Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Custom amplicon sequencing with dual indexing

    I am trying to use the Caporaso protocol for 16S amplicon sequencing but want to modify it to have dual indexing. I am designing the PCR primers to have the following format:
    Forward:
    5' Adaptor-Index-Primer Pad-Linker-16S Primer Sequence-3'
    Reverse:
    5'-RC Adaptor (P7)-Index-Primer Pad-Linker-16S Reverse sequence-3'

    I would like to just use the Nextera or Truseq barcode sequences but when I spoke to the Illumina tech they said that the index sequences posted are the sequences as read by the MiSeq. I believe this means that the Index 1 (p7 side) should be reverse-complemented on my custom primer. But I'm not sure is the second Index (P5 side) also needs to be reverse complemented. Does anyone have experience with custom primers and how to insert the barcode sequence?

    Also, I am looking at a different region V4 through V5 so my reverse primer sequence is different. I am trying to increase the Tm to be as close to 65 or 70C as possible. I have it up to 63 with a new primer pad. Is this sufficient?

    Thanks for any help on these matters

  • #2
    For a dual index approach, have a look at:

    Comment


    • #3
      custom amplicon dual indexing

      Thanks for this link! It is just what I was looking for and is very helpful.

      The reverse v5 sequence has a lower melting temperature than the reverse v4 so I need to modify the pad to increase the Tm. Can I just change some of the A's and T's to G's and C's in a non complementary way to the target group? Does the pad sequence need to be anti complementary?


      Originally posted by RCJK View Post
      For a dual index approach, have a look at:
      http://www.mothur.org/w/images/0/0c/..._MiSeq_SOP.pdf

      Comment

      Latest Articles

      Collapse

      • seqadmin
        Essential Discoveries and Tools in Epitranscriptomics
        by seqadmin




        The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist...
        04-22-2024, 07:01 AM
      • 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

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by seqadmin, Today, 08:47 AM
      0 responses
      10 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 04-11-2024, 12:08 PM
      0 responses
      60 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 04-10-2024, 10:19 PM
      0 responses
      57 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 04-10-2024, 09:21 AM
      0 responses
      53 views
      0 likes
      Last Post seqadmin  
      Working...
      X