Unexpected results when using a standardized and widely adopted approach

Where does this story come from?
Archeology - this story was told us by Kirsten Ziesemer M.Sc.

Tell us your horror story, what happened?
We had this great sample set composed of more than 100 archaeological samples (that is a lot considering that samples decay over time and usually result in smaller sample sets). So, with this dataset, we wanted to look deeper into the ancient DNA in the dental calculus. We used a standardized and widely used approach that targeted a specific region in microbial DNA (V3 region of the 16S rRNA gene). But, our results showed highly skewed microbial profiles, which looked nothing like we (biologically) expected. Such as exceptionally high frequencies of the human-associated archaeon Methanobrevibacter that cannot be explained by exogenous contamination. So we conducted a series of in silico and in vitro experiments to test whether this was an actual biological finding or that it was caused by the characteristics of ancient DNA (different than modern DNA).

Did you find a solution? How did this situation end?
It turns out, the specific region that we targeted has extensive length polymorphisms (or variations), and due to characteristics of ancient DNA, the microbial species that had the longest V3 region dropped out! Resulting in a highly skewed microbial profile, thus anyone that would like to study ancient microbial DNA should use a different method than the widely accepted and standardized approach that has been used so far. 
My research failure turned into a great publication in Scientific Reports.

Was there a lesson learned? How could this horror be avoided?
Sometimes you just gotta ‘dig’ a little deeper to find out what is going on. You might think that you’ve done something wrong, but there is another possibility; it might be that there is something wrong with the widely accepted methodology.

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Photo provided by Kirsten Ziesemer

Data horror_10b

Image from Warinner C, Rodrigues JF, Vyas R, Trachsel C, Shved N, Grossmann J, Radini A, Hancock Y, Tito RY, Fiddyment S, Speller C, Hendy J, Charlton S, Luder HU, Salazar-García DC, Eppler E, Seiler R, Hansen LH, Castruita JA, Barkow-Oesterreicher S, Teoh KY, Kelstrup CD, Olsen JV, Nanni P, Kawai T, Willerslev E, von Mering C, Lewis CM Jr, Collins MJ, Gilbert MT, Rühli F, Cappellini E. Pathogens and host immunity in the ancient human oral cavity. Nat Genet. 2014 Apr;46(4):336-44. doi: 10.1038/ng.2906. Epub 2014 Feb 23. PMID: 24562188; PMCID: PMC3969750. https://pubmed.ncbi.nlm.nih.gov/24562188/

The stories for this Data Horror Week were collected by the Research Data Management (RDM) Support desk at VU Amsterdam.

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