SEQanswers

Go Back   SEQanswers > General



Similar Threads
Thread Thread Starter Forum Replies Last Post
Second-strand cDNA synthesis question eab Sample Prep / Library Generation 0 12-13-2011 02:50 PM
Strand-specific library appears not strand-specific oligo Illumina/Solexa 7 12-08-2011 09:54 AM
strand specific RNAseq Pepe Sample Prep / Library Generation 5 10-27-2011 07:58 AM
cuffdiff not strand-specific? burkard Bioinformatics 0 11-15-2010 10:30 AM
strand-specific Illumina protocol PFS Bioinformatics 1 06-28-2010 07:53 AM

Reply
 
Thread Tools
Old 11-27-2013, 06:29 AM   #21
TonyBrooks
Senior Member
 
Location: London

Join Date: Jun 2009
Posts: 298
Default

You digest the second strand cDNA before PCR using an enzyme that cleaves wherever there is a uracil base (dUTP is used in place of dTTP in the second strand mastermix). This means when you do the PCR you are only amplifying from the first strand cDNA, hence all reads will from the same strand as the mRNA. The first strand cDNA will be complementary to the mRNA that produced it. The PCR product is still double-stranded
TonyBrooks is offline   Reply With Quote
Old 11-27-2013, 06:58 AM   #22
chaomeizhang
Junior Member
 
Location: Tampa, Florida

Join Date: Aug 2011
Posts: 7
Default

Appreciate...

my understanding is:
when you do PCR with two primers using the first strand cDNA as template, at first cycle it will use one of the primers (complimentary to the first strand cDNA) to generate a reverse complimentary strand that is actually is the second strand cDNA, and the following cycles will use the double strand DNA as template. How does the PCR only amplify the the first strand cDNA????

Thanks.
chaomeizhang is offline   Reply With Quote
Old 11-27-2013, 07:11 AM   #23
TonyBrooks
Senior Member
 
Location: London

Join Date: Jun 2009
Posts: 298
Default

Both strands are created in the PCR, but by digesting the second strand you ensure that the P5 adapter is always on the 5' end of the mRNA and P7 is always on the 3' end. This means all reads from the sequencer will be the same sequence as the mRNA that made the library (Illumina read 1 are read P5->P7)

http://www.rna-seqblog.com/wp-conten...reparation.jpg

Last edited by TonyBrooks; 11-27-2013 at 07:12 AM. Reason: including image
TonyBrooks is offline   Reply With Quote
Old 11-27-2013, 07:34 AM   #24
chaomeizhang
Junior Member
 
Location: Tampa, Florida

Join Date: Aug 2011
Posts: 7
Default

The point is that dUTP digestion of second strand is performed before PCR amplification. The following PCR cycles use the normal dNTP, there is no way to use the dUTP digestion to get rid of the second strand??? Thanks.
chaomeizhang is offline   Reply With Quote
Old 11-27-2013, 08:09 AM   #25
TonyBrooks
Senior Member
 
Location: London

Join Date: Jun 2009
Posts: 298
Default

You remove the second strand before PCR. This means the template for the reaction is the first strand only. In an unstranded assay, both strands are used as template - so library is created from both strands.
The PCR amplicon is not digested - it is double stranded DNA.
Look at the jpg in the link. Sequencing is always in the blue to red direction.
TonyBrooks is offline   Reply With Quote
Old 07-01-2016, 04:05 AM   #26
AmitChaurasia
Junior Member
 
Location: Delhi, INDIA

Join Date: Jan 2013
Posts: 7
Default

Thanks Tony & Chipper for your beautiful expression... I was also curious about the question asked by chaomeizhang. Thanks chaomeizhang.

Last edited by AmitChaurasia; 07-01-2016 at 05:08 AM.
AmitChaurasia is offline   Reply With Quote
Old 08-02-2017, 10:30 AM   #27
chaomeizhang
Junior Member
 
Location: Tampa, Florida

Join Date: Aug 2011
Posts: 7
Default

Quote:
Originally Posted by Chipper View Post
It is because you have two different adaptors (annealed in a Y-shape). Without dUTP you will get two different amplicons for each insert due to the Y-adaptors meaning that the first read can start from either end of the insert, but with dUTP you will only amplify one amplicon (which will be double stranded) so all reads from the same transcript will go in the same direction.
Thank you Chipper.

I would like to add some note to the explanation.

The enrichment PCR does regenerate the DNA strand which was degraded by UDG, but the adaptors on both sides of the strands can not hybridized with the lawn oligos on the flow cell, so these strands will be excluded during the cluster generation and eventually not be sequenced.
chaomeizhang 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 07:45 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO