SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
TruSeq smallRNA and chip-Seq libraries sajoshi Sample Prep / Library Generation 0 05-27-2016 05:21 AM
truseq libraries for nimblegen catpure mwt General 1 11-18-2011 08:08 AM
Help with smallRNA Truseq kit - Agilent results? Tali7 Illumina/Solexa 5 09-14-2011 10:04 AM
Help with smallRNA Truseq kit - Agilent results? (repost from Illumina forum) Tali7 RNA Sequencing 2 08-03-2011 12:05 AM
smallRNA v1.5 versus TruSEQ Fiji_surf Illumina/Solexa 0 05-02-2011 03:48 PM

Reply
 
Thread Tools
Old 06-10-2016, 07:07 AM   #1
sajoshi
Member
 
Location: cleveland

Join Date: Dec 2015
Posts: 26
Default TruSeq smallRNA libraries

I am doing a TruSeq smallRNA library prep. When I was doing Ligate 3' adapter, and came to adding the STP step, the tube in the kit was empty (Illumina glitch). Illumina said to keep samples at -80 and shipped me the STP. Then I continued. When I checked BioA traces (see attached) the band is not where it should be. So it did not work and I will have to repeat. Any suggestions/feedback if you have any experience with this library prep.
Attached Files
File Type: pdf June 9_O'tiernney-ginn_initial check_DNA HS.pdf (1.68 MB, 35 views)
sajoshi is offline   Reply With Quote
Old 06-10-2016, 07:20 AM   #2
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 415
Default

How did your RNA sample look before the library prep?
luc is offline   Reply With Quote
Old 06-10-2016, 07:38 AM   #3
sajoshi
Member
 
Location: cleveland

Join Date: Dec 2015
Posts: 26
Default small RNA library

See attached. Sample was purified when given to me and hence checked on smallRNA chip and BioA conc. was close to the conc. by Qubit microRNA assay. I used a human brain total RNA as control while preparing libraries.
Attached Files
File Type: pdf June 3_O'tiernney-ginn_smallRNA.pdf (1.33 MB, 26 views)
sajoshi is offline   Reply With Quote
Old 06-15-2016, 07:49 AM   #4
kerplunk412
Senior Member
 
Location: Bioo Scientific, Austin, TX, USA

Join Date: Jun 2012
Posts: 119
Default

The RNA looks good to me, but the libraries seem to be just adapter-dimer. How much RNA input did you use?

Also, I would like to mention that the TruSeq kit is about the worst Small RNA-Seq kit you can get, at least in terms of cost and data quality. I highly recommend you check out the NEXTflex Small RNA Sequencing Kit v3, as it uses randomized adapters to reduce ligase bias and therefore generate more accurate data and greater discovery/detection rates, it includes completely gel-free or low-input options, and it is much more cost effective.

As a disclaimer, I work for Bioo Scientific.
kerplunk412 is offline   Reply With Quote
Old 06-17-2016, 07:17 AM   #5
sajoshi
Member
 
Location: cleveland

Join Date: Dec 2015
Posts: 26
Default message

Quote:
Originally Posted by kerplunk412 View Post
The RNA looks good to me, but the libraries seem to be just adapter-dimer. How much RNA input did you use?

Also, I would like to mention that the TruSeq kit is about the worst Small RNA-Seq kit you can get, at least in terms of cost and data quality. I highly recommend you check out the NEXTflex Small RNA Sequencing Kit v3, as it uses randomized adapters to reduce ligase bias and therefore generate more accurate data and greater discovery/detection rates, it includes completely gel-free or low-input options, and it is much more cost effective.

As a disclaimer, I work for Bioo Scientific.
I just send you a message asking a question. Please let me know.
sajoshi is offline   Reply With Quote
Old 06-17-2016, 09:26 AM   #6
kerplunk412
Senior Member
 
Location: Bioo Scientific, Austin, TX, USA

Join Date: Jun 2012
Posts: 119
Default

Quote:
Originally Posted by sajoshi View Post
I just send you a message asking a question. Please let me know.
Response sent.

Also, to be fair I should mention some of the other kits out there and the pros and cons.

NEB: Cost effective, lower bias than TruSeq, but substantially more bias than NEXTflex (Bioo)

TriLink: Cost effective (I think), streamlined protocol with no gel size selection required, but similar bias as TruSeq

Clontech: Uses poly(A) tailing and template switching RT to eliminate ligation steps, thereby reducing bias. Shows slightly less bias than NEXTflex kit, but mapping rates to miRNA are very low, leading to lower overall detection/discovery rates in total RNA samples. This makes sense, as other than size selection there is no way to select for miRNA as there is with ligation-based kit. I think this protocol is also pretty streamlined.
kerplunk412 is offline   Reply With Quote
Old 09-20-2016, 05:57 AM   #7
anup.chugani
Junior Member
 
Location: Bangalore

Join Date: Jun 2014
Posts: 2
Default

Any idea how TriLink works?
They claim to have modified the 3' and 5' adapters.
We have observed the presence of adapter peak at ~60bp. Can it affect the clustering?
anup.chugani is offline   Reply With Quote
Old 09-20-2016, 11:00 AM   #8
kerplunk412
Senior Member
 
Location: Bioo Scientific, Austin, TX, USA

Join Date: Jun 2012
Posts: 119
Default

TriLink has indeed modified the adapters to greatly reduce adapter-dimer formation, but I am not sure exactly how it works. In my experience their kit works well but still has big issues with bias.

A peak at ~60 bp is most likely excess PCR primer and will not interfere with clustering, as it will not be able to amplify on the flow cell.
kerplunk412 is offline   Reply With Quote
Old 09-21-2016, 02:06 AM   #9
anup.chugani
Junior Member
 
Location: Bangalore

Join Date: Jun 2014
Posts: 2
Default

Thanks for the reply.
Since it does not cluster, could it occupy the oligoes on the flowcell and stay inert? We have actually observed less cluster numbers for this run.
anup.chugani is offline   Reply With Quote
Old 09-21-2016, 07:33 AM   #10
kerplunk412
Senior Member
 
Location: Bioo Scientific, Austin, TX, USA

Join Date: Jun 2012
Posts: 119
Default

Unless there is a large amount of excess PCR primer compared to actual library product it should not occupy enough primers on the flow cell to make any difference. The flow cell has enough primers on it to allow single library molecules to make clusters of about 1,000 strands (according to Wikipedia) through bridge amplification, so since the excess PCR primer is not amplified the number of primer sites it occupies on the flow cell will be negligible.
kerplunk412 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 02:37 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO