Table 3:
Summary of Sanger sequencing of plasmid-cloned adapter-dimer cDNAs.
| Buffer 4B | Read2:cRead1 Junction | primer duplex overhang | primer duplex paired | 1st cDNA nucle otide | 1st cDNA base paired | primer duplex dependent? | NTA dependent? | Detectable by Read1 sequencing? |
|---|---|---|---|---|---|---|---|---|
| 1Y primer duplex v2 & 3′rC no UMI adapter template | C|GAGATCGG | +1 C | No | G | 0 | No | Yes | Yes |
| C|GAGATCGG | +1 C | No | G | 0 | No | Yes | Yes | |
| T|GAGATCGG | +1 T | No | G | 0 | No | Yes | Yes | |
| C|-AGATCGG | +1 C | rC* | A | −1 | No | Yes | Yes | |
| C|-AGATCGG | +1 C | rC* | A | −1 | No | Yes | Yes | |
| T|-AGATCGG | +1 T | rC* | A | −1 | No | Yes | Yes | |
| T|-AGATCGG | +1 T | rC* | A | −1 | No | Yes | Yes | |
| T|-AGATCGG | +1 T | rC* | A | −1 | No | Yes | Yes | |
| T|-AGATCGG | +1 T | rC* | A | −1 | No | Yes | Yes | |
| T|-AGATCGG | +1 T | rC* | A | −1 | No | Yes | Yes | |
| T|-AGATCGG | +1 T | rC* | A | −1 | No | Yes | Yes | |
| T|-AGATCGG | +1 T | rC* | A | −1 | No | Yes | Yes | |
| T|-AGATCGG | +1 T | rC* | A | −1 | No | Yes | Yes | |
| T|-AGATCGG | +1 T | rC* | A | −1 | No | Yes | Yes | |
| T|-AGATCGG | +1 T | rC* | A | −1 | No | Yes | Yes | |
| T|-AGATCGG | +1 T | rC* | A | −1 | No | Yes | Yes | |
| T|-AGATCGG | +1 T | rC* | A | −1 | No | Yes | Yes | |
| T|-AGATCGG | +1 T | rC* | A | −1 | No | Yes | Yes | |
| C|------GG | +1 C | rG | G | −6 | Yes | No | No | |
| C|------GG | +1 C | rG | G | −6 | Yes | No | No | |
| C|------GG | +1 C | rG | G | −6 | Yes | No | No | |
| C|------GG | +1 C | rG | G | −6 | Yes | No | No | |
| T|------GG | +1 T | rG* | G | −6 | No | Yes | No | |
| T|-------G | +1 T | G* | G | −7 | No | Yes | No | |
| T|-------G | +1 T | G* | G | −7 | No | Yes | No | |
| T|-------G | +1 T | G* | G | −7 | No | Yes | No | |
| T|-------G | +1 T | G* | G | −7 | No | Yes | No | |
| T|-------G | +1 T | G* | G | −7 | No | Yes | No | |
| T|-------G | +1 T | G* | G | −7 | No | Yes | No | |
| T|-------G | +1 T | G* | G | −7 | No | Yes | No | |
| T|-------G | +1 T | G* | G | −7 | No | Yes | No | |
| +1 Y primer duplex v2 & 3′ddCRN 5 UMI adapter template | T|GTTTTCTAGATCGG | +1 T | No | G | 0 | No | Yes | Yes |
| C|GCGGCCCAGATCGG | +1 C | No | G | 0 | No | Yes | Yes | |
| T|--AGCTTAGATCGG | +1 T | A | A | −2 | Yes | Yes | Yes | |
| T|--AACTTAGATCGG | +1 T | A | A | −2 | Yes | Yes | Yes | |
| C|--GGCTAAGATCGG | +1 C | G | G | −2 | Yes | Yes | Yes | |
| T|--GGGCTAGATCGG | +1 T | A | G | −2 | Yes | Yes | Yes | |
| C|--GGTACAGATCGG | +1 C | G | G | −2 | Yes | Yes | Yes | |
| T|--GTCATAGATCGG | +1 T | A | G | −2 | Yes | Yes | Yes | |
| T|--GCGTAAGATCGG | +1 T | A | G | −2 | Yes | Yes | Yes | |
| T|--GAGAAAGATCGG | +1 T | A | G | −2 | Yes | Yes | Yes | |
| T|--GGGTTAGATCGG | +1 T | A | G | −2 | Yes | Yes | Yes | |
| T|---GGTTAGATCGG | +1T | N | G | −3 | Yes | Yes | Yes |
Notes: In sequences of Read2:cRead1 junction column, the “-“ symbol was used to emphasize sequence that was excluded from the junction. In the primer duplex paired column, the “*” symbol was used to emphasize that a mismatch was observed. Column seven provides defines whether the adapter dimer was dependent on a correct base-pair between the primer duplex +1 nucleotide and the 3′ nucleotide of adapter template. Column eight defines whether the adapter dimer was dependent on a NTA to the +1 primer duplex to template jump to the adapter template. Column nine defines whether the adapter-dimer cDNA could be sequenced on an Illumina instrument, as assessed by the presence of a complete cRead1 primer sequence.