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Old 02-23-2016, 01:31 PM   #1
amango
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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 01:33 PM. Reason: clarity
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Old 02-24-2016, 11:10 AM   #2
thermophile
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I use the Kozich 2013 set up so my index primer is the reverse compliment of my R2 sequencing primer
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