Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Problems of assembly polishing with RS_Resequencing.1 protocol

    Hi all,

    I’m experiencing a difficulty in getting a complete bacterial genome from PacBio sequencing data using the SMRT Analysis protocols.
    My usual workflow is as follows:
    De novo assembly by HGAP2 ->Circularization by circlator or minimus -> Polishing by RS_Resequencing.1 protocol until no variants are detected.
    This workflow has worked nicely. Usually, repeating RS_Resequencing several times resulted in 100.0% of Consensus Concordance and no variants.

    But, for a new bacterial genome we sequenced recently, the result of RS_Resequencing is different from usual. During repeating the RS_Resequencing 8 times, variants have been detected continuously at 10-20 positions and Consensus Concordance never reached 100.0%. At some positions, 1-bp insertion occurred repeatedly per each resequencing.


    In my opinion, it seems that more repetition of RS_Resequencing would not be helpful.
    What should I do to solve this problem?
    Does anyone have a similar experience?

    Thanks in advance!

  • #2
    Hi dladuswj,

    It seems likely that the bacterial genome that you are sequencing contains more/longer homopolymer/repetitive regions than the typical genomes you are sequencing, which could result in further iterations of the Resequencing algorithm "failing to converge" on a 100% consensus.

    You should investigate the alignments at the particular areas where the variants are being called to determine what is causing the variant calling to differ from round to round.

    Comment


    • #3
      It is possible that the data is from a population that is not perfectly clonal. This happens frequently in slow growing organisms, or things that mutate rapidly.

      Comment

      Latest Articles

      Collapse

      • 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
      • seqadmin
        Techniques and Challenges in Conservation Genomics
        by seqadmin



        The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

        Avian Conservation
        Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
        03-08-2024, 10:41 AM

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by seqadmin, Yesterday, 06:37 PM
      0 responses
      8 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, Yesterday, 06:07 PM
      0 responses
      8 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-22-2024, 10:03 AM
      0 responses
      49 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-21-2024, 07:32 AM
      0 responses
      66 views
      0 likes
      Last Post seqadmin  
      Working...
      X