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Old 10-12-2014, 05:50 PM   #1
HTS_Newby
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Location: Denmark

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Default ITS1 amplicon sequencing with Nextera indices

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!
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Old 10-13-2014, 06:25 AM   #2
cliffbeall
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Location: Ohio

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Quote:
Originally Posted by HTS_Newby View Post
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.
Quote:
Originally Posted by HTS_Newby View Post
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.
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