Metagenomic sequencing of gut microbiome: human gut biopsy
For library preparation, 1 ng of intact genomic DNA was processed using the Nextera XT DNA Sample Preparation Kit (Illumina). The target DNA was simultaneously fragmented and tagged by the Nextera Enzyme Mix containing transposome that fragments the input DNA and adds the bridge PCR (bPCR)-compatible adaptors required for binding and clustering on the flowcell. Next, DNA was amplified using a limited-cycle PCR program adding index 1(i7) and index 2(i5) (Illumina) in unique combinations and sequences specific for cluster formation. The thermal profile for the amplification had an initial extension step at 72°C for 3 minutes and initial denaturing step at 95°C for 30 seconds, followed by 12 cycles of denaturing of 95°C for 10 seconds, annealing at 55°C for 30 seconds, a 30 second extension at 72°C, and a final extension for 5 minutes at 72°C. The library DNA then was purified using the Agencourt® AMPure® XP Reagent. Each sample was quantified and normalized prior to pooling. The DNA library pool was heat denatured before loading on the MiSeq reagent cartridge and on the MiSeq instrument. Automated cluster generation and paired-end sequencing with dual reads were performed per the manufacturer’s instructions.