| Construction Protocol | The obtained indexed PCR products were pooled after purification with AMPure XP Kit (Beckman Coulter) and all amplicons were quantified using Qubit?? 2.0 Fluorometer. The quality of each amplicon was checked by Agilent 2100 Bioanalyzer using DNA 1000 Kit (Agilent Technologies, Santa Clara, CA, U.S.) according to the manufacturer???s instructions. Amplicons were pooled at equimolar ratio and then diluted to final concentration of 8 pM. 15% (v/v) of Phix control libraries (Illumina, San Diego, CA, USA) was combined with normalized library to increase diversity of base calling during sequencing. Amplicons library was subjected to sequencing using V3 reagents kits on Illumina MiSeq system (Illumina, San Diego, CA, U.S.A). Pair-end sequencing was carried out in 281 cycles per read (281 x 2). To prevent the potential demultiplexingerrors, we converted the raw MiSeq BCL data into FASTQ data byourselves using the bcl2fastq v1.8.4 program distributed by Illumina. |
FASTQ
