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-   -   Reverse Engineering of Primer mix by NGS (http://seqanswers.com/forums/showthread.php?t=75900)

sfranzenburg 05-08-2017 06:03 AM

Reverse Engineering of Primer mix by NGS
 
Dear All,

we have a primer mix of unknown sequences. Has anyone tried yet to create NGS libraries with this primer mix as templates to reverse engineer their sequence?
At first we thought about simply putting them into the normal A-Tail - Endrepair - Ligation pipeline, but of course that does not work because we deal with ssDNA here...

best regards,

Sören

cmbetts 05-08-2017 09:57 AM

One of the miRNA methods would probably work. The RNA ligase used in most preps can also act on ssDNA. You might need to do a PNK treatment first. I haven't looked at the library prep chemistry in a while, but they might rely on the miRNAs mostly being 5' phosphorylated.
You could sequence also sequence the amplicons. You'll be enriched for the primer sequences at the 5' ends if you do a non-nextera prep. You won't know the 3' ends, but you can make an educated guess by looking at Tms or feeding the assembled amplicons through primer3

nucacidhunter 05-08-2017 10:37 AM

If primers are longer than 40nt you can prepare library using Accel-NGS 1S Plus or any other ssDNA library prep kit. For shorter primers it might be possible to customise protocols.

cmbetts 05-08-2017 03:09 PM

The Meyer lab protocol for ancient DNA would also work http://www.nature.com/nprot/journal/....2013.038.html if you want to go full homebrew on your library prep

austinso 05-09-2017 07:44 AM

I second the Meyer protocol. In fact he alludes to that application in that protocol. Just remember to scale according to molarity and not to mass. Works well if everything being cobbled together is <50 nts.

However, the challenge I think will be in the recoveries of the bead purification given the expected product sizes. It will also be low diversity, so you'll probably need to spike in a lot of PhiX when you sequence.

sfranzenburg 05-15-2017 09:59 PM

Thank you all,
Meyer protocol seems to be exactly what we were searching for.


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