SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
n+1 cycles - how do you deal with it fanli Illumina/Solexa 3 08-14-2015 12:46 PM
How to deal with .wig files!! P Mohsina Epigenetics 1 03-03-2014 03:57 PM
how to deal with adjoining SNPs? cometarossa Bioinformatics 2 01-23-2013 01:12 AM
How to deal with the document of SAM luckylove Bioinformatics 4 06-26-2012 01:08 AM
clustering of very dilute libraries ps25 Illumina/Solexa 4 06-14-2010 03:23 PM

Reply
 
Thread Tools
Old 10-09-2015, 07:23 AM   #1
JBKri
Member
 
Location: Greece

Join Date: Jan 2014
Posts: 70
Default How to deal with too dilute libraries?

So my worst fear came true; we have a deadline, we spent the last of the library kit, and our PCR-free libraries all came out well below the minimum for loading to the instrument (MiSeq). The concentrations from qPCR range from 0.14 to 0.7 nM

I remember reading about an alternative denaturing protocol, with HCl used to neutralize the NaOH instead of the large dilution after denaturation. But I did not see any specific instructions about the quantities and concentration of HCl. Does anyone have experience with this?
Edit: I see in the NextSeq protocol they use Tris-HCl pH 7, which enables denaturation of libraries down to 0.5 nM with a final loading concentration of 20 pM. We will have about half of that, so we will probably end up with a rather low cluster density.

Another option is to concentrate the libraries with speedvac, but I am not too eager about this. We tried it before on a library, and we somehow lost everything. Even if we reduce the volume to 1/10th, some samples will still be a bit too dilute.

Another option is to do enrichment PCR, (see my post here, but I am not sure about the primers we have or the conditions.

Any help will be greatly appreciated!
Jon

Last edited by JBKri; 10-09-2015 at 08:31 AM.
JBKri is online now   Reply With Quote
Old 10-09-2015, 06:40 PM   #2
GA-J
Member
 
Location: USA

Join Date: Jul 2015
Posts: 28
Default

I would just use this library(0.7nM), denature with 0.1N NaoH, then it comes to 7pM after 100x dilution with HT1 buffter. If your library's size is about 400bp, you may load at this concentration or adjust using your library's size adjust factor.

If you still worry about the 0.1N NaoH,after denature, use same concentration and volume of HCl (0.1N and same volume that you used NaOH for denature), then you are good to go as usual.

Last edited by GA-J; 10-09-2015 at 07:01 PM.
GA-J is offline   Reply With Quote
Old 10-12-2015, 06:53 AM   #3
MU Core
Member
 
Location: Columbia, Missouri

Join Date: Apr 2008
Posts: 51
Default

You may find these instructions helpful.
MU Core is offline   Reply With Quote
Old 10-13-2015, 08:31 AM   #4
JBKri
Member
 
Location: Greece

Join Date: Jan 2014
Posts: 70
Default

Thanks for the responses. I ended up using the NextSeq protocol, which gave me a final concentration of 13.5 pM of the pool, and it worked just fine.
Jon
JBKri is online now   Reply With Quote
Old 06-13-2018, 12:54 PM   #5
ishap
Junior Member
 
Location: DC

Join Date: Mar 2014
Posts: 1
Default

HI, My library concentrations are between 0.2 to 0.8ng/ul. Could you please let me know how exactly you proceeded to pool and denature and load your samples when they were this low?
Thanks alot in advance!
ishap is offline   Reply With Quote
Old 06-14-2018, 03:46 AM   #6
JBKri
Member
 
Location: Greece

Join Date: Jan 2014
Posts: 70
Default

Quote:
Originally Posted by ishap View Post
HI, My library concentrations are between 0.2 to 0.8ng/ul. Could you please let me know how exactly you proceeded to pool and denature and load your samples when they were this low?
Thanks alot in advance!
What type of library is this? How did you measure the concentration? For many types of libraries it is best to measure the concentration by qPCR. The resulting concentrations are expressed in pM or nM, not ng/ul, and depend on the average fragment size of the library.

Normally, the MiSeq denaturation protocol needs a minimum of 2 or 4 nM library concentration, which limits the final loading concentration to maximum 10 or 20 pM. Let's say you have a concentration of 0.2 ng/ul, with fragment size 350 bp, then the concentration is 0.87 nM, which is not enough if using the MiSeq denaturation protocol.

The NextSeq protocol is here.
In short, you mix 1 volume of library with 1 volume of 0.2N NaOH, incubate for 5 min at room temp, then add 1 volume of 200mM Tris-HCl pH7 (room temp). Then add Hyb buffer to make the final volume 600 ul.
Let's say you want to load at a final concentration of 13 pM. Then you need to take a 0.87nM pool volume of 9 ul, to which you add 9 ul of 0.2 NaOH, incubate, then add 9 ul 200 mM Tris-HCl and 573 ul Hyb buffer. Final concentration should be 13.05 pM.

Edit: By chance, yesterday I used this protocol for the first time in several years. Our pool was just 130 pM, but with the NextSeq denaturation protocol we made the final concentration around 15 pM, and it worked just fine.

Last edited by JBKri; 06-14-2018 at 04:01 AM.
JBKri is online now   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 07:28 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO