Evaluation of Metabarcoding Primers for Analysis of Soil Nematode Communities

Md Maniruzzaman Sikder,Mette Vestergård,Rumakanta Sapkota,Tina Kyndt,Mogens Nicolaisen

doi:10.3390/d12100388

Md Maniruzzaman Sikder, Mette Vestergård + Show 3 more

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https://doi.org/10.3390/d12100388

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Abstract

While recent advances in next-generation sequencing technologies have accelerated research in microbial ecology, the application of high throughput approaches to study the ecology of nematodes remains unresolved due to several issues, e.g., whether to include an initial nematode extraction step or not, the lack of consensus on the best performing primer combination, and the absence of a curated nematode reference database. The objective of this method development study was to compare different primer sets to identify the most suitable primer set for the metabarcoding of nematodes without initial nematode extraction. We tested four primer sets for amplicon sequencing: JB3/JB5 (mitochondrial, I3-M11 partition of COI gene), SSU_04F/SSU_22R (18S rRNA, V1-V2 regions), and Nemf/18Sr2b (18S rRNA, V6-V8 regions) from earlier studies, as well as MMSF/MMSR (18S rRNA, V4-V5 regions), a newly developed primer set. We used DNA from 22 nematode taxa, 10 mock communities, 20 soil samples, 4 root samples, and one bulk soil. We amplified the target regions from the DNA samples with the four different primer combinations and sequenced the amplicons on an Illumina MiSeq sequencing platform. We found that the Nemf/18Sr2b primer set was superior for detecting soil nematodes compared to the other primer sets based on our sequencing results and on the annotation of our sequence reads at the genus and species ranks. This primer set generated 74% reads of Nematoda origin in the soil samples. Additionally, this primer set did well with the mock communities, detecting all the included specimens. It also worked better in the root samples than the other primer set that was tested. Therefore, we suggest that the Nemf/18Sr2b primer set could be used to study rhizosphere soil and root associated nematodes, and this can be done without an initial nematode extraction step.

Highlights

Nematodes are highly diverse and abundant metazoans with a worldwide distribution [1,2].Generally, nematologists have relied on classical morphology-based taxonomy along with biochemical or molecular methods for nematode identification [3,4]
Based on the present study of individual nematode taxa, mock communities, and soil samples without prior nematode extraction, we found that the JB primer set targeting the I3-M11 partition of the COI gene is not suitable for nematode metabarcoding, especially in a soil DNA background
The retrieval of a high nematode diversity in only 0.25 g homogenized soil samples shows that the employed metabarcoding strategy is applicable in, e.g., rhizosphere community studies where the total sample weight rarely exceeds a few grams. Both the MMS and Nemf/18Sr2b primer set (NEM) primer sets outperformed the JB and SSU primer sets based on the nematode detection of individual nematode taxa, mock communities, and soil samples

Summary

Introduction

Nematodes are highly diverse and abundant metazoans with a worldwide distribution [1,2].Generally, nematologists have relied on classical morphology-based taxonomy along with biochemical or molecular methods for nematode identification [3,4]. DNA-based identification has eased the task of taxonomic nematode identification in recent years [7,8,9,10]. The barcoding approach was introduced for species-level detection [11,12,13]. The mitochondrial cytochrome oxidase I gene (COI gene) has been successfully used as barcode for the identification of nematodes and for resolving taxonomic relationships among closely related species [14,15,16]. The potential of COI gene-based barcoding specific to particular groups of nematodes has been explored on pure DNA samples of various nematode taxa including root-knot nematodes [18], marine nematodes [16], Aphelenchoididae [19] and Pratylenchus [20]. A lack of suitable COI-based consensus primers for the detection of nematode diversity in comparison to 18S-based primers limits the implementation of COI-based metabarcoding for nematodes [21]

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