Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • amplicons

    Hi users,
    doese anyone share our problem with short reads (>80%) during amplicon (with shotgun adapter) sequ and did you find a reason for that? We use highly purified amplicons, so no dimers or sth.
    Thanks a lot. Best!

  • #2
    Are you using XLR70 or XL+ chemistry?

    Comment


    • #3
      Originally posted by klymiuk View Post
      Hi users,
      doese anyone share our problem with short reads (>80%) during amplicon (with shotgun adapter) sequ and did you find a reason for that? We use highly purified amplicons, so no dimers or sth.
      Thanks a lot. Best!
      What evidence do you have that your amplicons are contain no dimers? Very few methods would remove dimer strands that anneal back to the adapters of larger library strands.

      What are the length of your amplicons?

      --
      Phillip

      Comment


      • #4
        What signal processing pipeline did you use? Shotgun or amplicon?

        Comment


        • #5
          Hi everybody,
          thanks a lot for your questions and comments:
          - we are using XLR70 Titanium chemistry
          - our amplicons range from 400 - 500 bp, we purify them with gel cut or by HPLC
          - we are using the amplicon processing pipeline
          thanks a lot for any hint!!
          best.

          Comment


          • #6
            Do you have a bioanalyzer trace of the amplicons, to help show that there were no small fragments present? They recommend running on a DNA 1000 chip, but lower levels of primers/dimers will show up better on a HS DNA chip.

            Comment


            • #7
              Yes, I see a lot of cases of this type on this forum where it is claimed "there are no small fragments present" and then when they post the bioanalyzer images there are are small fragments present! They are small peaks -- but there is the issue of molarity vs mass here. Even weakly fluorescing low molecular weight peaks may have substantial molar concentration.

              --
              Phillip

              Comment


              • #8
                I don't buy the tired excuse of small fragment problems leading to bad amplicon sequencing. No matter how many times we SPRI purify and no matter how clean our traces look, we have had many months of highly inconsistent and discordant amplicon results, ALL with amplicons and samples that we have previously been able to sequence perfectly.

                Why the change? At the risk of being arrogant, I know it is not on us: our staff have not changed. We have had engineers out to look at our machine, we have wasted > $20k in failed reagents. I believe there are production problems with some of the reagents, probably the emulsion oil and maybe the emPCR reagents, that are causing failures.

                The lack of QC on the kits we are sent is pushing us away from 454. Unfortunately, switching to Illumina may not be much better if Roche takes them over and they start having the same R&D and production problems.

                Comment


                • #9
                  Thats an example of our BA....I have a little bit the assumption of a Roche problem too...but would be very happy for any other hint to solve our problem. What kind of cleanup do you use? What kind of adaptors?....does it work?
                  best.
                  Attached Files

                  Comment


                  • #10
                    And to address the small fragments real quick – we were told that we had them over and over again, until an FAS sat down and looked at the runs with us. What tech support was seeing were reads that were filtered out and included in the short quality category. If I remember which filters are included in this, it’s the Signal Intensity Filter, Valley Filter, and Q-score Trimming Filter. So it seems that a lot more than just real, short, products are included in the short quality filter, which was misleading to us for a long time….

                    Comment


                    • #11
                      A big, big issue for me is that there has been construction going on directly above and/or below the room with the 454 and BioAnalyzer for the entire time we've had it. I can't really rely on the BioAnalyzer results at all, which is why I run every sample in duplicate, at least.

                      Comment

                      Latest Articles

                      Collapse

                      • seqadmin
                        Exploring the Dynamics of the Tumor Microenvironment
                        by seqadmin




                        The complexity of cancer is clearly demonstrated in the diverse ecosystem of the tumor microenvironment (TME). The TME is made up of numerous cell types and its development begins with the changes that happen during oncogenesis. “Genomic mutations, copy number changes, epigenetic alterations, and alternative gene expression occur to varying degrees within the affected tumor cells,” explained Andrea O’Hara, Ph.D., Strategic Technical Specialist at Azenta. “As...
                        07-08-2024, 03:19 PM
                      • seqadmin
                        Exploring Human Diversity Through Large-Scale Omics
                        by seqadmin


                        In 2003, researchers from the Human Genome Project (HGP) announced the most comprehensive genome to date1. Although the genome wasn’t fully completed until nearly 20 years later2, numerous large-scale projects, such as the International HapMap Project and 1000 Genomes Project, continued the HGP's work, capturing extensive variation and genomic diversity within humans. Recently, newer initiatives have significantly increased in scale and expanded beyond genomics, offering a more detailed...
                        06-25-2024, 06:43 AM

                      ad_right_rmr

                      Collapse

                      News

                      Collapse

                      Topics Statistics Last Post
                      Started by seqadmin, Today, 11:09 AM
                      0 responses
                      16 views
                      0 likes
                      Last Post seqadmin  
                      Started by seqadmin, 07-19-2024, 07:20 AM
                      0 responses
                      148 views
                      0 likes
                      Last Post seqadmin  
                      Started by seqadmin, 07-16-2024, 05:49 AM
                      0 responses
                      124 views
                      0 likes
                      Last Post seqadmin  
                      Started by seqadmin, 07-15-2024, 06:53 AM
                      0 responses
                      111 views
                      0 likes
                      Last Post seqadmin  
                      Working...
                      X