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
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
Comment