Distinct Intestinal Microbial Signatures Linked to Accelerated Biological Aging in People with HIV
DNA was extracted from approximately 200 mg of stool or tissue using the Qiagen DNeasy PowerSoil Pro kit and quantified using the Quant-iT PicoGreen Assay Kit. Barcoded PCR primers targeting the V1-V2 region of the 16S rRNA gene were used for library generation. PCR reactions were performed in duplicate using Q5 High-Fidelity DNA Polymerase (NEB). For high microbial biomass samples such as fecal material, each PCR reaction contained 0.5 M of each primer, 0.34 U Q5 Pol, 1X Buffer, 0.2 mM dNTPs, and 5 l DNA in a reaction volume of 50 l. Cycling conditions were as follows: 1 cycle of 98C for 1 min; 20 cycles of 98C for 10 sec, 56C for 20 sec, and 72C for 20 sec; 1 cycle of 72C for 8 min. For low microbial biomass samples such as colon and ileum tissues, each PCR reaction contained 0.5 M of each primer, 0.34 U Q5 Pol, 1X Buffer, 0.2 mM dNTPs, and 10 l DNA in a reaction volume of 50 l. Cycling conditions were as follows: 1 cycle of 98C for 1 min; 25 cycles of 98C for 10 sec, 56C for 20 sec, and 72C for 20 sec; 1 cycle of 72C for 8 min. Samples were amplified in duplicates, pooled, and purified using a 1:1 volume of SPRI beads. DNA in each sample was then quantified using PicoGreen and pooled in equal molar amounts. The resulting library was sequenced on the Illumina MiSeq using 2x250 bp chemistry.