Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa

Similar Threads
Thread Thread Starter Forum Replies Last Post
Dual indexing construct for MiSeq - where to put the barcode for index2? Vinz Illumina/Solexa 50 12-12-2014 02:19 PM
Fungal ITS amplicon sequencing (NexteraXT vs TruSeq Custom Amplicon) chapadosj Metagenomics 0 06-07-2013 07:59 AM
custom amplicon sequencing spacesonics Illumina/Solexa 0 08-13-2012 11:54 AM
96 or 384 Nextera-style dual indexing pmiguel Illumina/Solexa 3 06-27-2012 09:49 AM
Custom dual index adapters...dark cycles unnecessary? koadman Illumina/Solexa 13 04-23-2012 11:10 PM

Thread Tools
Old 06-26-2013, 03:33 PM   #1
Junior Member
Location: California

Join Date: Feb 2013
Posts: 3
Default 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:
5' Adaptor-Index-Primer Pad-Linker-16S Primer Sequence-3'
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
tickseq is offline   Reply With Quote
Old 06-26-2013, 05:24 PM   #2
Senior Member
Location: Australia

Join Date: May 2009
Posts: 155

For a dual index approach, have a look at:
RCJK is offline   Reply With Quote
Old 06-27-2013, 09:46 AM   #3
Junior Member
Location: California

Join Date: Feb 2013
Posts: 3
Default 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:
tickseq is offline   Reply With Quote

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off

All times are GMT -8. The time now is 06:54 AM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO