对大肠埃希菌分离物的 RNA 进行直接 cDNA 和 PCR-cDNA 纳米孔测序的比较。

IF 4 2区 生物学 Q1 GENETICS & HEREDITY
Gillian Rodger, Samuel Lipworth, Lucinda Barrett, Sarah Oakley, Derrick W Crook, David W Eyre, Nicole Stoesser
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引用次数: 0

摘要

全转录组(长读数)RNA 测序(牛津纳米孔技术公司,ONT)有望用于病原菌不同基因表达的参考诊断分析,包括抗菌药耐药基因(ARGs)。然而,直接 cDNA ONT 测序需要高浓度的多聚腺苷酸化 mRNA,而且扩增方案可能会带来技术偏差。在此,我们评估了直接 cDNA 和基于 cDNA PCR 的 ONT 测序对临床大肠埃希菌转录组分析的影响。使用 ONT 直接 cDNA 测序 SQK-DCS109 和 PCR-cDNA 条形码 SQK-PCB111.24 试剂盒对 4 个与血流感染相关的大肠杆菌分离物(每个分离物 n=2 个生物重复)进行了测序。生物和技术重复序列分布在 8 个流式细胞中,使用 16 个条形码,以尽量减少批次/条形码偏差。在对低丰度基因和 rRNA 基因进行硅清除后,将读数映射到转录本参考,并对转录本丰度进行量化。我们发现,使用这两种试剂盒以及限制分析范围仅包括 ARGs 时,读数计数之间存在很强的相关性。我们强调,GC 含量较高的基因之间的相关性较弱。与 PCR-cDNA 试剂盒相比,直接 cDNA 试剂盒的读数长度更长,而 PCR-cDNA 试剂盒的总产量更高。我们对使用直接 cDNA 和 PCR-cDNA ONT 测序试剂盒测序的生物和技术重复株进行了小规模但方法严谨的评估,结果表明基于 PCR 的扩增大大提高了产量,对核心基因和 ARG 表达的评估基本无偏见。不过,PCR 检测试剂盒的使用者应注意技术偏差的小风险,这种风险在基因表达量异常高(>52%)/低(>50%)的情况下似乎更大。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Comparison of direct cDNA and PCR-cDNA Nanopore sequencing of RNA from Escherichia coli isolates.

Whole-transcriptome (long-read) RNA sequencing (Oxford Nanopore Technologies, ONT) holds promise for reference-agnostic analysis of differential gene expression in pathogenic bacteria, including for antimicrobial resistance genes (ARGs). However, direct cDNA ONT sequencing requires large concentrations of polyadenylated mRNA, and amplification protocols may introduce technical bias. Here we evaluated the impact of direct cDNA- and cDNA PCR-based ONT sequencing on transcriptomic analysis of clinical Escherichia coli. Four E. coli bloodstream infection-associated isolates (n=2 biological replicates per isolate) were sequenced using the ONT Direct cDNA Sequencing SQK-DCS109 and PCR-cDNA Barcoding SQK-PCB111.24 kits. Biological and technical replicates were distributed over eight flow cells using 16 barcodes to minimize batch/barcoding bias. Reads were mapped to a transcript reference and transcript abundance was quantified after in silico depletion of low-abundance and rRNA genes. We found there were strong correlations between read counts using both kits and when restricting the analysis to include only ARGs. We highlighted that correlations were weaker for genes with a higher GC content. Read lengths were longer for the direct cDNA kit compared to the PCR-cDNA kit whereas total yield was higher for the PCR-cDNA kit. In this small but methodologically rigorous evaluation of biological and technical replicates of isolates sequenced with the direct cDNA and PCR-cDNA ONT sequencing kits, we demonstrated that PCR-based amplification substantially improves yield with largely unbiased assessment of core gene and ARG expression. However, users of PCR-based kits should be aware of a small risk of technical bias which appears greater for genes with an unusually high (>52%)/low (<44%) GC content.

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来源期刊
Microbial Genomics
Microbial Genomics Medicine-Epidemiology
CiteScore
6.60
自引率
2.60%
发文量
153
审稿时长
12 weeks
期刊介绍: Microbial Genomics (MGen) is a fully open access, mandatory open data and peer-reviewed journal publishing high-profile original research on archaea, bacteria, microbial eukaryotes and viruses.
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