A novel metabarcoding strategy for studying nematode communities

Abstract

16Nematodes are widely abundant soil metazoa and often referred to as indicators of soil health. 17While recent advances in next-generation sequencing technologies have accelerated 18 research in microbial ecology, the ecology of nematodes remains poorly elucidated, partly due 19 to the lack of reliable and validated sequencing strategies. Objectives of the present study 20 were (i) to compare commonly used primer sets and to identify the most suitable primer set 21 for metabarcoding of nematodes; (ii) to establish and validate a high-throughput sequencing 22 strategy for nematodes using Illumina paired-end sequencing. In this study, we tested four 23 343 We are grateful to Andrea M. Skantar, United State Department of Agriculture; Barbara Geric 344 Stare, Sasa Sirca and Gregor Urek, Department of Plant Protection, Agricultural Institute of 345 Slovenia for contributing DNA samples of nematode species. We would like to thank Susana 346 Santos for data visualization. We would also like to thank Mathilde Schiøtt Dige and Simone 347 Ena Rasmussen for their laboratory technical assistance. 348

Optimising high-throughput sequencing data analysis, from gene database selection to the analysis of compositional data: a case study on tropical soil nematodes

“…Recently, SILVA and PR2 databases have been successfully used in a number of nematode studies (Sapkota and Nicolaisen, 2015;Kerfahi et al, 2016;Geisen et al, 2018;Schuelke et al, 2018;Bongiorno et al, 2019;Wilschut et al, 2019;Sikder et al, 2020;Gong et al, 2021). However, studies on how the choice of reference database may influence nematode taxonomic classification and phylogenic results are lacking entirely.…”

Current advances in the identification of plant nematode diseases: From lab assays to in-field diagnostics

“…The eDNA is susceptible to contamination during sampling, extraction, and storage; the availability of species-specific DNA barcodes relies on the mass of the available databases. The identification of PPNs species is difficult due to the lack of available data for DNA barcoding of most known plant nematodes ( Sikder et al., 2020 ). DNA barcoding is a tool with much potential for taxonomy.…”

Contrasting influences of two dominant plants, Dasiphora fruticosa and Ligularia virguarea, on aboveground and belowground communities in an alpine meadow

“…Nematode DNA extraction was consistent with that described above, with nematode sequences amplified from the genomic DNA. While using DNA extracted from 0.25 g soil sub-samples should be interpreted with cautions, recent papers have shown that insights can be provided using this approach (Baidoo et al, 2017;Sikder et al, 2020;Wu et al, 2021). Specifically, the approach appears robust in providing a fingerprint of nematode communities, including DNA from species that are present in the system at large but are not present in the sub-sample itself (i.e., extracellular nematode DNA) (Peham et al, 2017).…”

“…In order to improve amplification efficiency, we repeated DNA amplification before sequencing. The primers NemF and 18Sr2b (Sikder et al, 2020) were used in a pre-amplification step followed by amplification with primers NF1 and 18Sr2b in a semi-nested procedure (Sapkota and Nicolaisen, 2015) (Supplementary Table S1). NF1 and 18Sr2b were tag encoded using the forward primer 5 -CGTATCGCCTCCCTCGCGCCATCAG-MID-NF1-3 and the reverse primer 5 -CTATGCGCCTTGCCAGCCCGCTCAG-18Sr2b-3 (Sapkota and Nicolaisen, 2015).…”