al. MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis. Proc Natl Acad Sci U S A. 2015;112:6437-6442Rubin B. E., Wetmore K. M., Price M. N., et al. The essential gene set of a photosynthetic organism. Proc Natl Acad Sci U S A. 2015;112:E6634-6643Turner K. H., Wessel A. K., Palmer G. C., Murray J. L. and Whiteley M. Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum. Proc Natl Acad Sci U S A. 2015;112:4110-4115Wu M., McNulty N. P., Rodionov D. A., et al. Genetic determinants of in vivo fitness and diet responsiveness in multiple human gut Bacteroides. Science. 2015;350:aac5992