Generally, this project uses RCF assistance with the integration of new analytical technology into existing research efforts, enabling alternative research methods. Specifically, this project involves the development of new molecular identification protocols using a MinION on-site sequencer for entomological taxa that play a role during the process of decomposition.
As a corpse decomposes, different groups of arthropods and insect arrive on the remains to consume it, to pray of necrophagous species or to find shelters and potential mates. The successional pattern of the different groups is predictable and can be used to help build a timeline of decomposition and therefor can assist in the estimation of the minimum postmortem interval (mPMI). Among the first colonizers of a decaying corpse, there is the family of blow flies (Diptera: Calliphoridae); they are known to locate remains within minutes from exposure. Shortly after they arrive, blow flies start their colonization by laying eggs and once these hatch, the maggots start consuming the body. For this reason, the development cycle of blow flies has been abundantly used to estimate time of colonization (TOC) or mPMI. However, being poikilotherms, their growth rate depends on temperature; simply put the warmer the temperature, the faster they develop, and the colder the temperature, the slower their growth. Development though is also species specific; different species will develop at different rate and therefore, it is incredibly important to properly identify the species before conducting any type of forensic entomological analysis. The family Calliphoridae is comprised by thousands of species and their morphological identification is not always possible due to the lack of dichotomous keys or to damaged specimens whose morphology is no longer intact. In these cases, molecular identification can help. Molecular species identification is also known as barcoding and when it comes to insects of forensic interest, this method normally utilizes mitochondrial genes COI and COII. The traditional methodology consists in the enzymatic extraction of DNA, quantification normally via nanodrop, PCR, gel electrophoresis, sequencing and then comparison of the obtained sequences against the sequences of public databases such as the BOLD Systems (https://www.boldsystems.org). This process may be resource intensive and time consuming, imposing limits on the research.
The MinION sequencer, developed by Oxford Nanopore Technologies represents a way to obtain sequences of any taxa directly on site, without the need of all the traditional steps of molecular analysis. This technology requires an equipment investment, allows for preparation of sequence libraries, and the device itself is small and portable. The on-site sequencer connects to a laptop to download the sequences that can then be analyzed using the MinION software. This system would allow forensic scientists to perform on-site molecular analysis of immature specimens, damaged specimens and even insect fragments. In addition to being a great tool for forensic analyses, this technique will also help in environmental investigations and contribute greatly to biodiversity studies.
The project we propose seeks to build a library of sequences of insect taxa of forensic interest using the Oxford Nanopore MinION system and to validate our library in the field using samples collected from decomposing swine carcasses.