1. Sequencing process

16S metagenomics library was constructed using two rounds of PCR reactions. The PCR primers,

forward

(5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’)

and reverse

(5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT-3’),

were designed to amplify the V3V4 domain of bacterial 16S ribosomal DNA as well as adding adaptor sequences. Illumina index sequences were subsequently attached into the amplicons in the second PCR.

PCR amplification was performed in a 50 μl reaction volume containing 25 μl 2X Phusion High-Fidelity PCR Master Mix (New Enagland Biolabs), 0.2 μM of each forward and reverse primer, and 100 ng DNA template. The reaction conditions consisted of an initial 98°C for 30sec, followed by 30 cycles of 98°C for 10 sec, 55°C for 30 sec, and 72°C for 30 sec, as well as a final extension of 72°C for 5 min. The number of cycle for second PCR was 10.

Next, amplified products were checked by 2 % agarose gel electrophoresis and were purified using the AMPure XP PCR Purification Kit (Agencourt). Quantification was performed by using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) on Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and Light cycler 96 (Roche)- all according to respective manufacturer instructions. The final purified libraries were applied for cluster generation and sequencing on the MiSeq using V3 600 cycles kit.

2. Raw data process

3. Data analysis workflow

1. QC results

QC: # of sequencing reads passed by the quality threshold

Merge: # of merged sequencing reads

OTU.cluster: # of clustered sequencing reads

Alignment: # of sequencing reads mapped to Silva database

Represent.OTU: # of clusters

Mapped.OTU: # of clusters mapped to Silva database

Number.of.Tax: # of Taxonomy

Path: ./result/OTU & QC/

2. Taxonomy results