Developing the basic PrediCtoR system
The basic tool, delivered via a web-based interface, predicts the success of PCR amplification of DNA using a predictive model of DNA decay. PrediCtoR is a simple-to-use, free, non-destructive tool designed to enhance the conservation of museum and herbaria collections, by preventing unnecessary destructive sampling. It does this by predicting the success of PCR amplification based upon sample size and amplicon length
PrediCtoR normalises four of the key contributing factors to DNA (and collagen) decay, namely (i) age (ii) temperature (iii) specimen size and (iv) polymer (amplicon) length into a single parameter - thermal age (kyrs@10°C). ‘Thermal age’ normalises different samples and reports them on a common scale; in general the older the specimen the greater the projected accumulated DNA damage. The mathematics have been encoded in PHP programming language which is availavle open access.
Proof of Concept
The first part of the proof of concept was to establish that PrediCtoR successfully predicts temperatures for known sites. JRA1 compared PrediCtoR estimates of Teff with manually calculated values from actual sample sites and estimates taken directly from weather station data (n = 200). Outliers will be investigated further to establish reasons for any apparent anomalies. Two problems were obvious altitude and changing climate. JRA1 i) explored the problem of altitude, and incorporate the ICOA standard atmosphere lapse rate (of 6.4 °C km-1) into PrediCtoR and ii) produced estimates of Pleistocene temperature fluctuations. In order to achieve the latter long-term estimates of Pleistocene air temperature fluctuation (fixed to MAT estimates for the site) will be scaled using estimates of maximum depression at Last Glacial Maximum to estimate Teff for pre-Holocene samples.
The second part of the proof of concept was to use large datasets of geographically located samples with age, DNA success and amplicon length, to derive our first real estimate of the rate of decay of DNA in archive bone. This provided PrediCtoR with its first useful output, namely, the first prediction of percentage success (as a function of thermal age).
Task 1.3 Enhancement
The basic core of PrediCtoR will be extended to increase functionally. Initially this will be the implementation of a new collagen prediction algorithm, using an activation energy of 173 kJ mol-1. Collagen is another helical biopolymer, which is also prone to hydrolysis and subsequent melting, but as collagen makes up almost 50% of the total volume of bone, it is much easier to measure using non-invasive methods than DNA.
PrediCtoR normalises thermal age for amplification length and subsequently report to the User both the (a) the maximum amplicon lengths and (b) minimum sample sizes for which 25% 50% and 75% success rates are predicted. This can be computed using standard probabilistic modelling; observed fragment length patterns from non-targeted pyro-sequencing are consistent with random hydrolysis, and DNA concentration, as a general rule, directly scales with sample size. PrediCtoR aims to improve thermal age estimate by taking into account burial depth of the bone (this would invoke an exponential decay function on seasonal fluctuation) and soil type (by including a thermal diffusivity term).
The final version of the PrediCtoR Website will contain the facility to upload results of DNA extraction successes (which will also include details of PCR and ultimately sequencing success as the three elements are all part of the same result process to attain meaningful data). Equally importantly, records of failures will also be invited to enable wider understanding of the challenges and provide a platform to highlight where positive outcomes have been achieved for both User and collections managers. All potential users of museum archive specimens will be able to input information on a given specimen (including geographic location, type, size, extraction protocol, amplification protocols along with projected amplicon length and PCR amplification success.
Each of the Thermal-age.eu enhancements will be made available as soon as they are developed so the tool can be accessed by researchers.
Individuals may request to upload (current and archive) comparative data on temperatures in their museum stores, which can be used to estimate the effect of storage temperature on long-term DNA viability. Data from other fossil and bone collections will be sought as part of other projects.
For more information contact firstname.lastname@example.org.