牛津纳米孔文库制备试剂盒的偏差及其对微生物组和基因组分析的影响。

IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Ziming Chen, Chian Teng Ong, Loan To Nguyen, Harrison J Lamb, O González-Recio, M Gutiérrez-Rivas, Sarah J Meale, Elizabeth M Ross
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引用次数: 0

摘要

背景:牛津纳米孔测序是一种不依赖于聚合酶产生序列数据的长读测序技术。牛津纳米孔测序中使用的测序文库制备方法依赖于添加与适配器序列结合的马达蛋白,该适配器序列使用基于连接的方法(连接测序试剂盒)或基于转座酶的方法(快速测序试剂盒)添加。然而,这些方法的酶促步骤可能容易受到基序偏倚的影响,包括腺嘌呤-胸腺嘧啶(AT)序列由于连接和转座的偏倚而代表性不足。本研究旨在比较这些文库制备方法的识别基序和相对相互作用频率,并评估它们对相对测序覆盖率、微生物组和甲基化谱的影响。研究了DNA提取试剂盒和碱基调用模型对微生物组分析的影响。结果:利用结扎和快速文库试剂盒产生的测序数据,我们在快速试剂盒中鉴定出与MuA转座酶一致的识别基序(5’-TATGA-3’),在结扎试剂盒的序列末端鉴定出低频率AT。快速试剂盒在鸟嘌呤-胞嘧啶(GC)含量为40-70%的区域产率降低,而结扎试剂盒在不同GC含量区域的覆盖分布相对均匀。由于读取时间较长,与快速方案相比,结扎试剂盒显示出更高的分类分类效率。不同的文库制备方法导致不同分类水平的瘤胃微生物谱和模拟群落谱存在显著差异。结扎试剂盒在随后的细菌DNA甲基化统计中优于快速试剂盒,尽管没有显著差异。结论:我们的研究结果表明,由于牛津纳米孔文库制备过程中酶促反应引起的系统偏差,谨慎和一致的文库制备方法选择对于牛相关微生物组分析等定量方法至关重要。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Biases from Oxford Nanopore library preparation kits and their effects on microbiome and genome analysis.

Background: Oxford Nanopore sequencing is a long-read sequencing technology that does not rely on a polymerase to generate sequence data. Sequencing library preparation methods used in Oxford Nanopore sequencing rely on the addition of a motor protein bound to an adapter sequence, which is added either using ligation-based methods (ligation sequencing kit), or transposase-based methods (rapid sequencing kit). However, these methods have enzymatic steps that may be susceptible to motif bias, including the underrepresentation of adenine-thymine (AT) sequences due to ligation and biases from transposases. This study aimed to compare the recognition motif and relative interaction frequencies of these library preparation methods and assess their effects on relative sequencing coverage, microbiome, and methylation profiles. The impacts of DNA extraction kits and basecalling models on microbiome analysis were also investigated.

Results: By using sequencing data generated by the ligation and rapid library kits, we identified the recognition motif (5'-TATGA-3') consistent with MuA transposase in the rapid kit and low frequencies of AT in the sequence terminus of the ligation kit. The rapid kit showed reduced yield in regions with 40-70% guanine-cytosine (GC) contents, while the ligation kit showed relatively even coverage distribution in areas with various GC contents. Due to longer reads, ligation kits showed increased taxonomic classification efficiency compared to the rapid protocols. Rumen microbial profile at different taxonomic levels and mock community profile showed significant variation due to the library preparation method used. The ligation kit outperformed the rapid kit in subsequent bacterial DNA methylation statistics, although there were no significant differences.

Conclusions: Our findings indicated that careful and consistent library preparation method selection is essential for quantitative methods such as bovine-related microbiome analysis due to the systematic bias induced by the enzymatic reactions in Oxford Nanopore library preparation.

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来源期刊
BMC Genomics
BMC Genomics 生物-生物工程与应用微生物
CiteScore
7.40
自引率
4.50%
发文量
769
审稿时长
6.4 months
期刊介绍: BMC Genomics is an open access, peer-reviewed journal that considers articles on all aspects of genome-scale analysis, functional genomics, and proteomics. BMC Genomics is part of the BMC series which publishes subject-specific journals focused on the needs of individual research communities across all areas of biology and medicine. We offer an efficient, fair and friendly peer review service, and are committed to publishing all sound science, provided that there is some advance in knowledge presented by the work.
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