16S rRNA gene sequencing of SIV infected Rhesus Macaque (Macaca mulatta): Rectum & Jejunum fecal samples
DNA samples were sent to the University of Minnesota Genomics Center for 16S rRNA gene paired-end sequencing on an Illumina MiSeq. Two primer sequences were used to sequence the V4-V6 region of the bacterial 16S rRNA gene. Meta V4 515F (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA) and V6R Nextera (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCGACRRCCATGCANCACCT) were used with 16S-specific portions in bold. Indexing primers were added to add both the index and the flowcell adapters. The forward indexing primer (AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC) and reverse indexing primer (CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG) were used with the p5 and p7 flow cell adapters in bold. PCR reactions were performed using KAPA HiFidelity Hot Start Polymerase. Prior to sequencing pooled, size-selected sample was denatured with NaOH, diluted to 8 pM in IlluminaÍs HT1 buffer, spiked with 20% PhiX, and heat denatured at 96C for 2 minutes immediately prior to loading. A MiSeq 600 cycle v3 kit was used to sequence the sample. Nextera adapter sequences were used for post-run trimming (Read 1: CTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG, Read 2: CTGTCTCTTATACACATCTGACGCTGCCGACGANNNNNNNNGTGTAGATCTCGGTGGTCGCCGTATCATT).