Bacterial 16S of lake water
The 16S rRNA V3 (bacteria only) and V4-V5 (bacteria & archaea) hypervariable regions were amplified using the primer pair 341F CCTACGGGNGGCWGCAG and 534R ATTACCGCGGCTGCTGG and the primer pair 515F GTGYCAGCMGCCGCGGTAA and 926R CCGYCAATTYMTTTRAGTTT, respectively (Garca-Lpez et al., 2020; Klindworth et al., 2013). An additional 5 bases of inline barcode and partial Illumina adapter were incorporated at the 5 end of the primers to enable inline barcoding (Glenn et al., 2019). Different samples were amplified using different combinations of the forward and reverse inline primers. PCR was performed using Rediant II PCR mastermix (Apical Scientific, Malaysia) using the PCR profile of: 95C for 2 minutes followed by 30 cycles of 95C for 15s, 50C for 30s and 72C for 30s. The purified amplicons were used as the template for 8 cycles of index PCR to incorporate the complete Illumina adapter and Illumina-compatible dual-index barcodes. The constructed libraries were subsequently size-selected using 0.8 X vol of SPRI bead and pooled into a single tube. Quantification of the pooled libraries used Denovix high sensitivity assay. Sequencing of the pooled libraries was performed on a NovaSEQ6000 (Illumina, San Diego) using the 2x150 bp paired-end sequencing configuration.
SUB15088126
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