生物
清脆的
基因组
遗传学
计算生物学
基因组编辑
DNA测序
基因
Cas9
全基因组测序
基因组学
作者
Guanqing Liu,Yiping Qi,Tao Zhang
出处
期刊:Methods of Molecular Biology
日期:2021-01-01
卷期号:2238: 145-172
被引量:1
标识
DOI:10.1007/978-1-0716-1068-8_10
摘要
The CRISPR/Cas systems have become the most widely used tool for genome editing in plants and beyond. However, CRISPR/Cas systems may cause unexpected off-target mutations due to sgRNA recognizing highly homologous DNA sequence elsewhere in the genome. Whole-genome sequencing (WGS) can be used to identify on- and off-target mutation. Here, we describe a pipeline of analyzing WGS data using a series of open source software for analysis of off-target mutations in CRISPR-edited rice plants. In this pipeline, the adapter is trimmed using SKEWER. Then, the cleaned reads are mapped to reference genome by applying BWA. To avoid mapping bias, the GATK is used to realign reads near indels (insertions and deletions) and recalibrate base quality controls. Whole-genome single nucleotide variations (SNVs) and indels are detected by LoFreq*, Mutect2, VarScan2, and Pindel. Last, SNVs and indels are compared with in silico off-target sites using Cas-OFFinder.
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