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Old 07-22-2014, 01:08 PM   #1
kzarn
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Question Degraded gDNA & TruSeq Nano Kit

Hi All,

I am hoping to prepare libraries with Illumina's TruSeq Nano kit and although Illumina recommends only using high-quality genomic DNA, I am wondering if anyone has experience using degraded gDNA with this kit. Most of our samples are degraded to some extent, but some are high quality. I suspect that all or most of the DNA degradation occurred as a result of poor environmental conditions in the field prior to collection of muscle tissue from carcasses.

In hopes of keeping our sample size as high as possible, I'm wondering if it might be worth it to try to prepare libraries with the degraded DNA samples. Could I size select the samples to remove the smaller degraded fragments? I know that DNA damage is a considerable factor here, and want to avoid factoring that into data analysis further downstream...

Thanks! Replies are much appreciated!
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Old 07-23-2014, 02:43 AM   #2
nucacidhunter
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Quote:
Could I size select the samples to remove the smaller degraded fragments?
Smaller fragments will be cleaned after shearing and also after end repair, so an extra size selection will not make much difference.

I had some trouble interpreting your gel run. The agarose concentration and run voltage are too high for genomic DNA resolution. I am not sure if some DNA has stocked in the wells or not and it seems there is no DNA in some lanes.

The best that one can do with degraded DNA is to repair it first using DNA repair enzyme mix. Shearing condition in Illumina user guide is for high molecular weight DNA, so you might need to optimise shearing condition for your typical degraded sample to achieve fragment size distribution similar to recommended input DNA. Input DNA also can be increased to 2x to compensate for low quality and fragments that will not contribute to library because of excess damage.

Library can be prepared with Nano kit using even pg amount of DNA. The downside of preparing a library from very low amount and degraded DNA is that the library may not have high diversity or representation of some region might be affected. Sometimes these effects can be tolerated depending on the aim of experiment.
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Old 07-23-2014, 03:44 AM   #3
WhiteSeal
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Hi,

I have been using the TruSeq DNA/TruSeq Nano kit on degraded FFPE material for quite some time now. For the FFPE material we use 150ng qubit measured DNA as input and we know from experience that this gives us good results.
As we are using samples below the optimal standard, we have found that diluting the adapters 1:9 helps (use 5 ul of that dilution in the procedure). For FFPE material we add all of the cleaned-up ligation product into the pcr reaction (for good quality DNA we use half) and we run the pcr with 10 cycles (as for good quality DNA we only do 8 cycles).
After sampleprep we always measure the sample with the BioAnalyzer, but with low quality samples there might be problems with adapter-dimers in the final prep result (this is the reason we also dilute the adapter, but sometimes it doesn't help). If this is the case we do an AMPure XP beads cleanup and measure again before running on the MiSeq/HiSeq.


** update 20140724**
I did perform the prep with end-repair, but as I did not change anything in that part of the protocol I did not mention it.

Last edited by WhiteSeal; 07-24-2014 at 04:34 AM.
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Old 07-23-2014, 08:48 AM   #4
kzarn
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nucacidhunter - I did not see any DNA in the wells while looking at the gel on our UV transilluminator, but it would have been a bit difficult to see as I added so little sample to each well and the well indentations make it difficult to visualize remaining DNA. I was thinking that the quantification could have been inaccurate which could have resulted in over dilution of some of the samples, or perhaps they are so degraded that the smear is difficult to visualize when only using 100 ng. Thinking about it now, though...the high agarose gel percentage seems more likely to have been the problem. My mistake! Thanks for posting your thoughts on this!

I'll be running the rest of our samples (and rerunning these) on a new TapeStation that our Genomics Core just installed, so hopefully those results will be a bit more informative. I will post the results in case there is any current or future interest. We are waiting for Genomic DNA Screen Tapes to arrive so results will not be posted for at least a couple of days.

WhiteSeal - Thanks for your input! Very helpful. Do you perform DNA repair on your samples? (Assuming you don't because it wasn't mentioned, but want to double check.) What is your target insert size? Have you run your degraded gDNA samples on a gel / do they look similar to the samples that we can see on our gel? I have never worked with FFPE samples and am not familiar with how FFPE storage affects / damages DNA.
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Old 07-23-2014, 10:12 AM   #5
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As a side note, we will be looking to determine variable SNPs, genotyping individual variation, and using individual variation to examine population structure and landscape trends. So we are hoping to avoid miscoding errors as much as possible given our starting material...

Any favorite or recommended DNA repair enzyme mixes?
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Old 07-23-2014, 05:06 PM   #6
nucacidhunter
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Quote:
After sampleprep we always measure the sample with the BioAnalyzer, but with low quality samples there might be problems with adapter-dimers in the final prep result (this is the reason we also dilute the adapter, but sometimes it doesn't help).
Presence of adapter in final library even after diluting adapters is a good indication that library preparation method has been less robust and only sub-nanograms of input has been converted into library, which is expected from low quality input.

Quote:
Any favorite or recommended DNA repair enzyme mixes?
I am only aware of NEB PreCR Repair Mix which repairs few common damages.
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Old 07-23-2014, 05:17 PM   #7
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Genomic Screen Tapes arrived today! For a (small) comparison...agarose gel samples 1 and 2 are the same as Tape Station samples F2 and H2, respectively.

While the Tape Station results still suggest pretty significant degradation, they give me a bit more hope than the agarose gel results did...
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Old 09-24-2014, 02:15 AM   #8
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Hi, did you shear your FFPE gDNA using covaris? In my opinion, even the FFPE DNA was degraded, it is still way more larger than the shared DNA fragments we used for WGS library construction.

Last edited by wangwde; 09-24-2014 at 11:59 PM.
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Old 09-24-2014, 06:56 AM   #9
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In my current protocol we share our gDNA with the covaris (duty cycle=10%,intensity=5.0, burst/sec=200, t=240sec, mode=frequency sweep) and we our getting good results. Without the shearing we also find that there are still very large fragments in the 'degrade' FFPE gDNA.

I have received a tip recently, but I have not tried it yet, where it was said that the shearing of the gDNA with the covaris throws the gDNA up into the covaris tube, thus leaving some long fragments to remain in the tube... recommendations: remove tube each minute and give it a short spin, thus making sure all the gDNA is fragmented.
Does anyone has any experience with this?
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Old 09-24-2014, 01:56 PM   #10
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WhiteSeal - I was originally trying to fragment 52Ál of DNA in the Covaris MicroTubes (snap-tops), and was worried about sample splashing up on the sides of the tubes during fragmentation. Our Genomics Core manager said that completely filling the tube with 132Ál of DNA sample will lead to more efficient and even fragmentation. I have been using 132Ál of DNA sample in my fragmentation protocol and have seen much narrower size ranges post fragmentation.

If you have enough DNA sample you might try to completely fill the tubes rather than try to fragment samples in partially filled tubes and deal with spinning them down midway through the fragmentation protocol.
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