Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • V4-V5 Region Using 2x250 - Enough Overlap?

    Hi All,

    We've been looking at the V4 region (515/806) in environmental samples for some time successfully. We'd like to move to the V4-V5 region using the 515/926 primers. That would result in a larger amplicon (~411 not including primer/barcode sequences).

    Our sequencing provider runs 2x250 regularly but 2x300 only sporadically so the cost would be significantly more unless we have lots of samples. Does anybody have experience using 2x250 with this region? Will there be enough overlap of the paired reads to assemble?

    Thanks for any insight!

  • #2
    I think the critical factor is that at a cluster density where you produce the most sequence (even the most Q30 bases), the quality of single-plex amplicon runs deteriorates badly over the last 50 bases of a 250 cycle read. Whereas if you keep the density much lower--such that you may only generate 10 million or fewer clusters--the quality can be high all the way to the ends of the reads.

    At these lower cluster densities we see pair merging occurring successfully at rate approaching 90% even with 450bp inserts.

    600 cycle cassette? I don't know, is anyone getting reasonable 300 base reads with those?

    --
    Phillip

    Comment


    • #3
      Sorry for the late reply. Thanks! This helps a lot. We're discussing with our service provider and will be doing some test runs to evaluate the data.

      Comment


      • #4
        I wouldn't move to the longer region on the 2x250 kit. Allegedly the 2x300 is better in the past ~year but I haven't tested it out so continue offering amplicons that are <300bp long.
        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
        10 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, Yesterday, 06:07 PM
        0 responses
        9 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
        67 views
        0 likes
        Last Post seqadmin  
        Working...
        X