Genjet gel extraction kit

The locus specificity of each marker tested by capillary electrophoresis was checked by the Sanger sequencing method. All markers were amplified in multiple copies, separated in 1% (w/v) agarose gel, and excised by clean scalpel after 1 h separation. DNA purification was performed using the GenJet Gel Extraction kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manual’s recommendations. The clean amplicons, together with the specific primers, were sent to the Eurofins Genomics (Eurofins Genomics Germany GmbH, Ebersberg, Germany) sequencing service and were bidirectionally sequenced. The succeeding quality evaluation, assembly, alignment with reference sequence, and necessary manual correction of all sequences was done by Sequencing Analysis Software v5.2 (Thermo Fisher Scientific, Waltham, MA, USA), BioEdit Software v7.0.9.0 [58 ] and an online version of MUSCLE software [59 (link)] available on webpages of The European Bioinformatics Institute (EMBL-EBI).

We further investigated the Hammarskog picorna-like virus (HPLV) with PCR and Sanger sequencing. Viral hits were inspected and retrieved in Megan, overlapping contigs that mapped to the same virus were manually assembled into longer sequences. The extended sequences were subsequently used to design primers pairs to fill gaps and cover as much as possible of the genome using the primer3 software. The original RNA extractions of interest were reverse transcribed to cDNA using SuperScript^® III (Invitrogen, Carlsbad CA, USA). PCR reactions were carried out with KAPA2G Robust Hotstart ReadyMix (KAPABiosystems, Wilmington, MA, USA). PCR products were analysed with electrophoresis and bands of correct size were cut out and extracted with a GenJet gel extraction kit (Thermo Fisher Scientific, Waltham, MA USA). Purified PCR products were sent to Macrogen for Sanger sequencing and the returning sequences were analysed in DNASTAR, SeqMan Pro (Version 2.1.0.97).