Miseq dna sequencing

Participants reporting antibiotic use during the week prior to sample collection were excluded from the analysis (n=3). The study group allocation (i.e., 5 participants received the intervention and 7 received usual care) was not tested in this pilot study due to the small sample size and the need for participants to experience different magnitudes of cardiorespiratory fitness change over time. Fecal wipe samples were collected and processed according to the protocol for pre-moistened wipe fecal samples outlined by Kumar et al. [20 (link)]. Briefly, samples from the assessed participants were collected and shipped overnight to the University of Alabama at Birmingham (UAB) Microbiome Resource Laboratory. Samples were stored at −80 °C until analysis. Each sample was dissolved in a buffer solution, then processed with a DNA Miniprep Kit to obtain isolated fecal DNA. Polymerase chain reaction (PCR) was used to amplify the V4 region of the 16S rRNA gene, which was then analyzed using Illumina MiSeq DNA sequencing. 250 base paired-end sequences were obtained with raw data processed, integrated, and analyzed using the Quantitative Insights into Microbial Ecology (QIIME) bioinformatics software and QWRAP program. Microbiome analyses, including quality control, OTU picking, sequence databases and sequencing primers, were performed as previously described [20 (link)].

Strains IU7476, IU7477, IU7567, IU7570 and IU7765 containing suppressor mutations that allowed the growth of a Δpbp2b deletion mutant were isolated as described in Results (Table S1). Overnight cultures still in exponential phase were diluted into 5 ml of BHI broth to an OD₆₂₀ ≈0.01 and grown to an OD₆₂₀ ≈ 0.3 to 0.4. Cells were collected by centrifugation (10,000 xg for 10 min). Genomic DNA was purified from collected cells using a MasterPure Gram-positive DNA purification kit (Epicenter Biotechnologies) according to the manufacturer’s protocol. DNA library construction, Illumina MiSeq DNA sequencing, and bioinformatic analyses are described in procedures in Supplemental Information.