Table 2.
Primers for LAM- HTGTS
| Use | Name | Sequences |
|---|---|---|
| Bridge adapter (step 20) | Adapter-upper* | GCGACTATAGGGCACGCGTGGNNNNNN-NH2 |
| Adapter-lower* | /5-Phosphorylation/CCACGCGTGCCCTATAGTCGC-NH2 | |
| Nested PCR (step 28) | I5-Nested** | ACACTCTTTCCCTACACGACGCTCTTCCGATCT BARCODE NESTEDPRIMER |
| I7-Blue | CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGACTATAGGGCACGCGTGG | |
| Tagged PCR (step 41) | P5-I5*** | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT |
| P7-I7*** | CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTC |
End modifications can be produced by Integrated DNA Technologies, the synthesis code for “/5-Phosphorylation/” is “/5Phos/”, for “-NH2” is “/3AmMO/”, and for “/5-biotin/” is “/5BiosG/”, “N” means random nucleotide.
“Nested primer” is the locus-specific nested primer (in bold), and “barcode” means the DNA sequence to differentiate samples with the same locus-specific nested primer (underlined), thus these samples can be sequenced in the same Miseq run. Barcodes can be any non-tandem DNA sequences between 0 and 10 bp, or use the Miseq index following the manufacturer's instructions.
Sequences from the Miseq primers are marked in italics. Note that I5 and I7 primers share 14-bp homologies at the 3’ end (compare the italic sequences of I5-nested to that of I7-Blue), thus the Tm in step 42 is 62°C to reduce cross-template amplification. Alternatively, P5-I5 can be further shortened from the 3’ end to avoid annealing to the 3’ region of I7 with same sequences.