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

Similar Threads
Thread Thread Starter Forum Replies Last Post
MiSeq Custom Sequencing Primer Tm? pmiguel Illumina/Solexa 43 11-12-2015 11:52 AM
Miseq Custom primer for 16s V1V3 region lmma Illumina/Solexa 12 02-26-2015 01:13 PM
Using MiSeq with both standard and custom sequencing/index primers at the same time? kevin_bryant Illumina/Solexa 1 02-27-2014 12:57 PM
Validation of Custom Sequencing Primer Akira Illumina/Solexa 1 11-05-2012 09:22 AM
custom sequencing primers athos Illumina/Solexa 5 02-04-2011 10:47 AM

Thread Tools
Old 02-23-2016, 12:31 PM   #1
Location: New York

Join Date: Dec 2009
Posts: 17
Default Index sequencing primer for use with custom primers (16S metagenomic)

I'm trying to determine the correct index sequencing primer to use with a set of custom primers designed to amplify a locus (16S rDNA) while also adding adapters (for Illumina Miseq) and index sequences. The primers are constructed as shown below and have the index on the forward primer.

I'm trying to fully convince myself that primer 2 is correct, not primer 1. Primer 2 makes sense because replication would start there and head in the 3' to 5' direction to the index. Also, I know of index sequences for other primers with indexes on the reverse primer that are oriented in the corresponding location as primer 2, but on the reverse primer. But I've seen protocols out there, for use with these exact primers, that call for primer 1. And the sequence for primer 1 matches Illumina's published nextera read 2 index primer (below, from p10 of this Illumina manual).

Can anyone weigh in?

Last edited by amango; 02-23-2016 at 12:33 PM. Reason: clarity
amango is offline   Reply With Quote
Old 02-24-2016, 10:10 AM   #2
Senior Member
Location: CT

Join Date: Apr 2015
Posts: 243

I use the Kozich 2013 set up so my index primer is the reverse compliment of my R2 sequencing primer
thermophile 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 10:12 PM.

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