TABLE 4.
2-Step protocol PCR primersa
| Primer | Sequence (5′→3′) |
|---|---|
| Step 1a | |
| Forward | ACACTGACGACATGGTTCTACA + heterogeneity spacer (0–7 bp) + ACTCCTRCGGGAGGCAGCAG |
| Reverse | TACGGTAGCAGAGACTTGGTCT + heterogeneity spacer (0–7 bp) + GGACTACHVGGGTWTCTAAT |
| Step 2b | Illumina 3′ flowcell linker + index + CS1/CS2 |
| Forward | AATGATACGGCGACCACCGAGATCTACAC + index (8 bp) + ACACTGACGACATGGTTCTACA |
| Reverse | CAAGCAGAAGACGGCATACGAGAT + index (8 bp) + TACGGTAGCAGAGACTTGGTCT |
a
See Data Set S1 for full oligonucleotide sequences. For step 1, primer sequences are presented as Illumina 5′ sequencing primer (CS1/CS2) + heterogeneity spacer + 16S rRNA gene V3-V4 primer.
b
See Data Set S1 for full forward and reverse oligonucleotides, respectively. For step 2, primer sequences are presented as Illumina 3′ flow cell linker + index + CS1/CS2.