SEQanswers

Go Back   SEQanswers > Applications Forums > Sample Prep / Library Generation



Similar Threads
Thread Thread Starter Forum Replies Last Post
SMARTer Ultra low RNA Kit Primers are found in sequencing data and Tophat questions TEFA Sample Prep / Library Generation 12 12-02-2015 07:48 AM
SMarter Low RNA Input Kit vanillasky Sample Prep / Library Generation 5 12-01-2015 05:09 AM
Best method/kit for rRNA depletion memento Illumina/Solexa 0 07-31-2013 06:49 PM
Need a RNA 6000 Pico Total RNA Kit? Jason507 Core Facilities 3 05-30-2013 02:06 AM
New Ribo-Zero™ Kit (Epidemiology):rRNA removal from microbial-infected mammalian RNA epibio Vendor Forum 0 12-12-2012 01:24 PM

Reply
 
Thread Tools
Old 08-18-2015, 11:51 PM   #1
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 438
Default SMARTER Pico RNA-seq kit - new ribo-depletion technique

The SMARTER Pico RNA-seq kit seems to implement a new ribo-depletion technique - ribodepletion of an almost complete sequencing library. (http://www.clontech.com/US/Products/...10020:22372:US).

Has anybody tried this kit or perhaps has an explanation how it could work?
The ribodepletion happens here after a some initial PCR amplification of the library, thus it should be double-stranded at this point. The they add some reagents (ZapR; likely including a bait library) and incubate at 37C one hour and at 72C for 10 minutes. During this treatment the rRNA-sequence containing library fragments are supposedly cut and thus will not be amplified in the following round of PCR which adds the complete Illumina adapters.
luc is offline   Reply With Quote
Old 08-19-2015, 12:51 AM   #2
Simone78
Senior Member
 
Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 206
Default

Quote:
Originally Posted by luc View Post
The SMARTER Pico RNA-seq kit seems to implement a new ribo-depletion technique - ribodepletion of an almost complete sequencing library. (http://www.clontech.com/US/Products/...10020:22372:US).

Has anybody tried this kit or perhaps has an explanation how it could work?
The ribodepletion happens here after a some initial PCR amplification of the library, thus it should be double-stranded at this point. The they add some reagents (ZapR; likely including a bait library) and incubate at 37C one hour and at 72C for 10 minutes. During this treatment the rRNA-sequence containing library fragments are supposedly cut and thus will not be amplified in the following round of PCR which adds the complete Illumina adapters.
very interesting, thanks for sharing!
from the manual it seems they have a buffer, an enzyme and a set of oligos that are specifically binding the rDNA. The oligos must be RNA oligos (they need to be stored at -80). They might anneal the rDNA to form RNA/DNA hybrids perhaps? In that case the enzyme would be RNAse H. Could it be something similar to this?
PMID: 22900061
In the paper, a set of DNA oligos was hybridized to the rRNA before any amplification, though. This is the same (or very similar) set of oligos that Clontech has in its RiboGone kit.
Simone78 is offline   Reply With Quote
Old 08-19-2015, 04:03 AM   #3
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,232
Default

Quote:
Originally Posted by luc View Post
Has anybody tried this kit or perhaps has an explanation how it could work?
This sounds similar to “Insert Dependent Adaptor Cleavage (InDA-C)” that has been adapted to SMART technology. This technology was released in some RNAseq kits early this year. Its performance is not as good as RNAseH or hybridization based methods (according to manufacturer data), but is one the post-cDNA synthesis methods for reducing rRNA reads in libraries with ultra-low inputs which former methods are not applicable.

Edit:
Comparison of protocols indicates that different mechanisms are used in each method. InDA-C method hybridises carefully designed oligos to ssDNA, extends and then uses BspQI (recognition site: GCTCTTCN^NNN) that cleaves adapter making that ssDNA un-amplifiable.

Last edited by nucacidhunter; 08-20-2015 at 04:32 PM.
nucacidhunter is offline   Reply With Quote
Old 08-19-2015, 04:29 PM   #4
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 438
Default

Quote:
Originally Posted by nucacidhunter View Post
This sounds similar to “Insert Dependent Adaptor Cleavage (InDA-C)” that has been adapted to SMART technology. This technology was released in some RNAseq kits early this year. Its performance is not as good as RNAseH or hybridization based methods (according to manufacturer data), but is one the post-cDNA synthesis methods for reducing rRNA reads in libraries with ultra-low inputs which former methods are not applicable.
Hi nucacidhunter,
On the first look it sounds similar, but the InDA-C process requires denaturation of the ds library to get the ribo-sequence oligos to anneal; the Pico RNA-seq kit strangely does not?
(As mentioned below: Perhaps the library is only amplified with a single primer in the first step and thus mostly single-stranded? This might allow for InDA-C style extension and digestion?)

Last edited by luc; 08-19-2015 at 04:50 PM.
luc is offline   Reply With Quote
Old 08-19-2015, 04:40 PM   #5
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 438
Default

Hi Simone,
thanks for your thoughts. I have not found any mention that RNAse H can be somehow motivated to actually digest DNA, which it would need to do in this protocol.
Somehow they induce a sequence-specific destruction of the library molecules (using some kind of bait sequences) without denaturing the library. Perhaps the library is only amplified with a single primer in the first step and thus mostly single-stranded? Otherwise recombinase would perhaps be an option?

Quote:
Originally Posted by Simone78 View Post
very interesting, thanks for sharing!
from the manual it seems they have a buffer, an enzyme and a set of oligos that are specifically binding the rDNA. The oligos must be RNA oligos (they need to be stored at -80). They might anneal the rDNA to form RNA/DNA hybrids perhaps? In that case the enzyme would be RNAse H. Could it be something similar to this?
PMID: 22900061
In the paper, a set of DNA oligos was hybridized to the rRNA before any amplification, though. This is the same (or very similar) set of oligos that Clontech has in its RiboGone kit.

Last edited by luc; 08-19-2015 at 04:48 PM.
luc is offline   Reply With Quote
Old 08-19-2015, 10:30 PM   #6
Simone78
Senior Member
 
Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 206
Default

Quote:
Originally Posted by luc View Post
Hi Simone,
thanks for your thoughts. I have not found any mention that RNAse H can be somehow motivated to actually digest DNA, which it would need to do in this protocol.
Somehow they induce a sequence-specific destruction of the library molecules (using some kind of bait sequences) without denaturing the library. Perhaps the library is only amplified with a single primer in the first step and thus mostly single-stranded? Otherwise recombinase would perhaps be an option?
You are right! I read the protocol in detail and I was also wondering that there is no denaturation step. Anyway, being interested in single-cell RNA-seq I also realized that this protocol is probably not for me. They say that one needs at least 250 pg of clean RNA. While the protocol can probably be scaled down to single cells, I will never be able to get clean RNA, the genomic DNA will always be present (DNAse treatment on single cell seems not feasible), so the random hexamers will amplify it along with the tot RNA.
Simone78 is offline   Reply With Quote
Old 08-20-2015, 05:40 AM   #7
HESmith
Senior Member
 
Location: Bethesda MD

Join Date: Oct 2009
Posts: 506
Default

I wonder if the mechanism is triplex DNA-mediated cleavage by poisoned/crippled topoisomerase. The PCR amplification that precedes rRNA removal is only five cycles and includes both forward and reverse primers, so it's not producing ssDNA. Triplex oligos can provide target specificity, and the trapped DNA/topo intermediate blocks amplification in the second PCR. Just guessing, but it's consistent with the protocol.
HESmith is offline   Reply With Quote
Old 08-20-2015, 05:06 PM   #8
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 438
Default

Thanks HESmith!
I will have to read up on this.

Quote:
Originally Posted by HESmith View Post
I wonder if the mechanism is triplex DNA-mediated cleavage by poisoned/crippled topoisomerase. The PCR amplification that precedes rRNA removal is only five cycles and includes both forward and reverse primers, so it's not producing ssDNA. Triplex oligos can provide target specificity, and the trapped DNA/topo intermediate blocks amplification in the second PCR. Just guessing, but it's consistent with the protocol.
luc is offline   Reply With Quote
Old 11-11-2015, 01:50 PM   #9
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,232
Default

Quote:
Originally Posted by HESmith View Post
I wonder if the mechanism is triplex DNA-mediated cleavage by poisoned/crippled topoisomerase. The PCR amplification that precedes rRNA removal is only five cycles and includes both forward and reverse primers, so it's not producing ssDNA. Triplex oligos can provide target specificity, and the trapped DNA/topo intermediate blocks amplification in the second PCR. Just guessing, but it's consistent with the protocol.
Or it could be the method described in this paper: http://www.febsletters.org/article/0...307-9/abstract
nucacidhunter is offline   Reply With Quote
Old 11-13-2015, 03:49 PM   #10
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 438
Default

Thanks Nucacidhunter,
RNAs are scary complex/capable.
luc is offline   Reply With Quote
Old 04-17-2016, 10:06 PM   #11
BioKiwi
Member
 
Location: KiwiTeritory

Join Date: Sep 2014
Posts: 19
Default

There is some anecdotal evidence that libraries prepared with this kit in input range (250pg-10ng) do not show any correlation with 100 ng or higher input libraries prepared with the same input total RNA using TruSeq kit, even though libraries prepared with the kit show good concordance with each other. I wonder if someone has done any comparison and how one can interpret result of a gene expression if it shows different values depending on what kit was used for library prep.
BioKiwi is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 05:00 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO