P1 and A adater trimming and mapping

Sergio.pv · 11-19-2013, 02:04 AM

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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11-19-2013, 02:04 AM	#1
Sergio.pv Member Location: Berlin Join Date: Jul 2013 Posts: 20	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

11-25-2013, 09:29 AM	#3
jonathanjacobs Member Location: Rockville, MD Join Date: Apr 2011 Posts: 23	doing a second round of both 5' and 3' trimming of P1 and A adapters (almost always) improves read mapping for Ion data (IME). __________________ @bioinformer http://www.linkedin.com/in/jonathanjacobs

11-25-2013, 06:02 AM	#2
trexbob Member Location: Atlanta, GA Join Date: Feb 2013 Posts: 17	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.