Abstract
Our knowledge of gene function has increasingly lagged behind gene discovery, hindering our understanding of the genetic basis of microbial phenotypes. Recently, however, massively parallel sequencing has been combined with traditional transposon mutagenesis in techniques referred to as transposon sequencing (Tn-seq), high-throughput insertion tracking by deep sequencing (HITS), insertion sequencing (INSeq) and transposon-directed insertion site sequencing (TraDIS), making it possible to identify putative gene functions in a high-throughput manner. Here, we describe the similarities and differences of these related techniques and discuss their application to the probing of gene function and higher-order genome organization.
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Acknowledgements
T.v.O. was supported by a postdoctoral fellowship from the Netherlands Organization for Scientific Research (Rubicon-NWO) and the Charles H. Hood Foundation. A.C. is an investigator of the Howard Hughes Medical Institute.
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Glossary
- Bacterial artificial chromosome
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A bacterial plasmid that contains a large eukaryotic DNA insertion (typically >150 kb) and can be used for cloning, genetic manipulation and transformation.
- ChIP–seq
-
(Chromatin immunoprecipitation followed by sequencing). A method that uses crosslinking of a protein to DNA followed by immunoprecipitation of the complex and subsequent sequencing of the bound DNA to reveal the binding site of the protein.
- Complementation assays
-
Assays in which a wild-type copy of a gene is reintroduced into a cell or organism that lacks the gene. This can confirm that the phenotype is caused by disruption or deletion of the gene in question, and that this phenotype can be reversed.
- Degenerate genes
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Genes that once had a function, but through the accumulation of mutations, became inactive.
- Digital-counting applications
-
Methods that count the total number of reads obtained for a particular sequence after massively parallel sequencing (in contrast to hybridization-based methods of quantification).
- DNA microarray
-
A glass slide (or other surface) on which oligonucleotides or PCR products of defined sequence are spotted. These microarrays are used to quantify the nucleic acids within a sample by hybridization.
- Haploinsufficiency
-
The inability of a single functional copy of a gene to produce a wild-type phenotype in a diploid organism. This occurs when the second copy of the gene is inactivated by mutation.
- Overlap analysis
-
An analysis that indicates a putative function for a hypothetical virulence gene using fitness data for the gene obtained during growth in defined in vitro conditions.
- Promoter assays
-
Assays that measure the transcriptional activity of a gene promoter by converting the RNA transcripts to cDNAs and then using massively parallel sequencing to determine the number of cDNA molecules present.
- Signature-tagged mutagenesis
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A technique in which a transposon is tagged with a specific DNA sequence (a bar-code) that is used to determine the presence of the transposon in a DNA pool (as the amplified and labelled tag hybridizes to a probe on a membrane).
- sRNA-seq
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(Small-RNA sequencing).The discovery of non-coding sRNAs through direct sequencing of their cDNAs by massively parallel sequencing.
- Transductional crosses
-
Experiments in which DNA is transferred from one bacterium to another by means of a bacteriophage.
- Type IIS restriction endonuclease
-
An enzyme that cleaves DNA at a defined distance from an asymmetrical recognition site.
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van Opijnen, T., Camilli, A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol 11, 435–442 (2013). https://doi.org/10.1038/nrmicro3033
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DOI: https://doi.org/10.1038/nrmicro3033
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