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-   -   P1 and A adater trimming and mapping (http://seqanswers.com/forums/showthread.php?t=35845)

Sergio.pv 11-19-2013 02:04 AM

P1 and A adater trimming and mapping
 
Hi eveyone,
i have a few very basic questions that are buging me:
- The emulsion PCR performed to create a template results in amplicons attached to a bed, which in turn goes to the pico-well and sequencing is performed afterwards. Such amplicons habe a forward strand attached to the bead and a reverse strand "free" that detaches after denaturation, leaving only the forward (5-3) starnd as the sequencing template, with he P1 adapter at 5' and the A adapter at 3'. Correct?

- Sequencing will ocurr after a primer anneals to the 3' end of the template. That region corresponds to the A adapter and the sequence occurs towards the bead. As a result, bases are called from the primer annealed to the A adapter and will finish in the last nucleotide of the P1 adapter (on the bead). Is this correct?

- Adapter trimming therefore should be performed at the 5' end (A adapter) and at the 3' end (P1 adapter)?

- Why is always talked about trimming only the 3' end?


I know is quite a long post, however i am new at NGS.

Thanks in advance,
Sergio

trexbob 11-25-2013 06:02 AM

Hi Sergio,
I'm also relatively new at this also, but... I can help somewhat. The Ion Torrent software usually trims off the 5' adapter, so unless you have a primer, you generally don't have to do 5' trimming. I've found however that there are usually base pairing issues in the first 10 bases or so, similar to thread http://seqanswers.com/forums/showthread.php?t=17521 . You should use FastQC or a similar program to analyze the quality of the sequencing and trim accordingly. You can use fastxtoolkit to trim Ion Torrent data, just be sure to include the parameter -Q 33 at the end to account for the quality score issue.

jonathanjacobs 11-25-2013 09:29 AM

doing a second round of both 5' and 3' trimming of P1 and A adapters (almost always) improves read mapping for Ion data (IME).


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