ITS1 amplicon sequencing with Nextera indices

[HTS_Newby]

Hi folks,

Sorry for putting very basic questions here. I am very new to NGS and I have planned to perform ITS1 amplicon sequencing on MiSeq. I have combed through several articles and found that 2x250 PE would be a suitable option for me. Anyway, I have some questions about indexing.

1. I have ca. 50 samples in which I would like to multiplex them. In this case, single or double index is better? And may you briefly explain differentations between the two systems or suggest a publication for further reading?

2. In case of double index, is it possible to use Nextera indices or other custom indices generated for bacterial 16s metagenomics for fungal ITS? I understand that they should be fine as long as I have both color channels in each base positions. Can you suggest if there is any I should be aware of?

Thank you in advance!

[cliffbeall]

Hi folks,

Sorry for putting very basic questions here. I am very new to NGS and I have planned to perform ITS1 amplicon sequencing on MiSeq. I have combed through several articles and found that 2x250 PE would be a suitable option for me. Anyway, I have some questions about indexing.

1. I have ca. 50 samples in which I would like to multiplex them. In this case, single or double index is better? And may you briefly explain differentations between the two systems or suggest a publication for further reading?

Dual indices will save on primer synthesis - you can do more samples with fewer primers because of the combinatorial effect.

2. In case of double index, is it possible to use Nextera indices or other custom indices generated for bacterial 16s metagenomics for fungal ITS? I understand that they should be fine as long as I have both color channels in each base positions. Can you suggest if there is any I should be aware of?

Thank you in advance!

The Illumina 16S metagenomic library prep guide says:

"The method described in this 16S Metagenomics protocol can be used for any targeted amplicon sequencing, relevant to virus research, mutation detection, or other microbiology‐ related studies."

I think you could look at that protocol and substitute in your gene specific sequences for the ones there (bacterial 16S V3V4). I think you need to sign up on their website, but you probably want to do that anyway.