SEQanswers

SEQanswers (http://seqanswers.com/forums/index.php)
-   Illumina/Solexa (http://seqanswers.com/forums/forumdisplay.php?f=6)
-   -   2 peaks in the library for RNA-seq (http://seqanswers.com/forums/showthread.php?t=82858)

circA 06-11-2018 11:58 PM

2 peaks in the library for RNA-seq
 
1 Attachment(s)
Hi all,

I used KAPA RNA hyperprep kit to generate libraries and checked the distribution by Tapestation4200. However, there were 2 peaks in all my 6 samples(B1,C1,D1 and D2, E2, F2), one around 320nt, the other ~470nt.
I tried to heat and re-anneal these samples, however, after heating, it seemed that all peaks were gone(attached pic, lane E1, F1, G1,A2, B2,C2).

Is it OK to use these libraries for sequencing?


Another question is that I made a terrible mistake and heated the original libraries. Now all I have is ~8uL of diluted libraries. Can I amplify these libs again by PCR, because the RNA used for lib. preparation is quite limited?

Any suggestions are appreciated.
Thank you.

nucacidhunter 06-12-2018 01:01 AM

I would suggest to do 1-2 cycle PCR using all volume of denatured library as template and re-analyse. It is possible that libraries has not re-annealed so is not detectable by the Tape.

Amplification of diluted samples will distort the results.

circA 06-12-2018 06:22 AM

Quote:

Originally Posted by nucacidhunter (Post 217982)
I would suggest to do 1-2 cycle PCR using all volume of denatured library as template and re-analyse. It is possible that libraries has not re-annealed so is not detectable by the Tape.

Amplification of diluted samples will distort the results.

Hi, thank you for your reply.
I did the reannealing by heating them to 95degrees for 2mins and then cooling to 45 degrees at 0.1deg/sec and then to RT by removing the heat block of the thermocycler.
Do you think this is OK for annealing?

I use 10mM tris(pH8.0) to store the libs and I think it should be OK to heat to 95 degs. However, it looks like all sequences are gone. If it is the reason you mentioned, do you think if it's possible to recover them by heating again and annealing again?

I did the quantification of the library by qPCR and the undiluted samples are quite concentrated(~100nM). Is it OK to use them as template and pcr for several cycles?

Thank you.

nucacidhunter 06-13-2018 01:54 AM

It should be enough for annealing low diversity short fragments such as adapter oligos. Denaturing should not destroy the DNA as in PCR this happens in every cycle. The fact that your fragments are not detected on Tape indicates that they have not been re-annealed. This is the reason that I suggested to do 1-2 PCR cycles.

Doing 2 cycles on majority of your library (denatured) will be better than several cycles of diluted library.

pmiguel 06-13-2018 09:37 AM

After heat denaturation you will probably still be able to visualize them on an RNA chip. Actually, I'm surprised the the tape station DNA fluor would be double-strand specific. The bioanalyzer DNA chip fluor isn't.

--
Phillip


All times are GMT -8. The time now is 08:10 AM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.