Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Why is a seventh base needed with MiSeq 6-base indices?

    The MiSeq sample prep guide indicates that a seventh base is needed after the 6th base. What is the reason for that?


  • #2
    Possibly because with the Illumina technology the accuracy of the base calling for any particular cycle (having to do with things like phasing/prephasing) depends partly on the next cycle, so I guess if you want to have 6-bases for the index read, you would actually do 7 cycles.

    Comment


    • #3
      @mastal is correct. Since phasing information can't be determined for the last base it is not used during (n-1 rule) downstream processing (demultiplexing).

      Comment


      • #4
        Originally posted by GenoMax View Post
        @mastal is correct. Since phasing information can't be determined for the last base it is not used during (n-1 rule) downstream processing (demultiplexing).
        This is true for the HiSeq, but the MiSeq will determine how many Index cycles to sequence based on the index length recorded in the sample sheet, without adding the the extra cycle. For standard TruSeq indexes (6bp) the MiSeq will only collect 6 cycles of index sequence and use all of them for demultiplexing. There is no way to tell the MiSeq to run and extra index cycle but ignore the last base when demultiplexing.

        Comment


        • #5
          @kmcarr: You are right.

          I tend to forget that we never use the on-board analysis on our MiSeq's. As a result we need to consider the n-1 rule while processing data externally.

          Comment

          Latest Articles

          Collapse

          • seqadmin
            Current Approaches to Protein Sequencing
            by seqadmin


            Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...
            04-04-2024, 04:25 PM
          • 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

          ad_right_rmr

          Collapse

          News

          Collapse

          Topics Statistics Last Post
          Started by seqadmin, 04-11-2024, 12:08 PM
          0 responses
          17 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 04-10-2024, 10:19 PM
          0 responses
          22 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 04-10-2024, 09:21 AM
          0 responses
          16 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 04-04-2024, 09:00 AM
          0 responses
          46 views
          0 likes
          Last Post seqadmin  
          Working...
          X