Sequencing of feces sample from Myotis dasycneme
Sample material for sequencing was nucleic acid extracted from selected bat faecal samples, collected in 2018 or from samples collected earlier (2014-2018) as described previously (Lazov et al., 2018) that had scored positive in pan-CoV RT-qPCR assays (Table 1). Briefly, bat faecal samples were collected from different locations in Denmark. The samples were homogenized in Eagles medium, the nucleic acids were extracted using a MagNaPure 96 robot (Roche) and tested using up to three different pan-CoV real-time RT-qPCR assays as previously described (Lazov et al., 2018). The nucleic acid samples were treated with DNAse I (Thermofischer) according to the manufacturers protocol and double-stranded cDNA was generated using a Superscript III first-strand synthesis kit (Invitrogen) and NEBNext mRNA second strand synthesis kit (New England Biolabs). The reactions were purified using the Gene Jet PCR purification kit with elution in two aliquots of nuclease free water (30l each) at 60 C. The samples were sequenced at the DTU MultiAssay Core in Kongens Lyngby, Denmark (DMAC). In brief, the samples were dried in a SpeedVac vacuum concentrator (Thermofischer) and resuspended in 5l nuclease free water to increase their concentration. Libraries were generated using the Nextera-XT DNA library Preparation kit and checked on the Bioanalyzer (note: often the DNA content was too low to pass the quality control standard but analyses were still continued). Finally, the libraries were run on the Illumina MiSeq platform using the Reagent Kit v3 600 bp (Illumina Inc, San Diego, CA, USA).