title: 16SrRNA-seq of human feces
design: total DNA is extracted from environmental or biological samples, such as soil, fecal matter, or tissue. The V3-V4 hypervariable region of the 16S rRNA gene is amplified using the specific primers 338F (5-ACTCCTACGGGAGGCAGCAG-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3). PCR is performed using high-fidelity DNA polymerase under the following cycling conditions: an initial denaturation at 95C for 3 minutes, followed by 3035 cycles of denaturation at 95C for 30 seconds, annealing at 55C for 30 seconds, and extension at 72C for 45 seconds, with a final extension at 72C for 5 minutes. After amplification, the PCR products are purified using magnetic bead-based purification to remove primer dimers and other contaminants, with the expected amplicon size being approximately 400-500 bp. Library preparation follows, involving the attachment of sequencing adapters and sample-specific barcodes, followed by a second PCR to incorporate these adapters. After a second round of purification, the library is quantified using fluorescence-based methods, such as Qubit or PicoGreen, and the fragment size distribution is checked using an Agilent Bioanalyzer. The libraries are then sequenced on an Illumina platform, with paired-end 250 bp or 300 bp reads to cover the V3-V4 region.