Testing for de novo mutation calling artefacts by re-sequencing ten siblings of a single mutation accumulation line in Arabidopsis thaliana

We conducted a mutation bias study in the plant Arabidopsis thaliana. To test for the possibility that our mutation bias results could in part be artefacts of a pooled-seedling sequencing approach, we resequenced entire rosettes of siblings (individual plants) of one mutation accumulation line (#73) and asked if the distribution of variation called (i.e., putative somatic mutations around transcription start and termination sites) was similar to the patterns seen with the seedling pools of the 107 individual lines described in the preceding section. Specifically, we grew 10 siblings of line #73 and extracted DNA from 3-week old whole rosettes. Barcoded PCR-free libraries for the 10 siblings were sequenced, with 150 bp paired-end reads, at approximately 60x depth each on a single lane of the Illumina HiSeq 3000 platform. Additionally, for one sibling, the same library was sequenced in an independent lane at approximately 600x depth.

我们针对拟南芥（Arabidopsis thaliana）开展了一项突变偏倚研究。为验证我们的突变偏倚结果可能部分源于混苗测序方法所引入的测序伪影这一可能性，我们对1个突变积累株系（#73）的姊妹植株（即单株个体）的完整莲座叶丛进行了重测序，并探究所检出的变异（即转录起始与终止位点附近的推定体细胞突变）的分布模式，是否与前文所述107个独立株系的混苗测序结果一致。具体而言，我们培养了株系#73的10株姊妹植株，并从生长3周的完整莲座叶丛中提取基因组DNA。我们为这10株姊妹植株构建了带条形码的无PCR扩增文库，采用Illumina HiSeq 3000测序平台的单泳道进行测序，单样本测序深度约为60×，读长为150 bp双端读段。此外，我们对其中1株姊妹植株的同一文库在独立泳道进行了额外测序，测序深度约为600×。