The Ketchum Report (Continued)

[Guest]

primers could effect the outcome of the sequencing by amplifying something other than that which was planned. Frequently when you do a pcr reaction, you get spurious bands, which are usually just labeled as artifacts - and usually have nothing to do with the intended target. I think the "unknowns" in the manuscript are just artifacts. If you sequence these spurious bands, you will come up with "unknown" sequences. Occasionally one set of primers might pick up other related genes or pseudo genes, but these would not be unknowns.

what process would be used to verify the amplified target was the only one amplified ?

[southernyahoo]

one more time, you do not use primers in Next gen sequencing!

Not all of the sequencing was next generation!

[Guest]

ridgerunner said:

very easy - they are just short pieces of DNA that are used to "prime" DNA synthesis in either PCR or sequencing reactions. Today it typically cost about 5-10 dollars and can be made often overnight. Once you know your target sequence, it is quite simple.

Okay, the primer Melba created dealt with the nuDNA, is that correct? And if that is correct wasn't it the nuDNA she called novel - and had develop the primer? If she developed a primer to isolate what she considered novel - would she have shared this primer with another lab so they could confirm her results?

Thanks

[Guest]

what process would be used to verify the amplified target was the only one amplified ?

One would typically analyze the size of the amplified product on a electrophoresis gel (like Figure 11). If there is one band, there is typically one product, where as if there are multiple bands, then this indicated multiple products. It is possible for two distinct product of similar enough mass that they run as one band. In this case, sequencing of the pcr product would lead to overlapping sequences. If it was two highly similar, but not identical sequences, you would get primarily one sequence, with any mis-matches showing up as a mixture of bases. If there are multiple bands, one can "gel purify" each product and sequence them independently.

[Guest Tyler H]

Okay stupid question time..

How could a primer adversely affect the outcome of sequencing?

In response to this, and to RR:

Well, at the time, I knew nothing about next-gen and was just throwing things out there to her, to see what she would say as to reasons our results differed so much.

[Guest]

Okay, the primer Melba created dealt with the nuDNA, is that correct? And if that is correct wasn't it the nuDNA she called novel - and had develop the primer? If she developed a primer to isolate what she considered novel - would she have shared this primer with another lab so they could confirm her results?

I believe all the novel or proprietary primers were for nuDNA - the mtDNA was 100% homologous to humans, and they used human primers and universal primers for the mtDNA sequencing.

Yes, it was the nuDNA that she is describing as novel, but exactly what she meant by "novel" I am unclear on - is that "non-human but primate like" or the unknown "doesn't match with anything".

I suspect she got the novel or unknown sequences initially by cross hybridization with human or universal primers (amplifying the product with some degree of mismatch of the primers). The pcr reaction and the products generated are very dependent on the conditions used - amount of "salt" in the reaction and the annealing temperature (the temp that you hybridize the primer to the DNA prior to new DNA synthesis). By lowering the temperature, you allow for less specific binding. Once you have the novel sequence, you can generate specific novel primers that are 100% homologous the that sequence.

To have another lab confirm her results, she would have to supply the primer sequences. But I am sometimes a bit uneasy with what is meant by confirm in this paper.

Edited March 12, 2013 by ridgerunner

[Guest thermalman]

Further to NDA's. They are not out of the question and promote secrecy for obvious reasons.

"So, those of you keeping your pulse on Bigfoot hunting reality entertainment should be aware that Spike has their own spin on the genre coming out this fall by the name of the Ten Million Dollar Bigfoot Bounty. In their answer to Finding Bigfoot, Sasquatch hunting teams from all over the county will be competing for the best, most definitive proof of the elusive beast in order to win the titular ten million bucks. Other than that, the specifics of the series have since been shrouded in secrecy and non-disclosure agreements."

[Guest]

Ridgerunner,

It's my understanding that when a scientist publishes a paper, they explain what they did to get their results - specifically to permit other scientists (the review panel as well as others who want to test the paper's findings and hopefully, replicate them) to fully evaluate their hypothesis, process and findings. So, since this was the paper submitted for peer review, shouldn't it be fully explanatory? It's already troublesome that the inadequate bits of data provided in her paper leave so many unanswered questions, according to most of the scientists who have commented on it so far, but why is there so much guesswork about how she did the work itself? Is it customary for scientists to have so many questions about how someone did their research once the paper is out? Just wondering.

[TimB]

Further to NDA's. They are not out of the question and promote secrecy for obvious reasons.

"So, those of you keeping your pulse on Bigfoot hunting reality entertainment should be aware that Spike has their own spin on the genre coming out this fall by the name of the Ten Million Dollar Bigfoot Bounty. In their answer to Finding Bigfoot, Sasquatch hunting teams from all over the county will be competing for the best, most definitive proof of the elusive beast in order to win the titular ten million bucks. Other than that, the specifics of the series have since been shrouded in secrecy and non-disclosure agreements."

Hmmm... I wonder if we have found the motivation for all the negativity? Present company excluded, of course...

[southernyahoo]

To have another lab confirm her results, she would have to supply the primer sequences. But I am sometimes a bit uneasy with what is meant by confirm in this paper.

I would think that in order to confirm her results, the other lab would have to arrive at the same sequence for a locus independently and without knowing what combination of primers she used.

[Guest njjohn]

How would this motivate negativity? The contestants are the only ones capable of winning the $10million. Anyone outside of the show isn't eligible. Ketchum's results wouldn't jeopardize the money of someone that's actually on the show.

And NDA's aren't new. They've been around for a long time. First one I ever signed was in '99 I believe. But trust me, if it's not in the NDA, it's not covered.

[Guest]

actually they did some more standard sequencing of several of the nuDNA genes described for which they generated new primers. These were targeted genes as SY stated.

RR, can you explain the difference between standard sequencing and next gen sequencing?

Further to NDA's. They are not out of the question and promote secrecy for obvious reasons.

"So, those of you keeping your pulse on Bigfoot hunting reality entertainment should be aware that Spike has their own spin on the genre coming out this fall by the name of the Ten Million Dollar Bigfoot Bounty. In their answer to Finding Bigfoot, Sasquatch hunting teams from all over the county will be competing for the best, most definitive proof of the elusive beast in order to win the titular ten million bucks. Other than that, the specifics of the series have since been shrouded in secrecy and non-disclosure agreements."

Yes, but in that case, the consideration is the possibility of winning $10 million for the contestants, or as a condition of employment if the crew had to sign them. In MK's case, I'm not seeing any such consideration.

Edit: removing brain fart whereby I thought PCR was sequencing, instead of amplification.

Edited March 13, 2013 by leisureclass

[Guest thermalman]

@ LC Even though we currently lack the knowledge and insight of MK's total NDAs, it wouldn't be to farfetched to think that consideration or compensation is not known by the all those who signed it. Otherwise, why sign off, if one doesn't feel fairly compensated?

I'll quote Plenipotentiary:

"Courts typically will not substitute their own judgment to replace the bargained for exchange to which the parties agreed. The individuals signing the NDA must have subjectively believed that they were receiving some benefit in the transaction. I haven't read that there was any duress or noticed evidence of something other than an arms length negotiation. Where the parties are in relatively equal position when entering into a contract, unconscionability is typically not a valid defense."

Edited March 13, 2013 by thermalman

[Guest BartloJays]

Bart Cutino

Alright best quote of the day by yours truly:

I told Justin, "You see I told you I'd take care of everything, I got you off murder charges and I've made these suckers still believe these things could be apes"

March 1 at 2:14pm

Posted on Team Tazer's FB page on march 1st.....

pretty much a nail in the coffin of the ''bear sample'' theory.

Darn.... you got me lol.

Everyone else is so ignorant they thought I was merely making a facetious comment on a public facebook page laughing at people so shallow and desperate to believe both in Justin making himself look like a hoaxer so he wouldn't go to jail and me desperately holding onto this "ape theory," I'm supposedly attached to by a few nutcases.

Hey grayjay, would you be ok with me going back and saying "grayjay" took this post literally and posted it on the BFF, so we can all have a good laugh at your expense?

[Guest]

I would think that in order to confirm her results, the other lab would have to arrive at the same sequence for a locus independently and without knowing what combination of primers she used.

Ideally, yes the lab would independently confirm the full result. But given that I believe the confirming labs were "contract" labs, I imagine they were primarily meant to confirm a finding - a pattern from the PowerPlex16 analysis, or confirming a sequence. Again, given the lack of data, confirmation may have only be at the level of "it looked like human".

For example, this segment from the paper by Ketchum et al, 2013 (my bolding)

"Samples 28, 33, 35 and 37 had sufficient DNA extracted and were chosen for MC1R locus sequencing. The primers used in the sequencing of MC1R were designed by DNA Diagnostics and additional primers by SeqWright to correspond with great apes, humans and Neanderthals. Upon sequencing, it was found that the samples indicated normal human MC1R sequence and carried alleles for red hair color in humans. Sample 28 and 37 presented a C/T at base 478 of the MC1R control region and Sample 35 exhibited G/C at base 880. (Table 6)

Samples 28, 35 and others were then sent to SeqWright to have the sequences confirmed with the design of new MC1R primers. As with other loci analyzed, MC1R analysis at SeqWright found partial human sequences in some DNA samples, while others had novel sequences and still others failed to amplify. All human control DNA amplified and sequenced successfully as before."

In the first part, they found samples that indicated normal human MC1R, but the SeqWright analysis found partial human sequences in some samples, while other had novel sequences and others failed to amplify. Does the latter confirm the former? Only in that they found some human sequence. Granted that 28 and 35 are the only two samples named in both studies, but the descriptions don't match imo. Naming samples "and others" makes any interpretation difficult if not impossible. Why were new primers used? Unclear.