SEQanswers

SEQanswers (http://seqanswers.com/forums/index.php)
-   Sample Prep / Library Generation (http://seqanswers.com/forums/forumdisplay.php?f=25)
-   -   Adapter multimer problem (http://seqanswers.com/forums/showthread.php?t=77752)

LozWhite 08-25-2017 05:44 AM

Adapter multimer problem
 
2 Attachment(s)
Hi all,

I am trying to trouble-shoot a problem I am having with my library prep.

I am preparing samples following Meyer & Kircher (2010) for Illumina sequencing with home-made adapters identical to Illumina's.

Using an input of 200ng of sheared DNA, I am getting very low library yields and strange dimer mutlimer's (see attached bioA results).

I have been using SPRI beads to clean up between steps and after PCR, but the smaller peaks are still coming through, as are some larger peaks.

Any ideas what could be causing this? I have double checked my DNA (definitely 200ng going in, and properly sheared) and my SPRI beads (working as expected on DNA ladders). My next best guess was that the blunt-end repair and/or phosphorylation reaction is not working properly? Perhaps degraded ATP or enzymes?

Any help would be really appreciated.

adam.geber 09-14-2017 10:08 AM

Can you elaborate on the protocol and conditions (e.g. how many cycles of PCR) that you're using? Do you have a positive control reaction alongside your samples, and if so are you seeing low yield for that library as well?

provirus 10-18-2017 01:05 PM

It would also be informative with bioanalyzer profiles showing: a) input (the 200 ng) alone and no library prep, b) library prep with input but no adapters, c) library prep with adapter but no input, d)-f) library preps with 10x fold dilutions (1x, 0.1x, 0.01x) of the adapter, and finally g) your dna together with a "standard" commercial adapter using standard protocol.

Even though you are sure that everything "is fine" (mass and that it is covarized correctly etc) many things might have gone wrong that you did not think of - the controls above might point to steps that are not optimal.
Good luck.

Genetic Librarian 10-19-2017 12:25 AM

My wild guess would be on low ligation efficacy, giving the final PCR no target to amplify.
If that is true, you can check that by quantifying your library by Qubit and by qPCR (e.g. Kapa library quant for Illumina). Normally, with a ligation efficacy of ~50%, your Qubit value would be twice as high as your qPCR value. If your library does not contain adapters (or very little), qPCR value should be waaaaay lower.


All times are GMT -8. The time now is 12:05 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.