Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Spiking phiX into multiplexed libraries as well as barcode sequences

    Hi all, I have a two part question:

    I am wondering if anyone here has tried spiking phiX into their multiplexed library samples? I heard from a couple people (as well as an Illumina rep) that this is a good way to ensure good base distribution to help with the cluster separation and identification that occurs for the 1st two bases (1st four if you have the new pipeline.

    I am also wondering if anyone is multiplexing on a regular basis and has a good method for designing their barcodes? I have read all the prior posts on barcoding but the Cronn and Craig papers are almost 2 years old so I just thought I'd see if people were doing anything else to ensure good base distribution as well as avoiding any issues that might occur with mismatches.

    To give you a little background we're looking to multiplex Chip-Seq samples, about 10 samples per multiplexed lane.

    Thanks!
    Last edited by cmawhinney; 04-07-2010, 08:23 AM.

  • #2
    Hi all,

    we are about to order 12 self made Illumina adapters with barcodes inserted at the position used by Craig et al., 2008. Here are my criteria for choosing the barcodes:

    • 5 nucleotides long
    • all 4 different nucleotides should be included
    • indices differ at 3 or more positions
    • no significant change in secondary structure of original oligos by index introduction

    With such restrictions I could find a set of 12 indices but figured out that one needs to use 6 bp if one needs much more than 12. I used the sequences for the paired end adapters since they could be used for single read and paired end read.

    We do ChIP-Seq as well and since we work with bacteria multiplexing is a nice option to reduce the cost.

    Comment


    • #3
      Originally posted by superduper
      Hi clpX,
      Could you share the protocol you used for ChIP-library preparation? I am have some trouble with my ChIP-libraries. The main failure in my prep is that I do not get amplification after size selection. I could actually see DNA on the gel when I cut out the 180-300 bp slice. However, after PCR all I got was just primer dimer that migrates way below 100 bp.
      Thanks!
      I would double check your primers compared to what you are putting on as adapters. If you have material, but you cannot amplify it...there is a simple reason.

      1. Primers do not match/hybridize to target DNA...why this is happening is the game you will have to play. Either primers or adapters. Do you run a positive control for your PCR reactions? You should. Could be your Master Mix too.

      Comment


      • #4
        Hi cmawhinney,

        according to our experience you are only likely to loose (up to a quite substantial amount of) clusters if you are using a low number of multi-plex tags. We also performed a cluster density - low complexity sequence simulation program which supports our empirical findings that if you have 4 or more different sequence tags at the start of your sequences you should not run into any problems. Using 10 different barcodes should definitely provide enough variability in the start, thus spiking in a PhiX sample is not necessary.

        If you have any further questions about initial biased sequences feel free to send me an email, by now we have done quite a few rescues of runs which failed due to this reason.

        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
        51 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