Evaluation of the Bacterial Diversity in the Human Tongue Coating Based on Genus-Specific Primers for 16S rRNA Sequencing

Figure 2

Schematic diagram of the primer design and library preparation. All the 16S rRNA gene sequences from the same genera were aligned to generate 20 bp of the conserved forward sequence (CF) and conserved reverse sequence (CR) flanking the variable region. The forward primer (PF) and reverse primer (PR) were designed by adding 6 bp of Eco57I recognition site and 16/18 bp of auxiliary sequence at the 5′ end of CF and CR. The microbial genome DNA of each subject was amplified by all the pairs of primers from different genus, producing the amplicons containing the Eco57I cutting site (CS) which was 16 bp downstream of the recognition site. Followed by Eco57I digestion, products generated by all the genus-specific primers, conserving 4 bp of each primer sequence, were mixed together to produce the sequencing library. The results of ligation-based sequencing were millions of reads, which were the first 35 bases at the 5′ end from either forward strand or reverse strand of library.