Next-Generation Sequencing (NGS) of bat mitochondrial D-loop DNA and viral fragment

Multiple haplotypes of the viruses and their host were expected to be present within the same guano samples. Therefore, the NGS was used to sequence the host and viral PCR products. The PCR products mentioned above were purified with the Agencourt AMPure XP reagent (Beckman Coulter, Brea, Calif., USA). An NGS library was constructed using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England BioLabs, Massachusetts, USA). Sequencing of paired ends was performed using the MiSeq Reagent Kit v2 (300 cycles) (Illumina®, San Diego, CA, USA), wherein each read was exported from MiSeq Reporter software in FASTQ format, and the NGS data were analyzed using CLC Genomics Workbench software, version 10.1.1 (Filgen, Nagoya, Japan). The overlapped sequence from each end was connected (Mismatch cost = 2, Gap cost = 3, max unaligned mismatches = 0, Minimum score = 6). Then, the sequences were mapped onto reference sequences obtained from the NCBI database. The mapped sequences with 96.44%-100%, similarity to each other were defined as one haplotype of M fuliginosus D-loop DNA [32]. On the other hand, the viral sequences with 100% similarity to each other were defined as one haplotype of either BtAdV or bat alphaCoV. GenBank accession numbers of all sequencing data of the mitochondrial D-loop DNA, and both of the viruses were presented in Tables ​Tables11 and ​and2,2, respectively.