short reads at 5' strand (ampliseq)

[maoyanjun]

Hi,
these days I run some ampliseq libraries(prep by ampliseq custom panel) on PGM, and found that there were some short reads(it seems that they were trimmed from 5'strand) ,I don't know why? Are they from unspecific PCR products?

[IonTom]

The reads are actually much longer. If you open your aligned BAM/SAM in IGV and make soft clipping visible you should see it. Sometimes you even see things like 235S17M.

In a recent paper they showed that for randomly generated reads with 300 to 400 base pairs
TMAP was still aligning like 15% of these.

http://www.biomedcentral.com/1471-2164/15/264/

If TMAP finds subsequences that align to the reference (like 17bp of 300) it will soft clip from the 5' end and report the remaining part of the read.

I don't know if is this is a bug or on purpose, but it kind of made me stop using TMAP.

[snetmcom]

The reads are actually much longer. If you open your aligned BAM/SAM in IGV and make soft clipping visible you should see it. Sometimes you even see things like 235S17M.

In a recent paper they showed that for randomly generated reads with 300 to 400 base pairs
TMAP was still aligning like 15% of these.

http://www.biomedcentral.com/1471-2164/15/264/

If TMAP finds subsequences that align to the reference (like 17bp of 300) it will soft clip from the 5' end and report the remaining part of the read.

I don't know if is this is a bug or on purpose, but it kind of made me stop using TMAP.

These are short PCR products, so I doubt it's from alignment. He is likely seeing these in the histogram from the report.

There are no settings for 5' trimming. I'm guessing it's primer dimer or he means 3' trimming.

[IonTom]

These are short PCR products, so I doubt it's from alignment. He is likely seeing these in the histogram from the report.

There are no settings for 5' trimming. I'm guessing it's primer dimer or he means 3' trimming.

It should also be noted that Ion Torrent produces a relatively large range of different
read lenghts i.e 30-330.