Summary:\nThis protocol is for the submission of DNA to generate libraries for 16S rRNA sequencing, which can be used for bacterial community analysis and detect variations in the microbiota under differing conditions.DNA is aliquoted into 96 well plates, which are properly packaged and shipped to the MMPC and libraries are prepared for community analysis as previously described by Seekatz et al. (1). Briefly, barcoded dual-index primers specific to the V4 region of the 16S rRNA gene amplify the DNA (2). PCR reactions are composed of 5 \u03bcL of 4 \u03bcM equimolar primer set, 0.15 \u03bcL of AccuPrime Taq DNA High Fidelity Polymerase, 2 \u03bcL of 10x AccuPrime PCR Buffer II (Thermo Fisher Scientific, catalog no. 12346094), 11.85 \u03bcL of PCR-grade water, and 1 \u03bcL of DNA template. The PCR conditions used consisted of 2 min at 95\u00b0C, followed by 30 cycles of 95\u00b0C for 20 s, 55\u00b0C for 15 s, and 72\u00b0C for 5 min, followed by 72\u00b0C for 10 min. Each PCR reaction is normalized using the SequalPrep Normalization Plate Kit (Thermo Fisher Scientific, catalog no. A1051001). The normalized reactions are pooled and quantified using the Kapa Biosystems Library qPCR MasterMix (ROX Low) Quantification kit for Illumina platforms (catalog no. KK4873). The Agilent Bioanalyzer is used to confirm the size of the amplicon library (~399 bp) using a high-sensitive DNA analysis kit (catalog no. 5067-4626). Pooled amplicon library is then sequenced on the Illumina MiSeq platform using the 500 cycle MiSeq V2 Reagent kit (catalog no. MS-102-2003) according to the manufacturer's instructions with modifications of the primer set with custom read 1\/read 2 and index primers added to the reagent cartridge. The\u201cPreparing Libraries for Sequencing on the MiSeq\u201d (part 15039740, Rev. D) protocol was used to prepare libraries with a final load concentration of 5.5 pM, spiked with 15% PhiX to create diversity within the run. FASTQ files are distributed to the client when the 2 x 250 bp sequencing completes.References:1. Seekatz AM, Theriot CM, Molloy CT, Wozniak KL, Bergin IL, Young VB. 2015. Fecal Microbiota Transplantation Eliminates Clostridium difficile in a Murine Model of Relapsing Disease. Infect Immun 83:3838-3846. 10.1128\/IAI.00459-15.\n2. Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. 2013. Development of a dual- index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol 79:5112\u20135120. 10.1128\/AEM.01043-13.