title: 5R 16S rRNA sequencing of human: pancreas
design: The study employed a multiplexed 16S rDNA sequencing protocol that targets five short 16S regions (V2, V3, V5, V6, V8) to increase the coverage and resolution of bacterial species detection from FFPE-derived DNA (fragment length 250-400 bp) . Five regions of the 16S rRNA gene were amplified using 100 ng DNA as an input and a set of 10 multiplexed primers (0.2M each primer, F1-TGGCGAACGGGTGAGTAA, F2-ACTCCTACGGGAGGCAGC, F3-GTGTAGCGGTGRAATGCG, F4-GGAGCATGTGGWTTAATTCGA, F5-10 GGAGGAAGGTGGGGATGAC, R1-AGACGTGTGCTCTTCCGATCTCCGTGTCTCAGTCCCARTG, R2-AGACGTGTGCTCTTCCGATCTGTATTACCGCGGCTGCTG, R3-AGACGTGTGCTCTTCCGATCTCCCGTCAATTCMTTTGAGTT, R4-AGACGTGTGCTCTTCCGATCTCGTTGCGGGACTTAACCC, R5-AGACGTGTGCTCTTCCGATCTAAGGCCCGGGAACGTATT), and 2 MULTIPLEX TCR PCR Mix (Milaboratory, Moscow, Russia). Amplification was done with an initial heating step of 95C for 5 min, 30 cycles of 30 seconds at 94C, 90 seconds at 62C, 35 seconds at 72C followed by a final elongation step of 5 min at 72C. Barcodes and Illumina adaptors were added to the amplicon with a second PCR reaction with 5 forward primers (0.2M each primer, FF1-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGCGAACGG20 GTGAGTAA, FF2-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACTCCTACGGGAGGCAGC, FF3-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGTGTAGCGGTGRAATGCG, FF4-25 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGGAGCATGTGGWTTAATTCGA, FF5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGGAGGAAGGTGGGGATGAC) and one 8 nucleotide barcode-specific reverse primer (0.4M, RR5-CAAGCAGAAGACGGCATACGAGAT-NNNNNNNN-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT). The amplicon was diluted into the reaction (10-fold) and amplified with 6 cycles of 10 seconds at 98C, 15 seconds at 64C, 25 seconds at 72C. Libraries were purified from primer dimers using MagPure A4 XP Beads (Magen, Guangzhou, China) at a volume ratio of 1:0.85 (library : beads). Then libraries were combined into the final library (100-430 amplicons) and sequenced on SURFSeq 5000 (GeneMind, Shenzhen, China).