Mo bio powerlyzer ultraclean tissue cell dna isolation kits

Bacterial sequence analysis was performed at the Alkek Center for Metagenomics and Microbiome Research (CMMR) at BCM. Bacterial genomic DNA was extracted using the MO BIO PowerLyzer UltraClean Tissue & Cell DNA Isolation Kits (MO BIO Laboratories, Clardad, CA, USA). All DNA samples were stored at −80 °C until further analysis.
The 16S rRNA variable region 4 (V4) was amplified by PCR, using the barcoded Illumina adaptor-containing primers 515F and 806R and sequenced in the MiSeq platform (Illumina, San Diego, CA, USA) using the 2 × 250 bp paired-end protocol yielding pair-end reads that overlap almost completely [16 (link),17 (link),18 (link)]. The primers used for amplification contain adapters for the MiSeq sequencing and single-index barcodes so that the PCR products may be pooled and sequenced directly [16 (link)]. The samples were rarefied to 1648 reads resulting in the loss of all negative controls for the DNA extraction kit. Rarefication ensured that we sampled the most microbial diversity.

The sequence analyses were performed at the Alkek Center for Metagenomics and Microbiome Research (CMMR) at BCM. Bacterial genomic DNA was extracted from the biopsies using the MO BIO PowerLyzer UltraClean Tissue & Cell DNA Isolation Kits (MO BIO Laboratories, Clardad, CA, USA). All DNA samples were stored at −80 °C until further analysis.
The 16S rRNA gene sequencing methods were adapted from the published methods [25 (link),26 (link),27 (link)]. The 16S rRNA hypervariable region 4 (V4) was amplified by PCR using the barcoded Illumina adaptor-containing primers 515F and 806R and sequenced on the MiSeq platform (Illumina, San Diego, CA, USA) using the 2 × 250 bp paired-end protocol. The primers used for amplification contained adapters for the MiSeq sequencing and single-index barcodes so that the PCR products could be pooled and sequenced directly [27 (link)].