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Old 02-26-2014, 12:16 AM   #1
guntelo0545
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Location: Seoul, Korea

Join Date: Feb 2014
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Default Amplicon sequencing & clustering using Pacbio

Hi there,
It is my first post and hope anyone would help me.
I have PCR amplicon which was amplified using mixture of templates with different SNPs. (It is an analysis that is very similar to the phage display analysis using NGS.)
Since it is 1.7kb long, I used Pacbio for sequencing.
Basically I want to get final results with;
1. the clusters of reads which have same SNPs
2. the ratio between the clusters

I used resequencing protocol in BLSR and found just one variant and with rare variant tool, got 3 variants but couldn't get any cluster information and ratio. (I guess I used wrong tools.)
Now I am looking for a tool to get the right answer.
Currently, using RS_Resequencing_ReadsofInsert.1, I got 7,372 ccs reads which have more than 9 pass. Using those, I will try some clustering tools. (but I am not sure I can make it.)
If anyone has experience on this kind of work, please help me out.
Thanks in advance,
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Old 02-26-2014, 07:36 AM   #2
rhall
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Location: San Francisco

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Posts: 322
Default

I'm not an expect on this type of analysis, but given you have PacBio's SMRT Analysis installed, have you tried running the RS_LongAmpliconAnalysis workflow? This clusters and phases amplicons. I believe it is currently in Beta, unfortunately there is not much documentation.
Edit:
It has been pointed out to me, offline, that the LongAmplicon analysis will probably not give the correct results if there are not enough differences (SNPs) between the amplicons. Although it may still be worth a try.
A straight clustering from a multiple alignment of the high pass CCS reads might be the easiest approach.

Last edited by rhall; 02-26-2014 at 11:09 AM.
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