Equipment for the Quantitative Metabarcoding of Pollen for Security-Related Forensics
Abstract
This proposal requests an integrated set of equipment for enhancing quantitation in the metabarcoding of pollen. Pollen has substantial potential utility as a biomarker in forensic studies, including security-related forensics, due to its ubiquity, its durability, and the specificity of its geographic and temporal signatures. However, its potential has been unrealized because traditional visual microscopic identification is slow, low-resolution, and expensive. The development of pollen "DNA metabarcoding", the genetic identification of mixed-species samples, seems poised to transform this process. But metabarcoding suffers from a key shortcoming: it provides only data on pollen presence, and not on relative abundance. But knowing whether a particular pollen type represents 1% vs. 80% of grains in a sample is key for forensic applications, including much finer spatial and temporal resolution of pollen signatures, and improved limits of detection for low-abundance pollen types. The requested equipment will contribute to research currently proposed to the DoD that seeks to make metabarcoding quantitative. A key need in quantitation for pollen metabarcoding is to carefully quantify the counts of pollen going in to our samples, which we will accomplish with a (1) particle counter system. Even ideal quantification based on molecular genetic methods can only yield relative proportions of different species in a sample, but particle counting of pollen will also allow for us to conduct absolute as well as relative quantitation of pollen. To operate the particle counter system at peak precision, we request (2) a sonicator and needed accessories to disaggregate pollen samples, which otherwise have a strong tendency to clump together, reducing precision in particle counting systems. In addition to these particle counting-related components, we also request equipment that will allow us to conduct the molecular genetic work central to this proposal. This includes a (3) replacement set of standard manual pipettes and (4) an inexpensive robotic pipettor to greatly expedite the range of pipetting activities involved in our workload, including DNA extraction, PCR, sample purification, and preparation for next-generation Illumina sequencing. A dedicated fast-processor computer is required for operation of the robotic pipettor and is also requested as part of the robotic pipettor setup. Both manual and robotic pipettors are requested as some tasks- particularly with small sets of samples, and some steps of DNA extraction-are more efficient to conduct manually whereas others- including PCR setup for large sets of samples and setup for Illumina sequencing-are much more efficient (and accurate) to batch-process with a pipetting robot. The equipment proposed here is integral to the success of the ARO proposal in review and will allow us to unlock the potential quantitative power of DNA metabarcoding of pollen for both forensic and other applications.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Feb 14, 2019
- Source ID
- W911NF1810187
Entities
People
- Berry J. Brosi
Organizations
- Army Contracting Command
- Emory University
- United States Army