trooogdooor

Hi Charon, We will be doing some genotyping (SNP analysis, etc), but we will also be doing WGS, and the WGS part is crucial! You're right that it's a large number, we'll be doing hundreds, and thousands in a few years - that's also why I started out saying that my question is "high-stakes". The WGS aspect is the reason I am concerned about DNA integrity. Certainly, the main purveyors of the technology, like Oragene and Norgen, have used their RT saliva kits for WGS: http://media.completegenomics.com/documents/Saliva_Application_Note.pdf http://www.dnagenotek.com/ROW/pdf/MK-00014.pdf I think your answer is pretty much the same one as I would give, though - IE, go forth and have a test run with the preservative, as devised. It's what needs to be done no matter what, I just want to make sure that my proposed list of chemicals isn't crazy. 1% (v/v) Triton X-100 (lysis) 1% (w/v) sodium deoxycholate (lysis) 0.1% sodium azide (biocide) 1% sodium dodecyl sulphide (biocide, antiviral) 50 mM Tris Hcl, pH 8.0 (buffer) 5 mM EDTA (chelation buffer, PI) 1 mM AEBSF, also known as 4-(2-aminoethyl)-benzesulfonyl fluoride–HCl (PI) 2 μg/ml aprotinin (PI) 100 μM leupeptin (PI) 1 μg/ml cystatin (PI) 1 mM benzamidine (PI). I'll be doing that in a week or two. And maybe call you Chiron from now on, thanks for your advice.

We'll be using Illumina Hiseq, right. We'll actually be aiming for 4-5ml of saliva, rather than the industry standard 2ml, for the reasons you cite. I know for sure that next-gen sequencing is done using these kits, it's the norm for some sequencing studies. These RT saliva kits are also used by all the major genotypers (23andme etc), which of course use an order of magnitude less DNA; but the studies claim essentially unchanged DNA levels for years. I'm curious, what do you think that Norgen and Oragen do to get this result, if not something similar to the preservative I posted above? They seem to have published papers demonstrating stability at RT for years, how do you think they achieve this?

You guess correctly, it's for whole genome sequencing and genotyping, unfortunately we can't PCR it for that. They do show very slight degradation in their DNA over a period of years, right. For our purposes, we plan to store the DNA for ca. 3 months, though, that's really all we need. Reading up on this, I do agree that the addition of protease inhibitors would be wise for long term storage. All the solutions with PI's I read up on basically say "protease inhibitor cocktail", or something equivalent, and it seems that the default cocktail is something like: 1 mM4-(2-aminoethyl)-benzesulfonyl fluoride–HCl, 2 μg/ml aprotinin, 100 μM leupeptin, 1 μg/ml cystatin, and 1 mM benzamidine . So I guess this makes my preservative: 1% (v/v) Triton X-100 (lysis) 1% (w/v) sodium deoxycholate (lysis) 0.1% sodium azide (biocide) 1% sodium dodecyl sulphide (biocide, antiviral) 50 mM Tris Hcl, pH 8.0 (buffer) 5 mM EDTA (chelation buffer, PI) 1 mM AEBSF, also known as 4-(2-aminoethyl)-benzesulfonyl fluoride–HCl (PI) 2 μg/ml aprotinin (PI) 100 μM leupeptin (PI) 1 μg/ml cystatin (PI) 1 mM benzamidine (PI). Sensible? I've also seen mention of Bestatin, E64, and Pepstatin A. Do you think that adding more PI's is the safer course of action when in doubt?

@CharonT: You make some solid points. I've removed DTT and NaCl, and added a new biocide, aimed at virus: 50 mM Tris Hcl, pH 8.0 (buffer) 1 mM EDTA (chelation buffer) 1% (v/v) Triton X-100 (lysis) 1% (w/v) sodium deoxycholate (lysis) 0.1% sodium azide (biocide) 1% sodium dodecyl sulphide (biocide, antiviral) The process is like this: 1) Spit kit gets sent out to donor. 2) Donor spits into tube, adds preservative, sends it back. 3) Lab gets the kit and processes it, possibly 2+ months later. So, freezing just can't be done, unfortunately. * Apparently, there are many commercial services who have this problem cracked. Check this, for example: http://www.norgenbio...duct.php?ID=385 They've released papers showing virtually no DNA degradation after several years in this preservative. So, it's definitely doable, the question is only how?

Hello, noble nerds, I ask for your advice on a high-stakes problem. I'm going to be collecting a large number of saliva samples, adding a preservation liquid to them, and then transporting them by mail at room temperature. The liquid basically needs to do three things: 1) lyse the saliva cells 2) provide anti-microbial protection (gram positive, gram negative, fungal) 3) buffer the DNA for preservation. There are already some commercial vendors of such a preservation liquid. But they're extremely expensive. 15+ USD for a single sample! And I need to transport thousands, this is just beyond my budget. * So, I've stitched together the below lysis solution. It's meant to store the DNA for ca. 3 months. 50 mM Tris Hcl, pH 8.0 (buffer) 1 mM EDTA (buffer) 1% (v/v) Triton X-100 (lysis) 1% (w/v) sodium deoxycholate (lysis) 100 mM NaCl (lysis) 10 mM DTT (lysis) 0.1% sodium azide (biocide) What I'm not sure about is, will this be antimicrobial enough? Sodium Azide kills gram-negatives, but what about gram-positives and fungals, should I add something more for those? Am I missing some obvious and elementary reason why this wouldn't work? Of course, I intend to do testing before I use it on scale, but there's no sense in wasting time if I'm doing something obviously stupid. Thanks, guys.