Brodbeck, S. | Search bioRxiv

bioRxiv2026

Exploring nematode diversity in soils: comparison of extraction methods and sequencing protocols

Soil nematodes play key roles in regulating organic matter decomposition, nutrient cycling and trophic interactions in the soil ecosystem, and are very useful bioindicators of soil condition and soil health. But to characterise them, we need reliable measurements of nematode biodiversity in soils. DNA-based techniques such as metabarcoding have recently explored their biodiversity, with successful results. However, there are many methodological steps that can modify the outcome considerably and require informed decisions. In this study, we tested the effect on nematode alpha and beta diversity metrics of three methodological steps, namely i) type of extraction of genetic material from the matrix, either from soil directly with different starting amount of material or extracting nematodes first via the Baermann or Oostenbrink methods, ii) the choice of taxonomically-relevant DNA segment for amplification, with a nematode specific primer (NemF/18Sr2b) and a universal eukaryotic primer (3NDf/1132rmod), both within 18S rDNA, and iii) the selection of appropriate reference library for taxonomic assignment (comparing three nematode specialised databases and three broad taxonomic databases). Observed differences in alpha diversity were more pronounced when comparing the nematode extraction vs soil extractions. In particular, direct soil extractions co-extracted many non-nematode taxa, especially if amplified with a universal primer, which can significantly reduce the number of nematode reads obtained and therefore the accuracy of nematode community indexes and other nematode-based metrics. The choice of the reference database strongly affected the taxonomic identification, which calls for extreme caution when making this selection. Notably, specialised databases tended to over assign reads to nematodes that were detected as non-nematodes by broad taxonomic databases. However, beta diversity metrics were consistent across methods, highlighting these metrics as robust against methodological choices. Our study clearly demonstrates that the choices of methods for nematode biodiversity studies using molecular tools can have a big impact on the results, and decisions should be made consciously and carefully.

ecology↗

bioRxiv2018

Using transcriptome sequencing and pooled exome capture to study local adaptation in the giga-genome of Pinus cembra

Despite decreasing sequencing costs, whole-genome sequencing for population-based genome scans for selection is still prohibitively expensive for organisms with large genomes. Moreover, the repetitive nature of large genomes often represents a challenge in bioinformatic and downstream analyses. Here we use in-depth transcriptome sequencing to design probes for exome capture in Swiss stone pine (Pinus cembra), a conifer with an estimated genome size of 29.3 Gbp and no reference genome available. We successfully applied around 55,000 self-designed probes, targeting 25,000 contigs, to DNA pools of seven populations from the Swiss Alps and identified > 140,000 SNPs in around 13,000 contigs. The probes performed equally well in pools of the closely related species Pinus sibirica; in both species, more than 70% of the targeted contigs were sequenced at a depth [≥] 40x, i.e. the number of haplotypes in the pool. However, a thorough analysis of individually sequenced P. cembra samples indicated that a majority of the contigs (63%) represented multi-copy genes. We therefore removed paralogous contigs based on heterozygote excess and deviation from allele balance. Without putatively paralogous contigs, allele frequencies of population pools represented accurate estimates of individually determined allele frequencies. Using population genetic and landscape genomic methods, we show that inferences of neutral and adaptive genetic variation may be biased when not accounting for such multi-copy genes. Future studies should therefore put more emphasis on identifying paralogous loci, which will be facilitated by the establishment of additional high-quality reference genomes.

genomics↗