Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Illumina paired end poor quality in reverse reads

    We recently received paired end results sequenced on the HiSeq 2500, and the forward reads are good but the reverse reads are of extremely low quality (.1% pass generous quality filtering thresholds).

    I've attached the FastQC results for the forward (R1) and reverse (R2) reads (let me know if you can't access them). My question is- has anyone seen this issue before? I think it is strange that the R1 reads are so good and the R2 reads are so bad. What would you attribute this to? Overclustering on the flowcell? Something wrong during library prep?

    Thanks in advance!
    Attached Files

  • #2
    Let us start with over clustering hypothesis first. Read 1 looks good.

    Can you ask the facility that generated this data if something happened to read 2 for flowcell your sample was on? Ask them for the cluster concentration for the flowcell too. Ideally if something went bad with read 2 they should not have released this data.

    Comment


    • #3
      Thanks for your response- I was thinking that too, that I will just need to contact the facility.

      Comment


      • #4
        While you are waiting for their answer go ahead and do some analysis to ensure that things otherwise look good (i.e. you get expected alignments to the right genome etc).

        Comment


        • #5
          You can also ask them for some library statistics... concentration of the final library and fragment size range (usually Bioanalyzer/Tapestation results). Sometimes you will see good first-read results and poor second read results from libraries whose concentrations are quite low and/or fragment sizes are too large (i.e. >1kb for the HiSeq).

          Comment


          • #6
            I have not seen FastQC output as bad as this one. There are four possible causes to investigate:

            1- Over-clustering as has been suggested in previous posts
            2- Degraded Read2 sequencing primer
            3- Low diversity in 3' end of library fragments introduced by library prep method
            4- Issues with sequencing machine or reagents

            Comment


            • #7
              I've seen this several times. In my experience it's almost certainly due to overclustering so you should ask the staff. The reads will look fine in R1, but after the turnaround step the quality gets really bad presumably because the clusters do grow a little bit with the second round of bridge amp.

              Comment


              • #8
                I've seen this with overclustering on MiSeq runs. My understanding is that it's a combination of resolution between clusters on R2 and depletion of sequencing reagents.

                Have you received your cluster density from the sequencing facility?

                Comment


                • #9
                  I heard of something like this for a custom amplicon project using non-standard adapters/primers. What kind of library is this?
                  Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.com

                  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