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Old 07-05-2017, 02:13 AM   #1
danthescienceman
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Question What's the best strategy for sampling to get high molecular weight DNA

Hi all,

there's a lot of chat about extraction tekkers to get HMW DNA so i think we've got that covered; however what i can't find is anything on sampling strategies.

Specifically what is the best way to go from raw tissue to HMW DNA if you CAN'T extract immediately after collection and then sequence straight away.

Is it best to freeze tissues (and at what temp, does tissue type [plant/animal etc] affect the outcome) close to collection and store until extraction/sequencing?

Or is it better to sample and extract immediately then store the DNA (4/-20/-80/LN? and what buffer? or plugs?) until sequencing? And should you make the libraries before storage or when ready to sequence?

I've heard anecdotally that -20 freezing of DNA can reduce the length of fragments significantly....

any help/advice appreciated

this was not so much of a problem with illumina seq
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Old 07-07-2017, 12:02 PM   #2
FlausFSU
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Perhaps a stabilization reagent like AllProtect Tissue Reagent?

https://www.qiagen.com/us/shop/sampl...inginformation
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Old 07-10-2017, 02:15 PM   #3
Carcharodon
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There's a lot to unpack here, but I can give some advice!

Freezing immediately is great, so long as you have access to those temperatures at the time of sampling. If you can take along, say, some liquid nitrogen (I know people who do this), then you're golden. But the time between sampling/death of the organism and preservation is crucial. This can also depend on what type of tissue you're sampling. Liver gives enormous DNA yields, but tends to degrade much more quickly than, say, muscle tissue.

It's the timing here that is the critical factor. For example, I've sampled shark tissues on 2x consecutive dives where samples collected earlier in the dive stayed with me for ~60 min underwater. It was CA, so the water was chilly, but not that cold. But I tended to notice that samples collected on earlier dives - or even earlier in the dive - tended to give more degraded DNA than the ones that were more immediately preserved/frozen (we'd take the boat back to the lab, and freeze at -80 C). Sometimes the difference was small, even negligible. Other times it really seemed to make a big difference.

If you can't freeze the tissue straight-away (as is the case in most field-situations), a preservative is really the way to go. I saw a paper recently that showed that DMSO gives lower DNA yield than ethanol, but tends to yield higher-MW DNA overall. I'll try to find it.

(There's also RNA-Later, which I tended to use in the field. I used it with mixed success. With RNA-Later, it's important to cool/freeze the tissue gradually... -4 C, to -20 C, to -80 C.)

With regard to preserving in tissue vs. immediate extraction, it follows (from above) that preservation before extraction is still critical. That delay will cause degradation, unless you're literally walking outside, grabbing a live specimen, and walking it back into the lab with you to extract DNA.

Finally, with regard to freezing degrading DNA, what's important is limiting freeze-thaw cycles as much as possible to avoid shearing DNA. At least, that's the common wisdom. But I HAVE heard/read that there's very little evidence (beyond the anecdotal) to suggest that this really does cause significant shearing. Still, best to be cautious.

tl;dr: Unless you can freeze this stuff in the field, put it in a preservative. Some work better than others. I recommend preserving before extracting. Freeze-thaw is a contentious subject, but convention is to avoid too many cycles. An initial freeze most likely won't hurt much at all.
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Old 07-11-2017, 01:25 AM   #4
danthescienceman
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Default thanks, plus follow up question

great advice, it fits with what i was thinking

Quote:
Originally Posted by Carcharodon View Post
There's a lot to unpack here, but I can give some advice!

It's the timing here that is the critical factor. For example, I've sampled shark tissues on 2x consecutive dives where samples collected earlier in the dive stayed with me for ~60 min underwater. It was CA, so the water was chilly, but not that cold. But I tended to notice that samples collected on earlier dives - or even earlier in the dive - tended to give more degraded DNA than the ones that were more immediately preserved/frozen (we'd take the boat back to the lab, and freeze at -80 C). Sometimes the difference was small, even negligible. Other times it really seemed to make a big difference.

[/b]
Did you notice if the size, or rather dimensions, affected things, i'd assume a larger specimen would freeze slower but is this noticeably different or did you chop them up first?

btw a few years ago i did some (admittedly basic) experiments on the affects of freeze thaw and found no noticeable degradation (but this was only checked on a gel as there was no long-read tech back then to worry about)
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Old 07-11-2017, 01:31 AM   #5
danthescienceman
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Default thanks, could be worth a shot

Quote:
Originally Posted by FlausFSU View Post
Perhaps a stabilization reagent like AllProtect Tissue Reagent?

https://www.qiagen.com/us/shop/sampl...inginformation
this seems a good idea, although limited to 5mm pieces could make some field work a little messy

it's also a bit pricey (527 for 100ml)
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Old 07-11-2017, 09:15 AM   #6
FlausFSU
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Quote:
Originally Posted by danthescienceman View Post
this seems a good idea, although limited to 5mm pieces could make some field work a little messy

it's also a bit pricey (527 for 100ml)
You can always reach out to your QIAGEN rep for a discounted price offer.

I believe the reason for the limited size of the tissue is to ensure the reagent can penetrate the entire tissue section in-time to stabilize the DNA. When the tissue is a huge chunk, you tend to get variability in your data as the outer edges of the tissue stabilization while the inner core doesn't get penetrated in time to prevent degradation.
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Old 07-11-2017, 01:14 PM   #7
Carcharodon
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Quote:
Originally Posted by danthescienceman View Post
great advice, it fits with what i was thinking



Did you notice if the size, or rather dimensions, affected things, i'd assume a larger specimen would freeze slower but is this noticeably different or did you chop them up first?

btw a few years ago i did some (admittedly basic) experiments on the affects of freeze thaw and found no noticeable degradation (but this was only checked on a gel as there was no long-read tech back then to worry about)
I actually hadn't noticed, but the fin-clips I collected were of different sizes (up to the size of a thumb-nail for adults, and maybe as small as half the area of a pinky-nail for neonates.

I goofed up and hadn't scored/diced the tissue, so when extracting DNA, I tend to cut and subsample from the margins of the tissue (following the logic of FlausFSU re: preservative soaking and efficiency).

Also, good to know (about the freezing experiment)!

In terms of freezing, it's hard to say. Often the samples went into a -80 C freezer directly. At that temperature, things freeze very quickly. But other times they had to sit in a cooler of water-ice, or were in a home freezer.

In short, I just don't know. But it is generally good practice to cut tissue into small chunks before placing them in any type of perservative.

I did have a colleague who put whole gobies into vials of RNA-Later. Usually when people take a whole animal, they make sure to open them up a bit so the preservative/fixative can soak through cell layers inside and out.

Another consideration (my apologies if you already know all of this, but it came to mind): when dealing with a dehydrating agent like ethanol (or even RNA-Later), if you have BIG tissue chunks, it's a good idea to let them sit in the preservative overnight, then replace the preservative with a fresh batch. This is because the water content of the sample can actually dilute the preservative somewhat significantly!

Last edited by Carcharodon; 07-11-2017 at 01:16 PM.
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