[mira_talk] Re: a question about Nextera Kit vs TruSeq Kit
- From: Shaun Tyler <Shaun.Tyler@xxxxxxxxxxxxxxx>
- To: mira_talk@xxxxxxxxxxxxx
- Date: Wed, 5 Nov 2014 09:20:17 -0600
We've only used the TruSeq nano so I can't speak for the others (although I
now might look at the ChIP kit). We did start evaluating the NEBNext
version and it seemed OK but was going to need a bit of tweaking. But that
was right when Illumina lowered the price on the TruSeq kits so the cost
savings wasn't worth the hassle.
Shaun
From: Kevin Chen <wchen20@xxxxxxxxx>
To: mira_talk@xxxxxxxxxxxxx
Date: 2014-11-05 08:31 AM
Subject: [mira_talk] Re: a question about Nextera Kit vs TruSeq Kit
Sent by: mira_talk-bounce@xxxxxxxxxxxxx
Absolutely! I agree with you Shaun that understanding what’s happening at
the web bench is helpful to understand the nature of the data.
Can I assume you are using the normal TruSeq DNA kit? I saw there are
recommendation on TruSeq nano (using at low as 100ng), or TruSeq ChIP kit
(handling 5-10ng), and even Rubicon kit (using 10-50ng). How do you like
these kits?
Thanks again for your great insight, much appreciated!
On Tue, Nov 4, 2014 at 10:39 AM, Shaun Tyler <Shaun.Tyler@xxxxxxxxxxxxxxx>
wrote:
We've actually gone as high as 30 cycles. Typically if samples are below
quantitation range we try to move only half of the library forward to the
PCR step and do 15-20 cycles (depending on your mood that day). Based on
those results we may repeat the PCR with the remaining sample and
increase the cycles by some arbitrary amount. We do it that was to avoid
reamplifying the amplified material but we've done that too when we have
to. This isn't something that I'd say is routine. But it's come up a
number of times when we've been virus fishing in clinical samples. After
depleting the host as much as possible you are usually going on blind
faith that something is there.
The other curious thing that we've encountered when doing excessive
cycles is short insert contamination. We don't see this when doing lower
cycles and I'm assuming it is because the smaller fragments amplify more
efficiently. These are not always apparent on the trace but there is a
distinct population of reads between 50-250 bp (mode ~ 150 bp) in the
final data sets when the main library is typically 500+ bp. In fact we
Pippin the libraries with a gate range of approx. 500-800 bp so why the
hell there are still small fragments is beyond me. We're still trying to
arrive at a reliable and consistent way of removing this material. It's
not that it isn't good data but it often causes runs to fail due to over
clustering. The flow cells are loaded based on molar calculations and if
you are not accounting for the small stuff you can be loading 5 or 10
times more than you think.
I know this discussion has been somewhat outside of the scope of this
forum but then again understanding the wet lab side of things can give
you a better understanding on the nature of the data.
Shaun
Inactive hide details for Andrej Benjak ---2014-11-04 01:59:04 AM---We
are often dealing with small starting material and foundAndrej Benjak
---2014-11-04 01:59:04 AM---We are often dealing with small starting
material and found the TruSeq Chip library kit is much bet
From: Andrej Benjak <abenjak@xxxxxxxxx>
To: mira_talk@xxxxxxxxxxxxx
Date: 2014-11-04 01:59 AM
Subject: [mira_talk] Re: a question about Nextera Kit vs TruSeq Kit
Sent by: mira_talk-bounce@xxxxxxxxxxxxx
We are often dealing with small starting material and found the TruSeq
Chip library kit is much better than the standard TruSeq. It can handle
5-10 ng of DNA, but we have done it with less (even from what appeared to
be empty tubes!). We normally do 18 PCR cycles.
I have never checked the read coverage distribution in detail since we
mostly use our sequences in mapping analyses, but from looking at the
genome browser it looks ok.
What can happen with very small starting material is the higher
occurrence of duplicate reads (PCR duplicates), which has to be
considered when assessing the sequencing coverage, but perhaps this is
our special case because most of our DNA derives from the host, and the
bug we are interested in is poorly represented.
Best,
Andrej
On 11/04/2014 02:31 AM, Kevin Chen wrote:
Hi Shaun, thank you very much for your explanation. I talked to our
lab manager and we are very interested to test out the minimal
amount that TruSeq library can handle. Do you have any suggestion
on cycle number and the starting amount of material recommendation?
For example, what number of PCR cycles works for an amount of like
100ng of DNA? I understand it is empirical but it would be huge
help for us to start.
Another question is when you measure the depth of coverage, what
metrics you used to measure the skew in depth of coverage? I am
thinking of coefficient of variation, but it might be over
simplifying the measurement.
Thank you very much!
On Wed, Oct 29, 2014 at 2:02 PM, Shaun Tyler <
Shaun.Tyler@xxxxxxxxxxxxxxx> wrote:
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
Inactive hide details for Kevin Chen ---2014-10-29
11:17:41 AM---Nextera XT uses 1ng gDNA or cDNA. That is
a huge advantage. AdKevin Chen ---2014-10-29 11:17:41
AM---Nextera XT uses 1ng gDNA or cDNA. That is a huge
advantage. Adrian, can you give more details on the
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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