As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq data consist of millions of nucleotide sequence reads that are generated by PCR amplification of transposon-genomic junctions. Reads mapping to the junctions are enumerated thus providing information on the number of transposon insertion mutations in each individual gene. 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, and when including a nested PCR, in the enrichment step. Additionally, we present nCATRAs - a novel, amplification-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore MinION sequencing. nCATRAs achieved 54 and 23% enrichment of the transposons and transposon-genomic junctions, respectively, over background genomic DNA. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of TnSeq 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 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 putative essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, nCATRAs is comparable to traditional PCR-based methods (Kendall’s correlation=0.896–0.897) although the latter remain superior owing to their accessibility and high sequencing depth.

中文翻译：

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转座子测序 (TnSeq) 分析中是否存在扩增偏倚？采用基于 PCR 和无扩增富集方法的金黄色葡萄球菌 TnSeq 文库的案例研究

随着转座子测序 (TnSeq) 检测在微生物学领域变得多产，仔细研究它们的潜在缺点很有意义。TnSeq 数据由数百万个核苷酸序列读数组成，这些读数是由转座子-基因组连接的 PCR 扩增产生的。读取到连接点的映射被枚举，从而提供有关每个单独基因中转座子插入突变数量的信息。在这里，我们探讨了 TnSeq 文库中转座子插入的 PCR 扩增通过在插入检测和/或计数中引入偏差而扭曲结果的可能性。我们比较了在富集步骤中改变 PCR 循环数和包含嵌套 PCR 时映射插入的检测和频率。此外，我们还介绍了 nCATRAs——一部小说，无扩增 TnSeq 方法，其中通过 CRISPR/Cas9 靶向转座子切割和随后的 Oxford Nanopore MinION 测序来富集插入。nCATRAs 在背景基因组 DNA 上分别实现了 54% 和 23% 的转座子和转座子-基因组连接富集。这些基于 PCR 和无 PCR 的实验表明，总体而言，PCR 扩增不会显着偏向 TnSeq 的结果，因为无论 PCR 循环数和 PCR 扩增与否，我们文库中代表的大多数基因的插入都被类似地检测到被雇佣。然而，先前被描述为必需的一小部分基因的检测对 PCR 循环数很敏感。我们得出结论，在 TnSeq 测定中基于 PCR 的转座子插入富集是可靠的，但是对分析推定的必需基因感兴趣的研究人员应该仔细权衡其文库制备方案中使用的扩增循环数。此外，nCATRAs 可与传统的基于 PCR 的方法相媲美（Kendall 的相关性=0.896–0.897），尽管后者由于其可访问性和高测序深度而仍然具有优势。