Dsdna high sensitivity kit

The specimens of O.a.aterrimus were collected from Yangshuo county of Guangxi province, preserved in the 100% ethanol, and stored at -20 °C. Total DNA of a single adult specimen was extracted from the muscle tissues using the DNeasy DNA Extraction Kit (QIAGEN) in accordance with the manufacturer’s instructions. The concentration of genomic DNA in extraction product was assayed on a Qubit fluorometer using a dsDNA High-sensitivity Kit (Invitrogen).

A. chamaeleonis was collected from a Bufo pageoti infected with A. chamaeleonis in Shenzhen, China. All samples were thoroughly cleaned with sterile physiological saline (37 °C), quickly frozen, transported on dry ice, and kept at −80 °C until further use. By using the microscope, morphological identification was carried out (Olympus). All experimental designs and nematode handling were approved by the Institutional Animal Care and Use Committee of Northeast Forestry University. Sodium dodecyl sulphate/proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation were used to isolate whole genomic DNA [11 (link)]. The DNA quantity was estimated using a Qubit fluorometer with the dsDNA high-sensitivity kit (Invitrogen) and using agarose gel (1.0%) electrophoresis. Genomic DNA was purified for long-read library preparation according to the manufacturer’s instructions of the Nanopore platform, followed by long-read sequencing.

The M-RTPCR amplicon libraries were prepared using the Nextera XT DNA library preparation kit and protocol (Illumina, San Diego, California, USA), cleaned using the 0.8 × AMPure XP beads, quantified on the Qubit 3.0 fluorometer using the dsDNA High sensitivity kit (Invitrogen, Carlsbad, California, USA). Library size distributions were assessed using the Agilent Technology 2100 Bioanalyzer and the High Sensitivity DNA kit (Agilent Technologies, Santa Clara, California, USA). Samples with a broad fragment size spectrum (> 250 bp) were normalized manually to 2 nM. 5 μL per sample library were pooled, denatured using Sodium Hydroxide (NaOH), and diluted to 12.5 pmol. Diluted libraries were spiked with 5% Phi-X control (Illumina, San Diego, CA, USA) and sequenced using the Illumina MiSeq (Illumina Inc., San Diego, California, USA) generating 2 × 250 bp paired reads per sample.
We assessed sequencing efficiency based on the depth of coverage and number of gene segments recovered per swab.

Adult specimens of N. samayunkur were collected from Odisha State, India. The studied species are common pests of crops, thus no prior permission was required for collection. Specimens were morphologically identified by the second author (K.T.) with available taxonomic keys^{2 ,10 (link)}, and preserved in absolute ethyl alcohol at −30 °C in Centre for DNA Taxonomy, Molecular Systematics Division, Zoological Survey of India, Kolkata. Genomic DNA was extracted using DNeasy (QIAGEN) following the manufacturer’s standard protocol. Concentration of DNA was determined using a Qubit fluorometer with a dsDNA high-sensitivity kit (Invitrogen), and by agarose gel (0.8%) electrophoresis.

The specimen of Lyrognathus crotalus was collected from the Badarpur (24.85N 92.56E), Assam, Northeast India. The morphological identification of this specimen was done by published taxonomic keys^{19 (link)}, and stored in absolute ethyl alcohol at −80 °C in Centre for DNA Taxonomy, Molecular Systematics Division, Zoological Survey of India, Kolkata. DNeasy DNA Extraction kit (Qiagen, Valencia, CA) was used for genomic DNA extraction and quantified by dsDNA high-sensitivity kit (Thermo Fisher Scientific, MA, USA) in Qubit fluorometer. In this study, no prior permission was required for the collection as the species is neither endangered nor protected species in IUCN Red List or Indian Wildlife Protection Act, 1972.