Department of Bacteriology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Fukuoka, Japan.
Department of Medicine and Biosystemic Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Fukuoka, Japan.
DNA Res. 2019 Oct 1;26(5):391-398. doi: 10.1093/dnares/dsz017.
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.
在细菌基因组和宏基因组测序中，Illumina 测序仪由于其高通量的能力而被最常使用，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目前可用于 Illumina 测序的文库制备试剂盒产生的测序偏差。我们的分析表明，Nextera XT 试剂盒在低 GC 区域引入了强烈的测序偏差。偏差的引入程度取决于 GC 含量的水平；GC 含量越低，产生的偏差越强。分析的其他试剂盒则没有引入这种强烈的测序偏差。在模拟细菌群落的宏基因组测序中，Nextera XT 引入的与 GC 含量相关的测序偏差更为显著，严重影响了低 GC 物种相对丰度的估计。我们分析的结果强调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，特别是在宏基因组测序中，存在着具有不同 GC 含量的各种微生物物种。我们的数据还表明，在分析和解释公开可用的宏基因组数据时，应特别注意使用了哪种文库制备试剂盒。
