Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • amplicon runs using bcl2fastq v217

    Has anyone noticed issues with quality scores on amplicon runs since the new version of bcl2fastq came out? I've had a number of runs be questionable since late Oct, but I also changed machines at the same time. Trying to figure out if I need to adjust wetlab conditions to new machine or if it's related to the software update.
    Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

  • #2
    bcl2fastq v.2.x uses binned Q-scores. I assume you are referring to that here as "issues"? Have you seen this paper?

    @Brian has a way to re-calibrate those using BBMap. There should be a post on here that describes how to do that.

    Comment


    • #3
      The beginning of R2 qscores are really low. This last run, R1 looks great with ~10% phiX aligned. R2 is horrible, <1% phiX aligned. I'm not sure how this would be software related but the thumbnails look fine-the machine didn't lose focus, clusters are there.

      oh hadn't seen that paper, thanks. Haven't used bbmap but know it's on one of our clusters
      Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

      Comment


      • #4
        The qscore profiles look similar to what I was seeing at the beginning of the year. But the machine that I'm currently using is the one that actually lead us to blame the TCM of my old machine
        Bridged amplification & clustering followed by sequencing by synthesis. (Genome Analyzer / HiSeq / MiSeq)
        Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

        Comment


        • #5
          Originally posted by thermophile View Post
          Has anyone noticed issues with quality scores on amplicon runs since the new version of bcl2fastq came out? I've had a number of runs be questionable since late Oct, but I also changed machines at the same time. Trying to figure out if I need to adjust wetlab conditions to new machine or if it's related to the software update.
          Are you sure the issue is with bcl2fastq? QScores are assigned (binned or not) by RTA. bcl2fastq merely extracts the scores already stored in the BCL file by RTA.

          Comment


          • #6
            not sure of anything. so maybe rerunning older version of bcl2fastq isn't going to change anything?
            Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

            Comment


            • #7
              talked to tech support, bcl2fastq is very unlikely to be the issue.
              Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

              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
              7 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, Yesterday, 06:07 PM
              0 responses
              7 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