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

[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

[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?

[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

[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.

[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?

[MadsAlbertsen]

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

[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?

[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?

[westerman]

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

[Richa Sharma]

Illumina Miseq V4-V5 16S rRNAsequencing

Hi all,

This is Richa. I have sent my 16s V4-V5 amplicons for sequencing with illumina MiSeq. But there is a problem. PCR fragments cluster very poorly. There are very less no. of sequences that we are receiving and 50% are phix.

What could be the reason ? How to troubleshoot. Kindly help...!!!!


Thanks.
Richa

[rhinoceros]

Why is that the case? Is it just poor primer design?

Something about introducing staggered adapters to deal with the low diversity of the ends. They also spike their samples with PhiX. Sorry, I don't recall the specifics. I don't personally deal with the wet part of biology much, so I leave this to those who do

[DRYTCYV]

And this one ? - http://supportres.illumina.com/docum...15044223-b.pdf

V3-V4 region

[snetmcom]

To everyone doing 16S with MiSeq, I very much recommend that you check JGI's related protocols. Your average 454-primers are going to introduce huge bias into 16S MiSeq sequencing, especially so while studying GC-rich communities.

Why is that the case? Is it just poor primer design?

[rhinoceros]

To everyone doing 16S with MiSeq, I very much recommend that you check JGI's related protocols. Your average 454-primers are going to introduce huge bias into 16S MiSeq sequencing, especially so while studying GC-rich communities.

[firefly2280]

Hi,

I know I'm late to this conversation but we had the same issues. I managed to 'borrow' some Indexed primers for V4 and for V5-7 regions and compare just a few on a small set of my samples. The V5-7 gave better results.

I agree it's hard to know what to do and the best case scenario if you have money, sample and time is to try a few different ones! Sadly, that wasnt possible in my case but it worked out well.