Abstract
Assigning metagenomic reads to taxa presents significant challenges. Existing approaches address some issues, but are mostly limited to metabarcoding or optimised for microbial data. We present PIA (Phylogenetic Intersection Analysis): a taxonomic binner that works from standard BLAST output while mitigating key effects of incomplete databases. Benchmarking against MEGAN using sedaDNA suggests that, while PIA is less sensitive, it can be more accurate. We use known sequences to estimate the accuracy of PIA at up to 96% when the real organism is not represented in the database. For ancient DNA, where taxa of interest are frequently over-represented domesticates or absent, poorly-known organisms, more accurate assignment is critical, even at the expense of sensitivity. PIA offers an approach to objectively filter out false positive hits without the need to manually remove taxa and so make presuppositions about past environments and their palaeoecologies.
Highlights
Next-generation sequencing allows detailed metagenomic analysis of a wide range of ancient samples
We present Phylogenetic Intersection Analysis (PIA) as a taxonomic binner which, like MEGAN, works from gold-standard BLAST output and is not designed for microbial data, yet goes further to address the shortcomings of BLAST and databases
This study evaluates PIA by benchmarking its performance against MEGAN with empirical and simulated data
Summary
Next-generation sequencing allows detailed metagenomic analysis of a wide range of ancient samples. Identifying ancient metagenomic sequences is still a challenge, for shotgun data. Shotgun sequencing has three key advantages over metabarcoding for ancient metagenomics. It can capture information from anywhere in the genome, greatly increasing sensitivity. Damage accumulates in DNA over time (Kistler et al, 2017), so is important for authentication of ancient reads, and occurs most rapidly on the single-stranded overhangs at the ends of molecules. Shotgun sequencing can potentially sequence whole molecules, especially when fragments are short, as is the case for ancient DNA. Shotgun data has the potential to supply highly sensitive and informative metagenomic data
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