Sample 140_S24 from CS disease group
We followed the steps described in the Illumina 16S sample preparation guide to amplify the V3 and V4 region, add Illumina sequencing adapters and dual index barcodes to the amplicon target. Primers were designed according to the V3-V4 regions of bacteria (the upstream primer: PCR_341 F is 5-CCTACGGGNGGCWGCAG-3, and the downstream primer: PCR_785R is 5-GACTACHVGGGTATCTA ATCC-3) (14). The Illumina overhang adapter sequences used were as follows: Forward overhang: 5 TCGTCG GCAGCGTCAGATGTGTATAAGAGACAG-[PCR_341F]; Reverse overhang: 5 GTCTCGTGGGCTCGGAGA TGTGTATAAGAGACAG-[PCR_785R]. Primers linked to adapters were used for PCR amplifcation; the PCR products were purifed, quantifed, and then normalized to form a sequencing library. These were sequenced by Illumina MiSeq as outlined in the Illumina 16S sample preparation guide.