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.

中文翻译：

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评价用于土壤线虫群落的元条形码入门。

尽管下一代测序技术的最新进展加快了微生物生态学的研究，但是由于以下几个问题（例如是否包括初始线虫提取步骤，缺乏），高通量方法研究线虫生态学的应用仍未解决。关于最佳性能的引物组合的共识，以及缺乏经过整理的线虫参考数据库。该方法开发研究的目的是比较不同的引物组，以鉴定最适合无需初始线虫提取的线虫的条码编码的引物组。我们测试了四个用于扩增子测序的引物组：JB3 / JB5（线粒体，COI基因的I3-M11分区），SSU_04F / SSU_22R（18S rRNA，V1-V2区）和Nemf / 18Sr2b（18S rRNA，V6-V8区）从早期的研究中 以及新开发的引物对MMSF / MMSR（18S rRNA，V4-V5区）。我们使用了来自22个线虫类群，10个模拟群落，20个土壤样品，4个根样品和一个散装土壤的DNA。我们用四种不同的引物组合从DNA样品中扩增了目标区域，并在Illumina MiSeq测序平台上对了扩增子进行了测序。根据我们的测序结果以及在属和物种等级上对序列读数的注释，我们发现Nemf / 18Sr2b引物组比其他引物组更能检测土壤线虫。该引物组在土壤样品中产生了74％的线虫起源读数。此外，该引物组在模拟社区中表现出色，可以检测所有包含的标本。它在根样品中的效果也比其他被测试的引物更好。因此，