Fragment size limit

[Gina_P]

Hi all, I have some libraries with fragments that look a bit on the large side (650 - 700 bp including adaptors) for re-sequencing. Should I just use these libraries anyway? Or possibly redo gel extraction? I saved the original gels.

Thanks!

[DMO]

What was your ave bp size post sonication?

Do you have a picture of your gel or BioA QC of the final product?

Whether or not you should move into sequencing will depend upon whether this was caused by less than suspected efficiency in your shearing method or due to an overamplification in the final enrichment of the library.

[Gina_P]

Hi DMO,

Thanks for answering me. I have a BioA QC. There are a few libraries that need to be cleaned up.

Also, apologies for being vague - this is for Illumina 2x50 sequencing!

[DMO]

Do you have a BioA trace post-sonication?

[Gina_P]

Unfortunately I don't... For now, I have recut the gels below my original extractions. I used the same shearing protocol on Covaris that I have used in the past, with good results, and I have never done a BioA QC check on shearing products. However, now I will probably do so in the future to make sure I the shearing went well... Any other advice? Thanks for answering!

[DMO]

I see smaller peaks on at least 2 of your traces which may indicate the samples were quite overamplified.

Can you answer a few more questions?

What is you shearing protocol?

What amount of material did you start with?

How many cycles of amplification did you do for your final enrichment?

[Gina_P]

I used the whole-genome resequencing protocol for Covaris:
40 seconds, 10% duty cycle, 5.0 intensity, 200 bursts per second, frequency sweeping mode, 6 degree temperature.

I started with 1 ug DNA.

10 cycles of amplification. Should I reduce?

[DMO]

You certainly could since you are getting plently of material, but based on these parameters I am less likely to jump to the overamplification conclusion. What size did you cut from your gel?

[Gina_P]

I cut from about 500 - 600, which was by accident. I meant to cut from 400 - 500. I have additional gel slices from 400 - 500 range saved in my fridge.

[DMO]

I would extract the DNA from those and amp those. I think you would be fine sequencing what you have, but you would have to scale back your loading concentration on your run a bit. So, if you finished prepping the other cut, you could load your seq run at a more standard concentration and acheive a higher number of reads out doing so.

[Gina_P]

Hm, good point about the concentration. Thanks for your help. I'm going to prep the other cuts today.

[Gina_P]

Here's the new BA result with smaller fragment size. There's still some problem with sample 9. For now, I'm not going to put it on the sequencer. It looks like the DNA is dragging along some smaller molecule with it, because a similar bump is in both of my different gel extractions. Also, I used Kapa Biosciences' library amp kit instead of the Illumina one this time...