全外显子组测序分析中预处理方法和变异识别方法的比较.docxVIP

全外显子组测序分析中预处理方法和变异识别方法的比较闫瑾, 潘琦, 任红（重庆医科大学附属病毒性肝炎所，重庆 400010）【摘要】目的研究在外显子组数据分析中，使用不同的预处理方法和变异过滤方法对变异识别的影响。方法采用FASTX-Toolkit、Trimmomatic作为预处理方法，修饰后不同的不匹配读长（single-end reads）取舍策略，以及硬过滤（hard filter）和变异质量得分重新校正（VQSR）作为变异过滤方法对两例全外显子组数据进行变异识别，通过数据覆盖深度（DP）、识别变异的数目、Ti/Tv值和基因型一致性等数据进行比较其效果。结果Trimmomatic预处理后的读长的测序覆盖深度与未预处理的原始数据接近，但明显高于FASTX-Toolkit预处理方法。当DP≥10×、基因型质量分数（GQ）≥20时，经Trimmomatic预处理后识别到的SNV数量比FASTX-Toolkit高，与未预处理组接近。当包含single-end读长时，FASTX-Toolkit组多识别的SNV数量高于 (28%)Trimmomatic组 (5%)。当样本量较少时，在所有试验组中硬过滤方法滤掉的SNV要少于VQSR。结论Trimmomatic修饰（过滤）原始序列更温和，而FASTX-Toolkit可能过度过滤了原始数据。保留single-end读长有利于下游变异识别。硬过滤相较于变异质量得分重新校准表现出更高的容忍度。[关键词] 全外显子组测序；预处理；变异识别[中图法分类号] R857.3；Q344+.12Comparison of methods for pre-processing and variants filtering in analyzing whole exome sequencing dataYan Jin, Pan Qi, Ren Hong（Institute for Viral Hepatitis，Chongqing Medical University， Chongqing，400010，China）【Abstract】 ObjectiveTo investigate how different methods for pre-processing and variants filtering affect variants calling.Method Through the calculation of depth of coverage, number of variants, Ti/Tv ratio and non-reference concordance, we compare the effect of FASTX-Toolkit and Trimmomatic in preprocessing the exome data, the strategies of single-end inclusion and ‘Hard’ filter and variants quality score recalibration (VQSR) in variants filter by using whole exome sequencing data from two test samples.ResultTrimmomatic pre-processed reads showed similar depth of coverage to reads those without pre-processing, but significantly greater than those by FASTX-Toolkit pre-processed reads. With depth of coverage ≥10× and genotype quality ≥20, the number of called SNVs identified by Trimmomatic was greater than FASTX-Toolkit, but similar to those without pre-processing. With the inclusion of single-end reads, the number of variants increased significantly for FASTX-Toolkit pre-processing (~28%) than Trimmomatic pre-processing (~5%). In the all settings, ‘Hard’ filtering filtered less SNVs than VQSR filt