RELATE comparison of Bray-Curtis similarity matrices.

<p>The Bray-Curtis similarity matrices calculated from square root transformed abundance of DNA fragments generated based on full datasets and sub-sampled datasets.</p

Results of CAP model cross-validation of soil metabolic profiles discrimination generated from full sequencing datasets.

<p>Results of CAP model cross-validation of soil metabolic profiles discrimination generated from full sequencing datasets.</p

Random Whole Metagenomic Sequencing for Forensic Discrimination of Soils

<div><p>Here we assess the ability of random whole metagenomic sequencing approaches to discriminate between similar soils from two geographically distinct urban sites for application in forensic science. Repeat samples from two parklands in residential areas separated by approximately 3 km were collected and the DNA was extracted. Shotgun, whole genome amplification (WGA) and single arbitrarily primed DNA amplification (AP-PCR) based sequencing techniques were then used to generate soil metagenomic profiles. Full and subsampled metagenomic datasets were then annotated against M5NR/M5RNA (taxonomic classification) and SEED Subsystems (metabolic classification) databases. Further comparative analyses were performed using a number of statistical tools including: hierarchical agglomerative clustering (CLUSTER); similarity profile analysis (SIMPROF); non-metric multidimensional scaling (NMDS); and canonical analysis of principal coordinates (CAP) at all major levels of taxonomic and metabolic classification. Our data showed that shotgun and WGA-based approaches generated highly similar metagenomic profiles for the soil samples such that the soil samples could not be distinguished accurately. An AP-PCR based approach was shown to be successful at obtaining reproducible site-specific metagenomic DNA profiles, which in turn were employed for successful discrimination of visually similar soil samples collected from two different locations.</p></div

Comparison of the metabolic soil profiles generated on full datasets at the subsystems level 1 (A, B, C) and subsystems function (D, E, F) resolution levels.

<p>Bray-Curtis distance similarity matrix was calculated from the square-root transformed abundance of DNA fragments matching taxa in the SEED database (E-value <1×10⁻⁵). The Bray-Curtis matrix was used for generating CLUSTER dendrogram, NMDS and CAP ordination plots. <b>CLUSTER analysis (A and D).</b> Red dotted branches on the CLUSTER dendrogram indicate no significant difference between metagenomic profiles (supported by the SIMPROF analysis, p<0.05). <b>NMDS unconstrained ordination (B and E).</b> The NMDS plot displays distances between samples. Data points that are closer to each other represent samples with highly similar metagenomic profiles. <b>CAP constrained ordination (C and F).</b> CAP analysis tests for differences among the groups in multivariate space. The significance of group separation along the canonical axis is indicated by the value of the squared canonical correlation (δ₁²) and P-value. A contour line on the NMDS and CAP ordinations drawn round each of the cluster defines the superimposition of clusters from CLUSTER dendrogram at the selected level of similarity.</p

Results of CAP model cross-validation of soil taxonomic profiles discrimination generated from full sequencing datasets.

<p>Results of CAP model cross-validation of soil taxonomic profiles discrimination generated from full sequencing datasets.</p

General characteristics of full sequencing data.

<p>Statistical data represented as mean ± Standard Deviation (SD). Percentage of sequences matching to the M5NR, M5RNA and SEED Subsystems databases was determined with an E-value cut-off of E<1×10⁻⁵. QC = quality control.</p

Comparison of the taxonomic soil profiles generated on full datasets at the phylum (A, B, C) and species (D, E, F) resolution levels.

<p>Bray-Curtis distance similarity matrix was calculated from the square-root transformed abundance of DNA fragments matching taxa in the M5NR database (E-value <1×10⁻⁵). The Bray-Curtis matrix was used for generating CLUSTER dendrogram, NMDS and CAP ordination plots. <b>CLUSTER analysis (A and D).</b> Red dotted branches on the CLUSTER dendrogram indicate no significant difference between metagenomic profiles (supported by the SIMPROF analysis, p<0.05). <b>NMDS unconstrained ordination (B and E).</b> The NMDS plot displays distances between samples. Data points that are closer to each other represent samples with highly similar metagenomic profiles. <b>CAP constrained ordination (C and F).</b> CAP analysis tests for differences among the groups in multivariate space. The significance of group separation along the canonical axis is indicated by the value of the squared canonical correlation (δ₁²) and P-value. A contour line on the NMDS and CAP ordinations drawn round each of the cluster defines the superimposition of clusters from CLUSTER dendrogram at the selected level of similarity.</p