Quantitative Metabarcoding of Pollen for Security-Related Forensics 8.2
Abstract
RATIONALE: Pollen has substantial potential utility as a biomarker in forensic studies, 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 taxa. Here we propose to develop methods to make pollen DNA metabarcoding quantitative. Background: DNA metabarcoding involves (1) isolating DNA; (2) amplifying a particular barcoding marker from that DNA to make many copies; (3) sequencing those copies simultaneously; and then (4) matching those sequences to databases built from positively identified species. The lack of quantitation comes from three different sources of variation: isolation bias, amplification bias, and copy number variation. DNA isolation varies across species, due to different biochemistries and cell wall thicknesses, among other attributes. Barcoding markers may amplify differentially across species given variation in DNA sequences and biochemistry. Most genes have multiple copies per cell, and the copy number of the barcoding markers contained in pollen varies from species to species. OBJECTIVES: 1. Quantitative Bias Assessment: 1.1) assess the three major sources of quantitative bias (isolation, amplification, and copy number) across the seed plant lineage; 1.2) use phylogenetic imputation to predict bias values in unknown samples. 2. Bioinformatics Pipeline Development: 2.1) develop and test a bioinformatics pipeline to allow for quantitative correction from next-generation sequencing of mixed-species pollen samples; 2.2) to combine information from multiple barcode markers to improve taxonomic identification and quantitation; 2.3) test our pipeline against known constructed mixtures of pollen 3. Absolute Quantitation Exploration: explore the potential for absolute quantitation with particle counters.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- May 07, 2018
- Source ID
- W911NF1710382
Entities
People
- Berry J. Brosi
Organizations
- Army Contracting Command
- Emory University
- United States Army