fc8b0fc2-85c2-41f7-826c-c933b9f46a58
Development and validation of a high-throughput sequencing test for mitogenome and rDNA assembly and annotation, and its use in support of nematode identification of regulatory concern
Nematoda is a diverse phylum and representatives are found in most habitats, including in and on animals and plants. Nematodes are regarded as the most abundant group in terms of individuals in marine and terrestrial sediments. Plant parasitic nematodes are globally responsible for an annual yield loss of $125 billion. Reliable species identification is essential to take appropriate phytosanitary measures. The introduction of validated Sanger sequencing of 28S, 28S and cox1 barcode loci represented a powerful tool in support of nematode identification. However, technical challenges associated with PCR and Sanger sequencing and the need for additional loci for identification hamper the efficient use of sequence data. To overcome these challenges, we developed an automated bioinformatic pipeline for the assembly and annotation of mitochondrial genomes and ribosomal DNAs, and defined and validated a standardised test protocol including controls for routine diagnostics (i.e. High-through sequencing test = HTS test). The HTS test can be performed on single nematode specimens and outperforms the Sanger based sequencing by producing less ambiguous consensus sequences and by producing additional sequence data offering additional diagnostic resolution when needed. Compared to the Sanger Sequencing, the HTS test represents a reduction in hands-on time. The HTS test is regarded as fit for the purpose for the molecular identification of single nematode specimens in support of nematode diagnostics of regulatory concern.
Nematoda is a diverse phylum and representatives are found in most habitats, including in and on animals and plants. Nematodes are regarded as the most abundant group in terms of individuals in marine and terrestrial sediments. Plant parasitic nematodes are globally responsible for an annual yield loss of $125 billion. Reliable species identification is essential to take appropriate phytosanitary measures. The introduction of validated Sanger sequencing of 28S, 28S and cox1 barcode loci represented a powerful tool in support of nematode identification. However, technical challenges associated with PCR and Sanger sequencing and the need for additional loci for identification hamper the efficient use of sequence data. To overcome these challenges, we developed an automated bioinformatic pipeline for the assembly and annotation of mitochondrial genomes and ribosomal DNAs, and defined and validated a standardised test protocol including controls for routine diagnostics (i.e. High-through sequencing test = HTS test). The HTS test can be performed on single nematode specimens and outperforms the Sanger based sequencing by producing less ambiguous consensus sequences and by producing additional sequence data offering additional diagnostic resolution when needed. Compared to the Sanger Sequencing, the HTS test represents a reduction in hands-on time. The HTS test is regarded as fit for the purpose for the molecular identification of single nematode specimens in support of nematode diagnostics of regulatory concern.