In Silico Separation of in Vitro Transcription-Derived Duplicates From PCR Duplicates to Enhance Sequence Data Utilization.

IF 2.4 Q3 BIOCHEMICAL RESEARCH METHODS
Bioinformatics and Biology Insights Pub Date : 2025-08-26 eCollection Date: 2025-01-01 DOI:10.1177/11779322251365042
Ryoga Suzuki, Kenichi Horisawa, Kazumitsu Maehara, Yasuyuki Ohkawa, Atsushi Suzuki
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引用次数: 0

Abstract

The polymerase chain reaction (PCR) amplification process of deoxyribonucleic acid (DNA) libraries can introduce bias in the sequence ratios. Consequently, several recent genomic and transcriptomic methods employing next-generation sequencing (NGS) utilize in vitro transcription (IVT) to amplify template polynucleotide chains. IVT amplifies nucleic acid sequences linearly, making it less susceptible to bias than the exponential amplification of PCR. Chromatin integration labeling sequencing (ChIL-seq), a tool for analyzing transcription factor binding and histone modifications, has incorporated IVT by replacing PCR in the DNA amplification step, enabling the analysis of small sample sizes, including single cells. In this study, we discovered that many of the excluded sequences known as PCR duplicates during the pre-processing step of ChIL-seq data analysis contain amplification products derived from IVT. Furthermore, we developed an in silico method to selectively eliminate PCR duplicates from NGS data while retaining IVT-derived amplification products. The method prevents excessive data reduction and significantly improves the utilization efficiency of NGS data.

Abstract Image

Abstract Image

Abstract Image

体外转录衍生重复序列与PCR重复序列的硅分离以提高序列数据的利用。
脱氧核糖核酸(DNA)文库的聚合酶链反应(PCR)扩增过程会导致序列比例的偏差。因此,最近采用下一代测序(NGS)的几种基因组学和转录组学方法利用体外转录(IVT)来扩增模板多核苷酸链。IVT线性扩增核酸序列,使其比PCR的指数扩增更不易受偏差影响。染色质整合标记测序(ChIL-seq)是一种分析转录因子结合和组蛋白修饰的工具,它通过在DNA扩增步骤中取代PCR而纳入了IVT,从而能够分析包括单细胞在内的小样本量。在本研究中,我们发现在ChIL-seq数据分析的预处理步骤中,许多被排除的序列(称为PCR重复)包含来自IVT的扩增产物。此外,我们开发了一种计算机方法来选择性地从NGS数据中消除PCR重复,同时保留ivt衍生的扩增产物。该方法避免了数据的过度缩减,显著提高了NGS数据的利用效率。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Bioinformatics and Biology Insights
Bioinformatics and Biology Insights BIOCHEMICAL RESEARCH METHODS-
CiteScore
6.80
自引率
1.70%
发文量
36
审稿时长
8 weeks
期刊介绍: Bioinformatics and Biology Insights is an open access, peer-reviewed journal that considers articles on bioinformatics methods and their applications which must pertain to biological insights. All papers should be easily amenable to biologists and as such help bridge the gap between theories and applications.
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