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Old 06-10-2014, 10:49 AM   #1
luc
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Location: US

Join Date: Dec 2010
Posts: 429
Default increased bias in single-buffer library preps (NEB, KAPA,...) Reason for concern?

Hi All,

the latest generation of library prep kits seem very convenient (only a single cleanup) and promise higher complexity sequencing libraries (less DNA is lost in cleanups etc.). They are available from almost all vendors NEB (Ultra), Kapa (Hyper), Thermo Scientific, Affymetrix, .... etc.
Very likely the kits use similar enzymes and use Taq polymerase to carry out the A-tailing in the presence of all 4 nucleotides.

We have so far sequencing data for the Nebnext Ultra kit only and have observed a higher than usual bias in first bases of the reads - indicating a stronger A-tailing bias (`40% As [green] vs 20% Ts [red], 22% Gs [black] vs 17% Cs). Please see the attached pictures.

Did you observe such a similar increased biases with other single-buffer kits?
Did you see or would you expect any of this bias impacting sequencing of somewhat stranded nature (strand-specific RNA-seq ; exome-capture sequencing) ?
Attached Images
File Type: png SingleBuffer.png (9.4 KB, 41 views)
File Type: png Two-Buffer.png (10.8 KB, 28 views)
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bias, library prep, single-buffer, strand-specific

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