MiSeq: V1-2 vs V4 for 16S rRNA amplicon sequencing

[westerman]

I would talk to your service provider (the people doing the actual sequencing) about your problems especially in regards to the phiX.

[mvdg]

Microbial profiling is new to me but I understand you can now combine variable regions due to the longer read lengths on the MiSeq. Has anyone combined regions for 18S similar to 16S as described by Mads Albertsen?

[fibar]

Hi everyone,
I go back to Pat Schloss: http://blog.mothur.org/2014/09/11/Wh...nce-matrix%3F/
If you don't de-noise properly (ie. fully overlapped reads), you get too many unique sequences. Hence, you end having a huge matrix downstream.

But what has happened in the field during the last year? Are there improved methods to deal with this problem? MadsAlbertsen, which method do you use?

[MadsAlbertsen]

We have been using the UPARSE workflow for some time now and are in general very happy with it (http://drive5.com/uparse/).

[UserChristine]

I want to sequence the V4 region using illuminas 16S metagenomic sequencing library preparation protocol (dual indexing approach using nextera indexes). Anyone have experience doing this?

[Brian Bushnell]

But what has happened in the field during the last year? Are there improved methods to deal with this problem?

In fact, there IS an improved method for dealing with noise and false-positives. BBMerge has a much lower false-positive merge rate than any other overlap-based read merger, including usearch.

[elizondas]

Primers and barcodes for V1-V2

Hello,

I was wondering whether any of you has sequenced using Illumina primers for V1-V2 region....Does anyone have a list of barcodes I could used? We need to focus on this region, since it is the best approach to identify sp within oral samples. ...

Many thanks
Elisa

[ymc]

Suppose I can the sequence of the full 16S gene at high accuracy, which region(s) can give me the closest discriminating power to the whole gene sequence?

[Brian Bushnell]

I don't think that question's been answered. But, you might want to look at this paper, which compares V4 to full-length:

http://www.nature.com/ismej/journal/vaop/ncurrent/full/ismej2015249a.html