amplicon read lengths

SeqNerd · 06-06-2011, 10:14 AM

I realize this is a familiar theme, but I thought I would chime in that our lab has tried (so far unsuccessfully) to do some amplicon sequencing on 100 bp libraries. The problems we've seen tend to fall into two categories:

1. Amplicon inserts shorter than expected after emPCR. Perhaps an issue where even small amounts of shorter contaminants can carry over from the earlier library-generating PCR, and are then preferentially amplified during emPCR. That there would be some bias isn't terribly surprising -- but we were surprised that what appeared to be good libraries by gel and/or bioanalyzer still showed this effect strongly.

2. Read lengths less than expected, given number of cycles. Even in cases where the amplicon length appears to be the correct length (that is, not surprisingly short or truncated), read lengths produced even by doing 120 cycles doesn't seem to be enough to produce full-length 100 bp reads.

Curious to hear other people's experiences here in tinkering with amplicons of various size and trying more or less # of cycles.

krobison · 06-06-2011, 11:54 AM

My (mis)adventure with 150-210bp amplicons & 130 cycles are documented online

http://omicsomics.blogspot.com/2011/...crecy-tax.html

Our problem wasn't read length, just a shortage of reads. We did clean up the input using the LabChip XT device (a demo) & saw no primer-dimer reads. The unfiltered SFF yields tiny number of very short reads, but not with the structure to suggest primer dimers.

In an earlier 454 amplicon experiment (so, different system but very analogous) we saw extremely favorable amplification of primer dimers.

For the reads you got, can you post a graph of quality-vs-position ala
http://omicsomics.blogspot.com/2011/...is-pretty.html

(for a simpler curve, you might plot the dominant quality value vs. position).

Anne zhao · 06-08-2011, 11:50 PM

Hi krobison, I can't open your link, but I'm very curious about your result! We ever tried 170bp insert, also short of reads (~1M/run) with low live ISPs rate and very high poor signal rate. We found ~1/10 short reads (50-80bp) in a run. We are now analyzing trimmed reads so see if it's related.

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06-06-2011, 10:14 AM	#1
SeqNerd Junior Member Location: North America Join Date: Jun 2011 Posts: 6	amplicon read lengths I realize this is a familiar theme, but I thought I would chime in that our lab has tried (so far unsuccessfully) to do some amplicon sequencing on 100 bp libraries. The problems we've seen tend to fall into two categories: 1. Amplicon inserts shorter than expected after emPCR. Perhaps an issue where even small amounts of shorter contaminants can carry over from the earlier library-generating PCR, and are then preferentially amplified during emPCR. That there would be some bias isn't terribly surprising -- but we were surprised that what appeared to be good libraries by gel and/or bioanalyzer still showed this effect strongly. 2. Read lengths less than expected, given number of cycles. Even in cases where the amplicon length appears to be the correct length (that is, not surprisingly short or truncated), read lengths produced even by doing 120 cycles doesn't seem to be enough to produce full-length 100 bp reads. Curious to hear other people's experiences here in tinkering with amplicons of various size and trying more or less # of cycles.

06-06-2011, 11:54 AM	#2
krobison Senior Member Location: Boston area Join Date: Nov 2007 Posts: 747	My (mis)adventure with 150-210bp amplicons & 130 cycles are documented online http://omicsomics.blogspot.com/2011/...crecy-tax.html Our problem wasn't read length, just a shortage of reads. We did clean up the input using the LabChip XT device (a demo) & saw no primer-dimer reads. The unfiltered SFF yields tiny number of very short reads, but not with the structure to suggest primer dimers. In an earlier 454 amplicon experiment (so, different system but very analogous) we saw extremely favorable amplification of primer dimers. For the reads you got, can you post a graph of quality-vs-position ala http://omicsomics.blogspot.com/2011/...is-pretty.html (for a simpler curve, you might plot the dominant quality value vs. position).

06-08-2011, 11:50 PM	#3
Anne zhao Junior Member Location: china Join Date: Feb 2011 Posts: 5	Hi krobison, I can't open your link, but I'm very curious about your result! We ever tried 170bp insert, also short of reads (~1M/run) with low live ISPs rate and very high poor signal rate. We found ~1/10 short reads (50-80bp) in a run. We are now analyzing trimmed reads so see if it's related.