13_4
Faecal microbiota during 10 days of fasting
The partial sequences of the hypervariable region of 16S rRNA gene (V4 & V5) were PCR amplified using the primer 520 F (5- AYTGGGYDTAAAGNG-3) and 907 R (5- CCGTCAATTCMTTTRAGTTT-3). With indices and linker sequences, libraries have a mean sequence length of 528 bp. After purification with AMPure beads, quality checks for library sizes and DNA concentration were performed with the Aglient Bioanalyzer using Aglient DNA 1000 chips (Aglient Technologies, Waldbronn, Germany). Libraries were finally pooled in equivalent 100 pM for Ion Torrent. Libraries prepared for Ion Torrent sequencing template-positive Ion PGM template Hi-Q ion sphere particles were produced using the emulsion PCR technique in the Ion One Touch 2 and the Ion PGM Hi-Q OT2 Kit (Life Technologies, Inchinnan, UK) following the manufacturers instructions for PCR, recovery and quality control. The libraries got divided to be run on four ION 318 chip-kit v2 BC chips (Life Technologies, Inchinnan, UK) executing 850 sequencing flows on an Ion PGM sequencer (Life Technologies, Inchinnan, UK) following the manufacturers instructions.
13_4
SUB5422701
13_4