Comparison of the sequencing bias of currently available library preparation kits for Illumina sequencing of bacterial genomes and metagenomes

Abstract 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.