16SSIVpos10HSABuccal10
Primers 341F (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG(spacer)TGCCTACGGGNGGCWGCAG) and 806R (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG(spacer)CCGGACTACNVGGGTWTCTAAT were used to amplify the V3-V4 domain of the 16S rRNA using a two step PCR procedure. In step one of the amplification procedure, both forward and reverse primers contained an Illumina tag sequence (bold), a variable length spacer (no spacer, A, CA, or GCA for 341F; no spacer, G, TG, ATG for 806R) to increase diversity and improve the quality of the sequencing run, a linker sequence (italicized), and the 16S target sequence (underlined). Each 25 l PCR reaction contained 1 Unit Kapa2G Robust Hot Start Polymerase (Kapa Biosystems), 1.5 mM MgCl2, 0.2 mM final concentration dNTP mix, 0.2 M final concentration of each primer, 3 l KAPA 5X Enhancer 1, and 1ul of DNA for each sample. PCR conditions were: an initial incubation at 95C for 3 min, followed by 25 cycles of 95C for 45 s, 50C for 30 s, 72C for 30 s and a final extension of 72C for 3 min. In step two, each sample was barcoded with a unique forward and reverse barcode combination using forward primers (AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC) with an Illumina P5 adapter sequence (bold), a unique 8 nt barcode (N), a partial matching sequence of the forward adapter used in step one (underlined), and reverse primers (CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGG) ) with an Illumina P7 adapter sequence (bold), unique 8 nt barcode (N), and a partial matching sequence of the reverse adapter used in step one (underlined). The PCR reaction in step two contained 1 Unit Kapa2G Robust Hot Start Polymerase (Kapa Biosystems), 1.5 mM MgCl2, 0.2 mM final concentration dNTP mix, 0.2 M final concentration of each uniquely barcoded primer and 1ul of the product from the PCR reaction in step one diluted at a 10:1 ratio in water. PCR conditions were: an initial incubation at 95C for 3 min, followed by 9 cycles of 95C for 30 s, 58C for 30 s, 72C for 30 s and a final extension of 72C for 3 min.