Illumina 16S rDNA amplicon sequencing of bile sample from gallstone patients
Without centrifugation of bile samples, total DNA of 400 μL bile per patient was extracted using Invitrogen Purelink Genomic DNA mini kit (Life Technologies, Carlsbad, CA, USA), following manufacturer’s blood DNA extraction protocol. DNA concentration was quantified by Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and quality was examined by agarose gel electrophoresis using E-Gel electrophoresis system (L ife Technologies, Carlsbad, CA, USA). Universal primer pairs 356F (forward, 5’CCTACGGGNGGCWGCAG3’) and reverse 803R (backward, 5’GACTACHVGGGTATCTAATCC3’) targeting V3-V4 region of bacterial 16S ribosome DNA (rDNA) gene were used for amplification. We employed a two-step PCR 16S rDNA gene amplification protocol  used by Ottesen et. al. (2014) for amplification and library preparation. In brief, at the first step, target region of 16S rDNA was amplified using 16S primers with overhang adapters attached (forward primer overhang adapter 5’TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG3’ and reverse primer overhang adapter 5’ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG3’). These overhang adapters are compatible with the Illumina Nextera DNA indices. Then, P5/P7 adapters and sample barcodes in Illumina Nextera XT index kit (Illumina, San Diego, CA, USA) were added to the cleaned-up PCR products by the second-round PCR. The final PCR products were cleaned up and sequenced by Illumina MiSeq platform to generate 2x250 bp paired-end reads, which could cover amplified 16S V3-V4 region.