Author: Anastasia Khodakova
Khodakova, Anastasia, 2015 Comparison of methods for forensic DNA typing of soils, Flinders University, School of Biological Sciences
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Soil is encountered commonly during the course of a forensic examination yet rarely is it analysed. Currently, forensic comparison of soil evidence samples is limited to the analyses of mineral and elemental composition. Recent advances in high-throughput sequencing (HTS) opens the possibility for the most quantitative and accurate examination of genetic richness and diversity of soils. These methodologies provide the ability to generate unique metagenomic DNA profiles from a variety of soil types that can be applied to forensic soil discrimination. The focus of this study is to compare the ability of modern metagenomic approaches to examine genetic similarities and variations of soil biota and to discriminate soils taken from different geographical locations a few km apart. The three most commonly used methods to study the whole biota include: shotgun sequencing where random DNA fragments of the whole metagenome are analysed; whole genome amplification (WGA) which is performed for sequencing of limited amounts of available DNA material; and arbitrarily-primed PCR (AP-PCR) where a single primer selectively amplifies sections within an entire metagenome prior to sequencing. In addition, gene-specific sequencing based on the evaluation of 16S rRNA bacterial genes was carried out. Soil samples were taken from three different locations within the Adelaide urban residential area approximately 5 km apart. Two soils from similar land use and vegetation type could not be easily distinguished visually, while the third soil was of a distinctively different type. Replicate samples were collected to determine any variation during the year and reproducibility of data for samples collected from the same location. Initial studies determined the optimum extraction and storage processes for retrieval of the maximum amount of high purity DNA from each sample type. The extracted DNA was analysed by each of the four metagenomic approaches using the Ion Torrent sequencing platform. The sequencing data was then analysed using traditional comparisons with different reference databases. Data from random whole metagenomic sequencing was additionally analysed using reference independent comparative bioinformatics approaches. Multivariate statistical comparison of the soils’ metagenomic profiles, obtained by AP-PCR and 16S rRNA-based sequencing techniques, allowed for accurate discrimination of the soil samples according to their geographical locations. Shotgun and WGA sequencing approaches generated highly similar profiles such that the soil samples could not be distinguished. The AP-PCR-based approach was shown to be successful at generating reproducible site-specific DNA profiles for samples collected from the same location through the different seasons of the year. The results of our proof-of-concept study demonstrate for the first time that metagenomic PCR-based sequencing approaches are able to reliably discriminate between visually similar soil types sampled from close but different locations. This represents a significant step towards implementation of a metagenomic sequencing technique to discriminate soil samples for forensic practice.
Keywords: forensic DNA typing of soils, soil metagenomics, high-throughput DNA sequencing, arbitrarily-primed PCR
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Prof. Adrian Linacre