A High-Throughput Sequencing Method to Assess the Structure and Composition of Gut Bifidobacterium

The next-generation high-throughput sequencing techniques have introduced a new way to assess the gut’s microbial diversity on the basis of 16S rRNA gene-based microbiota analysis. However, the precise appraisal of the biodiversity of Bifidobacterium species within the gut remains a challenging task because of the limited resolving power of the 16S rRNA gene in different species. The groEL gene, a protein-coding gene, evolves quickly and thus is useful for differentiating bifidobacteria.

The objective of the present study was to develop a new method to assess the structure and composition of gut bifidobacterium. 

To identify Bifidobacterium species, a region of 487 or 496 base pairs (bp) located at positions 1066 to 1552 (B. animalis) or 1561 (B. breve) of the complete groEL gene of ca. 1600 bp was chosen as discriminative target sites. Degenerate primers BifgroEL-F (5-TCC GAT TAC GAY CGY GAG AAG CT-3)/Bif-groEL-R (5-CSG CYT CGG TSG TCA GGAACA G-3) for the genus Bifidobacterium were manually designed according to multiple sequence alignment. The detection sensitivity and accuracy of the primer set Bif-groEL-F/Bif-groEL-R were evaluated employing known DNA amounts, ranging from 0.01 to 40 ng, of the artificial sample from 10 different bifidobacterial taxa. Sequence reads were processed with the QIIME package version 1.9.1.

The novel designed primer set can specifically differentiate Bifidobacterium species from non-bifidobacteria, and as low as 10⁴ cells of Bifidobacterium species can be detected using the novel designed primer set on the basis of Illumina Miseq high-throughput sequencing.