Investigating PCR bias in TnSeq

As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq results are determined by counting transposon insertions following the PCR-based enrichment and subsequent deep sequencing of transposon insertions. Here we explore the possibility that PCR amplification of transposon insertions in a TnSeq library skews the results by introducing bias into the detection and/or enumeration of insertions. We compared the detection and frequency of mapped insertions when altering the number of PCR cycles in the enrichment step. In addition, we devised and validated a novel, PCR-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore sequencing. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of the TnSeq assay insofar as insertions in the majority of genes represented in our library were similarly detected regardless of PCR cycle number and whether or not PCR amplification was employed. However, the detection of a small subset of genes which had been previously described as essential is indeed sensitive to the number of PCR cycles. We conclude that PCR-based enrichment of transposon insertions in a TnSeq assay is reliable but researchers interested in profiling essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, we present a PCR-free TnSeq alternative that is comparable to traditional PCR-based methods although the latter remain superior owing to their accessibility and high sequencing depth. Twenty-two samples were sequenced. 18 of the samples were sequenced using Illumina HiSeq, the remaining 3 samples were sequenced using Oxford Nanopore Technologies (ONT). The 18 Illumina-sequenced samples comprise three groups, each with a unique transposon-genomic junction enrichment method. Each of the three groups includes two types of samples: 3 biological replicates of the TnSeq library grown to exponential phase; 3 biological replicates of the same libraries grown to stationay phase. 3 samples were subjected to amplification-free transposon-genomic junction enrichment and subsequent sequencing using MinION ONT- these are experimental replicates of one TnSeq library grown to stationary phase. One sample (labeled TnSeq_WGS) was sequenced on MinION ONT without enrichment of the transposon.