Libraries - Unusual Profiles on Tapestation

[SofiaGranja]

Hello,

I'm posting this hoping that you can help me understanding 3 unusual Tapestation Profiles. These profiles correspond to (DNA) libraries already indexed and purified with magnetic beads for NGS. Attached are 6 images (2 of each profile). All the images correspond to Agilent D1000 ScreenTape.

I'm working with really degraded DNA so I'm never expecting to get a very high concentration (however, sometimes it's possible) or fragments larger than 300-400 bp.

Please let me know if you have ever encountered these type of profiles and/or if you have any suggestion for what might be causing this.

P.S. - I'm not entirely sure if the 1st and 2nd profiles are indeed different profiles. Also, before someone suggests that the 3rd profile (profiles with higher peaks) corresponds to too much DNA loaded on the tape, I'm not convinced that is the case, since these ones correspond to approx 20000 (pmol/l) and I have several samples with values up to 61800 (pmol/l) with a perfect profile.


1st Profile https://ibb.co/dgjvdhh
1st Profile https://ibb.co/vYT1nxH
2nd Profile https://ibb.co/ncthmDP
2nd Profile https://ibb.co/BzRR6wX
3rd Profile https://ibb.co/s38P7mp
3rd Profile https://ibb.co/209mHFy


Thanks in advance! Sofia

[aciulia]

Hi Sofia, when you say "these ones correspond to approx 20000 (pmol/l) and I have several samples with values up to 61800 (pmol/l) with a perfect profile", how are you measuring this? Thanks!

[jteeee2]

Hi Sofia- Could you provide a bit more info on the specific library prep method you used? The first couple of traces look as though they represent a pooled amplicon-based approach. 3 dominant peaks representing 3 different amplicon sizes. Difficult to say for sure without additional sample/kit info.

[SofiaGranja]

Hi!

I'm using TapeStation for measurements in the region 150-1000 bp. So the Molarity (pmol/l) is automatically calculated by the TapeStation program.

[SofiaGranja]

Hi!

The protocol is a Blunt-End (Illumina library building) adapted from:

Kircher M, Sawyer S, Meyer M (2012) Double indexing overcomes
inaccuracies in multiplex sequencing on the Illumina platform.
Nucleic Acids Research, 40, 1–8.

Meyer M, Kircher M (2010) Illumina sequencing library preparation for
highly multiplexed target capture and sequencing. Cold Spring
Harbor Protocols, 5.