In bacterial genome and metagenome sequencing, Illumina sequencers are most frequently used due to their high throughput capacity, and multiple library preparation kits have been developed for Illumina platforms. Here, we systematically analysed and compared the sequencing bias generated by currently available library preparation kits for Illumina sequencing. Our analyses revealed that a strong sequencing bias is introduced in low-GC regions by the Nextera XT kit. The level of bias introduced is dependent on the level of GC content; stronger bias is generated as the GC content decreases. Other analysed kits did not introduce this strong sequencing bias. The GC content-associated sequencing bias introduced by Nextera XT was more remarkable in metagenome sequencing of a mock bacterial community and seriously affected estimation of the relative abundance of low-GC species. The results of our analyses highlight the importance of selecting proper library preparation kits according to the purposes and targets of sequencing, particularly in metagenome sequencing, where a wide range of microbial species with various degrees of GC content is present. Our data also indicate that special attention should be paid to which library preparation kit was used when analysing and interpreting publicly available metagenomic data.

中文翻译：

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当前用于细菌基因组和元基因组的Illumina测序的文库制备试剂盒的测序偏倚比较。

在细菌基因组和元基因组测序中，由于Illumina测序仪的高通量能力，因此最常使用，并且已经为Illumina平台开发了多种文库制备试剂盒。在这里，我们系统地分析和比较了当前可用的Illumina测序文库制备试剂盒产生的测序偏倚。我们的分析表明，Nextera XT试剂盒在低GC区域引入了强大的测序偏向。引入的偏倚水平取决于GC含量水平。随着GC含量的降低，会产生更强的偏差。其他分析试剂盒没有引入这种强烈的测序偏倚。Nextera XT引入的与GC含量相关的测序偏倚在模拟细菌群落的元基因组测序中更为显着，并且严重影响了低GC物种相对丰度的估计。我们的分析结果突显了根据测序的目的和目标选择合适的文库制备试剂盒的重要性，特别是在元基因组测序中，其中存在各种程度不同的GC含量的微生物。我们的数据还表明，在分析和解释可公开获得的宏基因组学数据时，应特别注意使用了哪种文库制备试剂盒。特别是在元基因组测序中，其中存在各种程度不同的GC含量的微生物。我们的数据还表明，在分析和解释可公开获得的宏基因组学数据时，应特别注意使用了哪种文库制备试剂盒。特别是在元基因组测序中，其中存在各种程度不同的GC含量的微生物。我们的数据还表明，在分析和解释可公开获得的宏基因组学数据时，应特别注意使用了哪种文库制备试剂盒。