Table 2.
Sequences of the PCR primers for amplicon library preparation and qPCR
| Primer ID | Primer sequences (5′–3′) | Amplicon length (bp)a | Number of CpGs quantified by massive parallel sequencing |
|---|---|---|---|
| Sequences of the template specific 3′-portion of the fusion PCR primersb | |||
| SDHA-F | AGTTATTTTAAGTTTGTTTATATGTGATTT | 236a | 14 |
| SDHA-R | ATAAACACCAACATTTTTAAAACCC | ||
| SDHB-F | TAGTTTGGTTAAGATGATGAAATTT | 302a | 15 |
| SDHB-R | CTTTCAAAAAATAAAACTAAAACTTAAATA | ||
| SDHC-F | GAAAATAATTAGTAAATTAGTTAGGTAG | 198a | 13 |
| SDHC-R | ACTAAAATCACCTCAACAACAAC | ||
| SDHD-F | TATTAAGGAAGGTGAAATTTTTTTT | 313a | 25 |
| SDHD-R | TCCTAAAAACTCAAAATCATCCAC | ||
| Sequences of the qPCR primers | |||
| SDHA-F | TGGGAACAAGAGGGCATCTG | 86 | |
| SDHA-R | CCACCACTGCATCAAATTCATG | ||
| SDHB-F | ACCTTCCGAAGATCATGCAG | 86 | |
| SDHB-R | CTTCGGGTGCAAGCTAGAGT | ||
| SDHC-F | TGGCACTGGTATTGCTTTGA | 78 | |
| SDHC-R | GACTCAAAGTTCCCAGGGAGT | ||
| SDHD-F | GGTGTGGAGTGCAGCACATA | 88 | |
| SDHD-R | ACAACCCTCTCGCTAGTCCA | ||
a
Template-specific portion of each amplicon is shown. Given additional auxiliary 35-mer sequences at both ends, real amplicon size is 70 bp longer for fusion PCVR primers.
b
The complete PCR primer sequence includes an additional 35-mer portion at 5′-end. It serves for binding to the DNA capture beads and annealing the emPCR amplification primers and the sequencing primer. In addition, it contains the sequencing tetranucleotide key and 10-mer multiple identifier (MID) for demultiplexing reads. A total of six MIDs (MID1-6) were used from the 454Standard MID set.