Pre-amplification DNA TruSeq Double Peak

[pmiguel]

I also noticed this phenomenon with TruSeq RNA libraries. (See here.)
(Note this should not be confused with the post-enrichment-amplification double peak phenomenon. This phenomenon, when we see it, occurs prior to enrichment-amplification.

• Below two genomic DNA samples (~1ug each) were Covaris S2-fragmented, then ampure concentrated/shaped to remove the smallest fragments and any contaminating junk from the DNA prep.
• Each was split into 2 replicates, with each of the replicates being carried through the rest of the protocol. (Different adapter index used on each replicate.)
• The legend tells the story. Peak initially at ~200 bp after blunting/a-tailing.
• After ligation the main peak appears to be converted into a double peak. Or, in the case of the second set of libraries, a main peak with a larger shoulder.
• Following 6 cycles of enrichment PCR only the right-most peak appears to be retained.

I have a couple of hypotheses as to what might be going on. What is your take?

--
Phillip

[pmiguel]

I have a couple of hypotheses as to what might be going on. What is your take?

The two that spring to my mind are:

• The two pre-amp peaks represent 2 adapter ligation states. That is, insert + single adapter construct (left peak) or a both adapter construct (right peak).
  
• The two pre-amp peaks represent a both adapter construct + ligase still bound to its substrate (left peak?) or a both adapter construct without ligase bound.

After enrichment PCR either only the 2 adapter construct amplified or the ligase released the substrate molecule during PCR denaturation.

Other hypotheses?

--
Phillip

[Riva]

I would pick the first one. We are doing trouble shooting of poor PCR enrichment and found that libraries with small size shift after ligation on Bioanalzer resulted in failed PCR. We uses the TruSeq HT DNA kit and get 30% failed libraries (not enough DNA amount < 1ug) in two batches of library preparation (12 samples per batch).