A New Cost Effective, Simplified Workflow for Transposon Insertion Sequencing (TIS)

Abstract

Transposon insertion sequencing (TIS) encompasses methods such as Transposon Directed Insertion sequencing (TraDIS) and Transposon-Sequencing (Tn-Seq); these methods are widely uses for genome scale screens of essential and conditionally essential genes. Early limitations centred on mutant generation, today the biggest factor is sequencing and data generation and the costs associated. Here, we present and evaluate four methods TIS sequencing library generation protocols compatible with Illumina whole genome sequencing (WGS) workflows to maximise sequence efficiency and minimise turnaround times and costs. Using E.coli BW25113 transposon mutagenesis libraries generated with Tn5 and mariner (Himar1) transposons, we compare the methods in terms of reagent cost, workflow complexity, and target enrichments for the recovery of unique insertion sites and essential genes identified. All methods generated TIS data suitable for gene essentiality analysis. Illumina Flex based protocols were 4-6 tomes cheaper than the traditional Illumina Nextera based approach with similar or superior Transposon-Chromosome (Tn-Chr) junction enrichment. Across all methods, sequencing depth and mutant library density were the dominant factor in useful biological insight. Subsampling demonstrated that for a good quality mutant library, five million reads were sufficient to identify essential genes in E.coli. Whereas deeper sequencing reduced the statistical power and included contaminating background noise, seen primarily with Tn5. We conclude that an Illumina Flex based approach, especially when integrating with routine WGS, provides an excellent balance of speed, cost and data quality. Assuming five million reads and a robust Illumina Flex approach, a TIS library can be sequenced for around {pound}40.