16S V3-V4 Multiplexing >24 samples

[sweetph3]

I have a colleague who wants to amplify 16S V3-V4 and then have me multiplex and run on MiSeq. She at this point has 26 samples, but using Illumina's protocol for indexing with Nextera index primers, I can only do 24 samples. We don't want to spend close to $1000 for the 96-sample index set since this is not something that we typically do, and the index primers would likely not get used again. Are there other options for multiplexing >24 samples? We have TruSeq LT single index primers, but again that's only 24 using sets A and B.

Thanks for any suggestions!

[MU Core]

You may consider a 12 base index as described in the article below. Primers designed and ordered through IDT may fit the budget.

Caporaso JG, Lauber CL, Walter WA, Berg-Lyons D, Losupone CA, Turnbaugh PJ, Fierer N, and Knight R. 2011. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. PNAS. 108: 4516-4522.

[pmiguel]

I have a colleague who wants to amplify 16S V3-V4 and then have me multiplex and run on MiSeq. She at this point has 26 samples, but using Illumina's protocol for indexing with Nextera index primers, I can only do 24 samples. We don't want to spend close to $1000 for the 96-sample index set since this is not something that we typically do, and the index primers would likely not get used again. Are there other options for multiplexing >24 samples? We have TruSeq LT single index primers, but again that's only 24 using sets A and B.

Thanks for any suggestions!

They are just PCR primers. Just order 1 more primer -- one from the 96 index set from an oligo company. Should cost less than $10.

--
Phillip

[sweetph3]

They are just PCR primers. Just order 1 more primer -- one from the 96 index set from an oligo company. Should cost less than $10.

I thought Nextera index primers had some sort of nucleotide modification. I didn't realize regular primers would work. If so, great! Thanks!

[pmiguel]

I thought Nextera index primers had some sort of nucleotide modification. I didn't realize regular primers would work. If so, great! Thanks!

I can tell you we just use normal de-salted oligonucleotides from IDT sometimes for the amplification step after tagmentation. Works fine.

--
Phillip

[thermophile]

If you're just starting out, go dual index

http://aem.asm.org/content/79/17/5112.long

I'm currently using up our original 12bp indexed primers (Caporaso et.al, index on i7) and have added the i5 indexes from Kozich et.al. So even though the i7 primers have 12bp indexes, I'm only sequencing 8. It's working fine and i no longer have to spend hours trying to shuffle primers for all the different groups that want to multiplex on a single run