[mira_talk] Re: a question about Nextera Kit vs TruSeq Kit
- From: Shaun Tyler <Shaun.Tyler@xxxxxxxxxxxxxxx>
- To: mira_talk@xxxxxxxxxxxxx
- Date: Wed, 29 Oct 2014 13:02:09 -0500
We've successfully made TruSeq libraries when amounts have been so low
we've been unable to quantitate. It just requires playing with the PCR
cycles and going on faith. So the minimum requirement isn't really the
"minimum". Something to keep in mind with Nextera is that it may only
require a small amount of input but you will still need to obtain larger
amounts from your samples for good results. The yields need to be high
enough to accurately quatitate. Because the tagmentation reaction is very
much ratio dependant if you stray from the recommended input amounts you
can shear it way too much or not enough. Either way the sequencing results
will be poor. This isn't as much of an issue with TruSeq as the sonicating
isn't affected by concentration that much. We've also found with the Qbit
that concentrations below about 1 ng/ul (using the low range standards)
really can't be trusted. So if you are anticipating very low yields from
your samples TruSeq might still be the best way to go for consistent
results.
We've done hundreds of both types of libraries primarily on bacteria and
there is a noticeable skew with Nextera. If you map the reads back to the
assembly (or are using an assembler that produces more than just a fasta
file) it is quite apparent. 2 to 3 fold differences in depth of coverage
are not uncommon (Bastien originally helped sort this out). Because of this
we've been primarily using SPAdes as it does not seem to be adversely
affected by the uneven distribution. However what we lack in the resulting
assemblies is any reliable information regarding copy number for repetitive
regions. The depth of coverage gives you some indication but because of
the skewed distribution even that can't be completely trusted.
Other than the input requirements between the two approaches the biggest
attraction to Nextera XT was the cost. It was significantly cheaper per
sample than TruSeq and when your planning on a couple of hundred samples
that was a significant concern. But Illumina recently slashed their prices
on TruSeq so that's not as big of a factor any more. They are more labour
intensive and involve more in consumable costs but we're starting to
migrate more towards TruSeq now because of the cleaner data.
BTW - cDNA was mentioned which implies transcriptome analysis in which case
I would definitely NOT use Nextera XT. With the distribution skew you
would never be able to trust anything other than genes that are
substantially up or down regulated and any quantitative measurements would
just be ball park estimates.
At least that's my opinion ;-)
Shaun
From: Kevin Chen <wchen20@xxxxxxxxx>
To: mira_talk@xxxxxxxxxxxxx
Date: 2014-10-29 11:17 AM
Subject: [mira_talk] Re: a question about Nextera Kit vs TruSeq Kit
Sent by: mira_talk-bounce@xxxxxxxxxxxxx
Nextera XT uses 1ng gDNA or cDNA. That is a huge advantage.
Adrian, can you give more details on the coverage consistency, what metrics
you used to measure it? Thanks!
Regards,
Bing
On Wed, Oct 29, 2014 at 10:41 AM, Chris Hoefler <hoeflerb@xxxxxxxxx> wrote:
How minute are we talking about? TruSeq Nano can start with ~100 ng
unsheared gDNA.
On Tue, Oct 28, 2014 at 7:03 PM, Kevin Chen <wchen20@xxxxxxxxx> wrote:
Hi, Bastian & Mira community
I am Bing from U of Maryland. When I read your Mira manual, I
notice this statement below. I am interested in getting more
information on that. Because Nextera kit has its own advantages
that TruSeq does not have, for example, working with minute amount
of material, I have high hope for Nextera. Do you have any prelim
data to support this statement? If this is true, I have to
re-think a lot of my experiment design using Nextera, that’ll
change a lot of things.
"For de-novo assemblies, do NOT (never ever at all and under no
circumstances) use the Nextera kit, take TruSeq. The non-random
fragmentation behaviour of Nextera leads to all sorts of problems
for assemblers (not only MIRA) which try to use kmer frequencies
as a criterion for repetitiveness of a given sequence."
Thank you in advance,
Bing
--
Chris Hoefler, PhD
Postdoctoral Research Associate
Straight Lab
Texas A&M University
2128 TAMU
College Station, TX 77843-2128
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