Technical considerations for cost-effective transposon directed insertion-site sequencing (TraDIS)
Abstract Transposon directed insertion-site sequencing (TraDIS), a variant of transposon insertion sequencing commonly known as Tn-Seq, is a high-throughput assay that defines essential bacterial genes across diverse growth conditions. However, the variability between laboratory environments often r...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2024-03-01
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| Series: | Scientific Reports |
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| Online Access: | https://doi.org/10.1038/s41598-024-57537-6 |
| _version_ | 1797247362172715008 |
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| author | Yasuhiro Kyono Madeline Tolwinski Stephanie A. Flowers |
| author_facet | Yasuhiro Kyono Madeline Tolwinski Stephanie A. Flowers |
| author_sort | Yasuhiro Kyono |
| collection | DOAJ |
| description | Abstract Transposon directed insertion-site sequencing (TraDIS), a variant of transposon insertion sequencing commonly known as Tn-Seq, is a high-throughput assay that defines essential bacterial genes across diverse growth conditions. However, the variability between laboratory environments often requires laborious, time-consuming modifications to its protocol. In this technical study, we aimed to refine the protocol by identifying key parameters that can impact the complexity of mutant libraries. Firstly, we discovered that adjusting electroporation parameters including transposome concentration, transposome assembly conditions, and cell densities can significantly improve the recovery of viable mutants for different Escherichia coli strains. Secondly, we found that post-electroporation conditions, such as recovery time and the use of different mediums for selecting mutants may also impact the complexity of viable mutants in the library. Finally, we developed a simplified sequencing library preparation workflow based on a Nextera-TruSeq hybrid design where ~ 80% of sequenced reads correspond to transposon-DNA junctions. The technical improvements presented in our study aim to streamline TraDIS protocols, making this powerful technique more accessible for a wider scientific audience. |
| first_indexed | 2024-04-24T19:57:29Z |
| format | Article |
| id | doaj.art-c659783f99e34eb3abf53f3d762404e0 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-24T19:57:29Z |
| publishDate | 2024-03-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-c659783f99e34eb3abf53f3d762404e02024-03-24T12:17:52ZengNature PortfolioScientific Reports2045-23222024-03-011411910.1038/s41598-024-57537-6Technical considerations for cost-effective transposon directed insertion-site sequencing (TraDIS)Yasuhiro Kyono0Madeline Tolwinski1Stephanie A. Flowers2Department of Pharmacy Practice, College of Pharmacy, University of Illinois at ChicagoDepartment of Pharmacy Practice, College of Pharmacy, University of Illinois at ChicagoDepartment of Pharmacy Practice, College of Pharmacy, University of Illinois at ChicagoAbstract Transposon directed insertion-site sequencing (TraDIS), a variant of transposon insertion sequencing commonly known as Tn-Seq, is a high-throughput assay that defines essential bacterial genes across diverse growth conditions. However, the variability between laboratory environments often requires laborious, time-consuming modifications to its protocol. In this technical study, we aimed to refine the protocol by identifying key parameters that can impact the complexity of mutant libraries. Firstly, we discovered that adjusting electroporation parameters including transposome concentration, transposome assembly conditions, and cell densities can significantly improve the recovery of viable mutants for different Escherichia coli strains. Secondly, we found that post-electroporation conditions, such as recovery time and the use of different mediums for selecting mutants may also impact the complexity of viable mutants in the library. Finally, we developed a simplified sequencing library preparation workflow based on a Nextera-TruSeq hybrid design where ~ 80% of sequenced reads correspond to transposon-DNA junctions. The technical improvements presented in our study aim to streamline TraDIS protocols, making this powerful technique more accessible for a wider scientific audience.https://doi.org/10.1038/s41598-024-57537-6Transposon insertion sequencingEscherichia coliElectroporationIllumina library preparation |
| spellingShingle | Yasuhiro Kyono Madeline Tolwinski Stephanie A. Flowers Technical considerations for cost-effective transposon directed insertion-site sequencing (TraDIS) Scientific Reports Transposon insertion sequencing Escherichia coli Electroporation Illumina library preparation |
| title | Technical considerations for cost-effective transposon directed insertion-site sequencing (TraDIS) |
| title_full | Technical considerations for cost-effective transposon directed insertion-site sequencing (TraDIS) |
| title_fullStr | Technical considerations for cost-effective transposon directed insertion-site sequencing (TraDIS) |
| title_full_unstemmed | Technical considerations for cost-effective transposon directed insertion-site sequencing (TraDIS) |
| title_short | Technical considerations for cost-effective transposon directed insertion-site sequencing (TraDIS) |
| title_sort | technical considerations for cost effective transposon directed insertion site sequencing tradis |
| topic | Transposon insertion sequencing Escherichia coli Electroporation Illumina library preparation |
| url | https://doi.org/10.1038/s41598-024-57537-6 |
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