Evaluating high‐throughput sequencing as a method for metagenomic analysis of nematode diversity

Abstract

Nematodes play an important role in ecosystem processes, yet the relevance of nematode species diversity to ecology is unknown. Because nematode identification of all individuals at the species level using standard techniques is difficult and time-consuming, nematode communities are not resolved down to the species level, leaving ecological analysis ambiguous. We assessed the suitability of massively parallel sequencing for analysis of nematode diversity from metagenomic samples. We set up four artificial metagenomic samples involving 41 diverse reference nematodes in known abundances. Two samples came from pooling polymerase chain reaction products amplified from single nematode species. Two additional metagenomic samples consisted of amplified products of DNA extracted from pooled nematode species. Amplified products involved two rapidly evolving ~400-bp sections coding for the small and large subunit of rRNA. The total number of reads ranged from 4159 to 14771 per metagenomic sample. Of these, 82% were > 199 bp in length. Among the reads > 199 bp, 86% matched the referenced species with less than three nucleotide differences from a reference sequence. Although neither rDNA section recovered all nematode species, the use of both loci improved the detection level of nematode species from 90 to 97%. Overall, results support the suitability of massively parallel sequencing for identification of nematodes. In contrast, the frequency of reads representing individual species did not correlate with the number of individuals in the metagenomic samples, suggesting that further methodological work is necessary before it will be justified for inferring the relative abundances of species within a nematode community.

Tree diversity and species identity effects on soil fungi, protists and animals are context dependent

“…Other studies of soil animals have typically relied on identification of organisms obtained from specifically extracted pools of individuals (Porazinska et al, 2009;Hajibabaei et al, 2011). That approach is more labour intensive and enables to address only specific group(s) of soil biota, usually resulting in capturing the most mobile subset of the target species that respond to light or bait.…”

“…The alternative DNA-based tools have been developed and increasingly used for identification of bacteria, protists and fungi since two decades ago. More recently, the massively parallel DNA metabarcoding technology has been adopted for large-scale community-level identification of fungi (Jumpponen and Jones, 2009), protists (Chariton et al, 2010;Medinger et al, 2010) and animals (Porazinska et al, 2009). For meiofauna, metabarcoding studies have focussed on specific order to phylum-level groups such as nematodes or certain arthropods (Porazinska et al, 2009;Hajibabaei et al, 2011).…”

“…More recently, the massively parallel DNA metabarcoding technology has been adopted for large-scale community-level identification of fungi (Jumpponen and Jones, 2009), protists (Chariton et al, 2010;Medinger et al, 2010) and animals (Porazinska et al, 2009). For meiofauna, metabarcoding studies have focussed on specific order to phylum-level groups such as nematodes or certain arthropods (Porazinska et al, 2009;Hajibabaei et al, 2011). The cytochrome I oxidase gene, the standard barcode for animals, has proven to be suboptimal for metabarcoding analyses owing to problems with primer coverage and large DNA insertions in certain taxa (for example, Creer et al, 2010;de Wit and Erseus, 2010;Deagle et al, 2014;Zhan et al, 2014).…”

An introduction to and a reflection on the “ecogenomics promise”

“…With routine biomolecular techniques, one can analyse very large samples to obtain a metagenomics picture of all the nematodes, their abundance and the on-site distribution of different nematode species. This way differences in exact nematode community composition can be observed and related to soil conditions (Holterman et al 2008;Porazinska et al 2009). But DNA-barcoding is also used in other settings, for instance to differentiate between tropical trees.…”

Integrating Different Organizational Levels in Benthic Biodiversity – Ecosystem Functioning (BEF) Studies

“…The three sets of taxa (nematodes, prokaryotes and diatoms) may be pyrosequenced after distinct sampling procedures. Nematodes are extracted from sediment by sieving (45 µm mesh) and floatation techniques and genotyped using both small and large rDNA subunits (Creer et al, 2010;Porazinska et al, 2009Porazinska et al, , 2010 and/or mitochondrial genes such as part of the cytochrome oxidase subunit I gene (Derycke et al, 2010). Diatoms are collected on a filter before DNA extraction and amplification using the small rDNA subunit (Cuvelier et al, 2010;Medinger et al, 2010;Quaiser et al, 2010).…”