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Metagenomics Pipeline IDs Smashed Bugs, Plants, and More from Windshield Samples

NEW YORK (GenomeWeb News) – Metagenomic analyses of bug, plant, and other debris stuck to a car after a long trip can provide information about the organisms and species diversity present in different regions, according to a paper appearing in the advance, online edition of Genome Research today.

Members of the Galaxy, a collaborative project funded by the National Science Foundation, Pennsylvania State University, and the Huck Institutes of the Life Sciences, developed a phylogenetic profiling pipeline for identifying species using comparisons between metagenomic sequence reads and existing sequence databases.

"[O]ur objective was to build a complete pipeline for homology-based taxonomic labeling of metagenomic reads that was self-contained, and guided the user from data acquisition and quality control, to database searchers, and finally, actual metagenomic analyses," lead author Sergei Kosakovsky Pond, an infectious disease researcher from the University of California at San Diego, and co-workers wrote.

For the current study, they applied this approach to DNA extracted from material stuck to the windshield, bumper, and sides of a car following two different road trips. Their results turned up sequences representing numerous species, including insects, plants, and bacteria. And, based on their results so far, the team says metagenomic studies of eukaryotic species are feasible.

Although most metagenomics studies have been used to identify microbial species, they noted, the metagenomics approach also holds promise for sampling other types of biodiversity in the environment. For instance, they explained, only a fraction of the estimated 30 million insect species on Earth have been identified and studied.

In an effort to both test their metagenomics pipeline and explore the potential of eukaryotic metagenomics, the researchers examined material stuck to a car with double-sided tape after two separate trips: one from Pennsylvania to Connecticut and another from Maine to New Brunswick.

After each trip, the researchers removed the insect and other remains from windshield, bumper, and sides of the car and extracted DNA, which they sequenced using the Roche 454 FLX platform.

They then plugged this sequence data into the Galaxy metagenomics pipeline, which had been validated using Sargasso Sea metagenomics data published in a 2004 paper by J. Craig Venter Institute researchers.

Their approach relied on comparing metagenomic sequence reads to sequence databases rather than using protein or gene-based identification methods that could be complicated by the taxonomic complexity in the windshield and bumper samples.

Most of the reads they detected were bacterial, with Gammaproteobacteria turning up most often in samples from both trips.

In terms of insect material, the researchers reported, reads representing insects from the Diptera and Hemiptera orders were most common, with Acyrthosiphon being the most common insect genus.

Since the Acyrthosiphon genus includes the pea aphid — an invertebrate that relies on an endosymbiotic bacterial species called Buchnera aphidicola — the researchers also decided to look at whether levels of Acyrthosiphon and B. aphidicola corresponded with one another. Indeed, they reported, both appear to have been more common on the Maine to New Brunswick trip than the Pennsylvania to Connecticut trip.

The team also detected different amounts of plant material on each of the trips. While they got 10,242 green plant reads in material from Pennsylvania to Connecticut trip samples, they found only 612 from the Maine to New Brunswick samples.

But because most of the green plant material was limited to one side of the car in the first trip, the team suspects their findings may not reflect real differences in plant diversity but simply reflects plant material that got stuck to the car.

The researchers concede that they may have missed some species that are closely related to other, better-represented species in sequence databases. And, they say, the method has some limitations, since it only detects species resembling partially or completely sequenced species. Still, as more species are sequenced, the resolution should improve, the team noted.

"The dramatic drop in sequencing costs and increase in throughput will make thorough cataloging of biodiversity possible," the researchers concluded. "We are optimistic that we will soon be able to sequence genomic DNA from most holotypes available from major museum collections."

Data from the study are available through the national Center for Biotechnology Information or the Galaxy Library System. The analysis steps and open-source software used in the study are available in supplementary information accompanying the paper.

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