Evaluation of targeted Massively Parallel Sequencing methods for forensic metagenomics

Area of Science:

• Microbiology
• Forensic Science
• Bioinformatics

Background:

• Soil microbial communities are crucial forensic indicators for geographic origin determination.
• Standard 16S rRNA sequencing lacks the species-level resolution needed for precise forensic analysis.
• Targeted sequencing panels offer enhanced resolution for forensic investigations.

Purpose of the Study:

• To evaluate and compare three target enrichment methods for forensic metagenomic soil analysis.
• To assess the performance of amplicon capture versus hybridization capture methods.
• To identify the most accurate method for forensic soil microbiome profiling.

Main Methods:

• Whole Metagenome Sequencing (WMS) data from 134 Polish soil samples were used to identify 200 informative markers.
• Prototype targeted sequencing panels were created using amplicon capture (AmpliSeq™, xGen™) and hybridization capture (KAPA HyperPlus).
• Machine learning classifiers trained on WMS data evaluated the classification accuracy of sample origins.

Main Results:

• Performance varied by technology and DNA concentration; hybridization capture (Roche KAPA HyperPlus) showed superior results.
• Roche KAPA HyperPlus demonstrated the highest correlation with WMS data across various DNA inputs.
• An F1 score of 0.94 at 5 ng DNA input was achieved with KAPA HyperPlus, significantly outperforming other methods.

Conclusions:

• Hybridization capture methods, specifically Roche KAPA HyperPlus, provide a robust and accurate approach for forensic soil microbiome profiling.
• Targeted sequencing is highly effective for determining the geographic origin of soil samples, especially with low DNA quantities.
• Roche KAPA HyperPlus offers a reliable tool for forensic laboratories to predict the geographic origin of soil evidence.