Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • 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,

  • #2
    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, 12:09 PM.

    Comment

    Latest Articles

    Collapse

    • seqadmin
      Current Approaches to Protein Sequencing
      by seqadmin


      Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...
      04-04-2024, 04:25 PM
    • seqadmin
      Strategies for Sequencing Challenging Samples
      by seqadmin


      Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
      03-22-2024, 06:39 AM

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, 04-11-2024, 12:08 PM
    0 responses
    25 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-10-2024, 10:19 PM
    0 responses
    27 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-10-2024, 09:21 AM
    0 responses
    24 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 04-04-2024, 09:00 AM
    0 responses
    52 views
    0 likes
    Last Post seqadmin  
    Working...
    X