Loss of entire sample set after SPRI cleanup

[AvantS]

Hello all,

I was prepping samples for WGS Using the Kapa HyperPrep workflow. After the final cleanup with 0.8x AMPure SPRI beads, my gel confirmation showed that most of my samples were lost.

I'm trying to mentally troubleshoot what could have gone wrong, but I don't have extensive experience with library prepping to know where to look for the problem.

I washed the samples+beads in 80% EtOH twice for 30s and eluted in 10mM TE+0.05% Tween20 buffer warmed to 55°C. I air dried the beads for 2 minutes between the wash and elution. I incubated in buffer for 3 minutes before removing the beads and used a plate shaker to assist with DNA re-suspension. The cleanup was done with a bead extractor to minimize sample loss.

I redid the cleanup using pre-warmed 10mM Tris-HCl + 0.05% Tween20 instead of TE as the buffer, but the outcome was the same.

Does anyone have suggestions on what could have happened to effect the affinity of the DNA and beads?

Any ideas are welcome.

[seqsuave]

How fresh was the 80% Ethanol, if ethanol evaporated off you could have had elution during the washes. It is recommended that 80% Ethanol is not more than a week old.

[AvantS]

@SeqSuave. It was made on the same day as the bead cleanup.

[itstrieu]

I am assuming you are doing the PCR-free workflow? What was the input concentration and the concentration after final clean-up. You might have to increase your input concentration or do a PCR step to amply your library.

Also during the drying step if the beads are over-dried and you see hairline cracks, I would incubate the beads for 5 - 6 minutes to help increase yield.

[AvantS]

@itstrieu The workflow includes 3 PCRs: End repair & A-tailing PCR, Adapter Ligation, and an Indexing. Input concentration was 100ng/uL per sample. Quantification after cleanup was not done, since the gel-confirmation showed few successful sample bands.

[itstrieu]

Is the workflow

ER,AT, Ligation --> 0.8X bead cleanup --> Elute in 25 uL follow by Library Amplification --> Post-amplification Cleanup using 1x bead? With the concentration being 100ng/uL, how much total DNA is going into library prep?

Was their a QC check after fragmentation? One common mistake I see is using elution buffer/water during the wash step instead of ethanol which could wash away your DNA. Not on purpose but accidental since both on them are clear liquids.

[AvantS]

Is the workflow

ER,AT, Ligation --> 0.8X bead cleanup --> Elute in 25 uL follow by Library Amplification --> Post-amplification Cleanup using 1x bead? With the concentration being 100ng/uL, how much total DNA is going into library prep?

Was their a QC check after fragmentation? One common mistake I see is using elution buffer/water during the wash step instead of ethanol which could wash away your DNA. Not on purpose but accidental since both on them are clear liquids.

Our post-amp cleanup used 0.8x bead. The samples will be diluted to 20nM for sequencing.

It's not possible that the elution buffer was used instead of EtOH because each U-bottom plate used for cleanup was labeled with the reagent it contains (Sample, EtOH W1, EtOH W2, Etc)and I added the warmed elution buffer to its plate while air drying the beads.

[itstrieu]

The only two things I can think of is DNA in buffers containing high concentrations of EDTA, other chelating agents or salts may inhibit the
end repair and A-tailing reaction. The other is something going wrong with library amplification (maybe your index primers).

If you can, check the concentration or run a gel/tape of the adapter-ligated library to see if the yield is reasonable going into library amplification.

[AvantS]

The only two things I can think of is DNA in buffers containing high concentrations of EDTA, other chelating agents or salts may inhibit the
end repair and A-tailing reaction. The other is something going wrong with library amplification (maybe your index primers).

If you can, check the concentration or run a gel/tape of the adapter-ligated library to see if the yield is reasonable going into library amplification.

Thank you for the great advice. I'll run a gel confirmation to see if my adapter ligated libraries still have sample.

[beancat]

I've used Kapa Hyperplus 3-4 times a week for well over a year and what causes me to decrease yield or lose samples have almost always been the the frag enzyme activity or sometimes beads.

Fragmentation: Under or over fragmentation will definitely mess up your yield. Make sure your kit is not expired, enzymes are always stored on ice and never vortexed, and buffers have no precipitation remaining. Kapa is also super temperature sensitive so maybe there was an issue there with frag enzyme activity? If i had to bet, I would say this is probably what happened to you.

Beads: Make sure the beads are well suspended and at the correct temperature. I personally elute in 10 mM Tris, ph 8.0 and think tween just gets in the way. I think its a good idea to never use TE for library preparation.

[AvantS]

I've used Kapa Hyperplus 3-4 times a week for well over a year and what causes me to decrease yield or lose samples have almost always been the the frag enzyme activity or sometimes beads.

Fragmentation: Under or over fragmentation will definitely mess up your yield. Make sure your kit is not expired, enzymes are always stored on ice and never vortexed, and buffers have no precipitation remaining. Kapa is also super temperature sensitive so maybe there was an issue there with frag enzyme activity? If i had to bet, I would say this is probably what happened to you.

Beads: Make sure the beads are well suspended and at the correct temperature. I personally elute in 10 mM Tris, ph 8.0 and think tween just gets in the way. I think its a good idea to never use TE for library preparation.

That sounds very likely to me as well. My team and I will redo the pipeline keeping those suggestions in mind. Thank you.

[jmc887]

I've used Kapa Hyperplus 3-4 times a week for well over a year and what causes me to decrease yield or lose samples have almost always been the the frag enzyme activity or sometimes beads.

Fragmentation: Under or over fragmentation will definitely mess up your yield. Make sure your kit is not expired, enzymes are always stored on ice and never vortexed, and buffers have no precipitation remaining. Kapa is also super temperature sensitive so maybe there was an issue there with frag enzyme activity? If i had to bet, I would say this is probably what happened to you.

Beads: Make sure the beads are well suspended and at the correct temperature. I personally elute in 10 mM Tris, ph 8.0 and think tween just gets in the way. I think its a good idea to never use TE for library preparation.

Hi,

Interestingly I had a similar problem with the HyperPlus, my post can be found here:

http://seqanswers.com/forums/showthread.php?t=88750&highlight=kapa

beancat, since you are an experienced user of KAPA Hyperplus I would like to ask you what was your average pre-capture yield? I got an average yield of 500 ng, however the protocol says we should get more than 1000 ng. I was also thinking I was loosing sample during the SPRI clean ups, I mean using the same 100ng input with other kits I get higher pre-capture yields.

In anycase I will be following this thread for any updates.
Thanks!

[beancat]

Hi,

Interestingly I had a similar problem with the HyperPlus, my post can be found here:

http://seqanswers.com/forums/showthread.php?t=88750&highlight=kapa

beancat, since you are an experienced user of KAPA Hyperplus I would like to ask you what was your average pre-capture yield? I got an average yield of 500 ng, however the protocol says we should get more than 1000 ng. I was also thinking I was loosing sample during the SPRI clean ups, I mean using the same 100ng input with other kits I get higher pre-capture yields.

In anycase I will be following this thread for any updates.
Thanks!

So I cut my reaction volume considerably with a lot of other deviations from the normal protocol (including PCR-free) so I don't think a direct comparison is useful. The only thing I can say is that it performs a lot better (nearly 25% better) than Qiagen's QIAseq FX kit with the same template input.