title: 16S rRNA gene amplicon sequencing of stool sample from hormonal contraceptives study participant 08 at day of cycle 08 [JMF-1903-9-0061A]
design: Stool samples were collected every other day during one menstrual cycle, starting at the onset of menses (1st day of the cycle). DNA from stool samples was extracted using the QIAamp DNA Stool Mini Kit (Qiagen) following the manufacturers instructions. Before extraction, samples were subjected to mechanical lysis in ASL Buffer, by beat beating the samples two times on the FastPrep-96 Instrument (MP Biomedicals) for 45 s at 1400 rpm. The V3-V4 region of the bacterial 16S rRNA gene was amplified (30 cycles) from the DNA using the 341F and 785R primers (https://www.ncbi.nlm.nih.gov/pubmed/22933715) which were modified with a linker sequence (https://www.ncbi.nlm.nih.gov/pubmed/26236305) and barcoded (8 cycles) in a unique dual (UD) setup. Thereafter, barcoded samples were purified and normalized over a SequalPrep Normalization Plate Kit (Invitrogen) using a Biomek NXP Span-8 pipetting robot (Beckman Coulter), and pooled and concentrated on columns (Anlaytik Jena). Indexed sequencing libraries were prepared with the Illumina TruSeq Nano Kit as described previously (https://www.ncbi.nlm.nih.gov/pubmed/26236305), and sequenced in paired-end mode (2x300 nucleotide, v3 chemistry) on an Illumina MiSeq system following the manufacturers instructions. The workflow systematically included four negative controls (PCR blanks, i.e., PCR-grade water as template) for each 90 samples sequenced. Amplicon pools were extracted from the raw sequencing data using the FASTQ workflow in BaseSpace (Illumina) with default parameters. Sample demultiplexing was performed with the python package demultiplex (Laros JFJ, github.com/jfjlaros/demultiplex) allowing one mismatch for barcodes and two mismatches for linkers and primers.