TruSight Tagmentation Step - Inserts are too short

[78Matthias87]

Dear SEQanswers forum,

this is the first question I am asking here, so excuse me please, if I am doing something wrong...

Last Month, I started using the Illumina TruSight Rapid Capture kit and I (think) that I have
problems with the very first Step of the Protocol, the Tagmentation.

When I first used the recommended 50ng (Qubit) of human, NaCl-extracted gDNA (260/280: 1,8-2,0),
my library looked way too small on a gel trace (after the 1st indexing PCR-Step and cleanup "peak" at
about 230-250bp) and the average insert size (as determined after sequencing) was about 150bp.
The Coverage of this run was abysmal, too.

Considering the principle of the Tagmentation reaction, I solved this problem by just increasing
the amount of gDNA to 80ng and my insert size increased up to 210bp which was very close to what
illumina suggests as a good size for those rapid capture kits.

However...

...lately I opened up a new vial of Tagment DNA Enzyme and my Inserts have shrunk.
Now, 80ng of input DNA resulted in ~150bp inserts. Aware of the problem, I increased the input to 120ng
and now I am getting sizes of about 170-180bp (the gel trace after the 1st PCR has its Peak at about
300bp now), which is better but still not good enough.

As I am already using more than twice the recommended DNA, I am a little bit hesitant to further increase
its amount.

So... my questions are:

Is my problem indeed related to tagmentation?

Are there lower fidelity batches of Tagment DNA enzyme (maybe like the one I used in the beginning)?

Will a further increase of initial Input (160ng or even 200ng) interfere with downstream steps like cleanup
or the indexing PCR?

Which amount of gDNA are you people using in your TruSight Rapid capture preparations?

Thanks in advance,

Mat

[kerplunk412]

Hi Mat. Welcome the the forum, and you didn't do anything wrong on the post, it is very clear. I have never used this kit, but some things you may want to consider are using less enzyme or shorter tagmentation time rather than more DNA.

[nucacidhunter]

So... my questions are:

Is my problem indeed related to tagmentation?

Are there lower fidelity batches of Tagment DNA enzyme (maybe like the one I used in the beginning)?

Will a further increase of initial Input (160ng or even 200ng) interfere with downstream steps like cleanup
or the indexing PCR?

Which amount of gDNA are you people using in your TruSight Rapid capture preparations?

It more likely related to tagmentation but sizing tagmented DNA PCR amplicons by gel is not accurate thereby is not recommended by Illumina. Posting a Tape or Bioanalyser trace of tagmented DNA PCR before clean up can reveal potential issues.

All batches are quality checked and batch to batch variation is less likely to be unnoticed by manufacturer.

50 ng input is the optimum quantity for input and if you have to use more DNA there must be some other issues. Using more DNA could result in non-optimal results for instance by depletion of primers during PCR resulting in artefacts.

[78Matthias87]

Problem solved.

I have never used this kit, but some things you may want to consider are using less enzyme or shorter tagmentation time rather than more DNA.

@kerlpunk412
I followed your suggestion and used half of the recommended amount of enzyme for 50ng of starting material. The reaction produced inserts of about 220bp in all preps.

50 ng input is the optimum qualtity for input and if you have to use more DNA there must be some other issues. Using more DNA could result in non-optimal results for instance by depletion of primers during PCR resulting in artefacts.

@nucacidhunter
The additional DNA indeed decreased the yield of my indexing-PCR. The reduction of input material resulted in significantly higher yield (150-200%).

Well, all I can do now is to say "thank you very much" kerlpunk412 and nucacidhunter. You helped me a lot. I'll post my new tagmentation protocol here in case someone else runs into a similar problem.

Thanks again,

Mat


Tagmentation for TruSight Rapid Capture:

- 10.0µl gDNA (5ng/µl) in buffer EB (Qiagen)
- 25.0µl tagmentation buffer
- 7.5µl enzyme
- 7,5µl bufffer EB (to 50µl)

- shake @1800RPM for one minute
- briefly spin to collect the samples
- incubate @58°C for 10 minutes (I use a PCR-block with the lid heated to 63°C)

- add 15µl Stop Tagment Buffer
- shake @1800RPM for one minute
- briefly spin to collect the samples
- incubate for four minutes at room temperature

-> proceed to bead cleanup and the indexing PCR

[kerplunk412]

Happy to help, and thanks for posting your results and protocol.