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
        Recent Advances in Sequencing Analysis Tools
        by seqadmin


        The sequencing world is rapidly changing due to declining costs, enhanced accuracies, and the advent of newer, cutting-edge instruments. Equally important to these developments are improvements in sequencing analysis, a process that converts vast amounts of raw data into a comprehensible and meaningful form. This complex task requires expertise and the right analysis tools. In this article, we highlight the progress and innovation in sequencing analysis by reviewing several of the...
        Today, 07:48 AM
      • 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

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by seqadmin, Today, 07:17 AM
      0 responses
      11 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 05-02-2024, 08:06 AM
      0 responses
      19 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 04-30-2024, 12:17 PM
      0 responses
      20 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 04-29-2024, 10:49 AM
      0 responses
      28 views
      0 likes
      Last Post seqadmin  
      Working...
      X