In designing reverse primer for amplicon Ion Torrent, should I use B adapter (CCTATCCCCTGTGTGCCTTGGCAGTCTCAG) or P1 adapter (CCTCTCTATGGGCAGTCGGTGAT)? I have two Ion Torrent application notes, both dated 2011, and they have conflicting information. Also, the B-adapter design contains the key sequence TCAG, but P1 does not. Is that right that when I use the P1 adapter, the B adapter and key are added in e-PCR?
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Originally posted by Retro View PostIn designing reverse primer for amplicon Ion Torrent, should I use B adapter (CCTATCCCCTGTGTGCCTTGGCAGTCTCAG) or P1 adapter (CCTCTCTATGGGCAGTCGGTGAT)? I have two Ion Torrent application notes, both dated 2011, and they have conflicting information. Also, the B-adapter design contains the key sequence TCAG, but P1 does not. Is that right that when I use the P1 adapter, the B adapter and key are added in e-PCR?
This strategy employs A and P1-tagged fusion primers.
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The version with P1 reverse primer is here:
The version with B reverse primer:
The version with P1 seems more recent, so probably correct. However, I guess the B and key will be added in the e-PCR. That will only make the 3' end adapter end longer by the 23 bp of the P1 sequence. This will further limit the size for the specific insert in the already limited range in Ion Torrent.
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Originally posted by Retro View PostOK, just to answer to myself, even when P1 primer is used, the primer B and "TCAG key sequence" are present at the ends of the reads. They must be present on the beads.
P1 and A are the current sequences.
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To SeAA:
You are right, P1 and A are the current sequences. Previous version of Ion Torrent amplicon method had primers A and B. Since my original question , we performed the sequencing with A-P1 amplicons. In reads that are long enough, the 3' end contains the P1 primer, followed by the TCAG key sequence and B primer sequence. That probably means that the beads contain B sequence with the TCAG key. Notice that the new version of the Ion Torrent protocol does not have the key sequence in the P1 primer, it did have it in the previous version with the B primer. For some reason they decided to switch the way the 3' end is amplified.
It is also interesting that all these primers are from other NGS technologies, A and B is from 454 and P1 from some other, not sure now. Probably to make it easier for users to switch to Ion Torrent, which is what we did from 454.
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