Material of 1X PBS, using sterile dissecting

Material of 1X PBS, using sterile dissecting

Material and methods:1-Sample collection and transportation: In JIRI GAISLER cave AOUKAS (données gps), and MELBOU cave (données gps) in Bejaia city in Algeria, directly beneath the bat colonies, 35 fresh bat guano samples were collected, in 2ml cryo-tubes containing 1ml of 1X PBS, using sterile dissecting forceps, the samples were transported in Nitrogen dry shipper and stored at -80ºC.

2- Nucleic acid extraction: all samples were first homogenized by adding two glass beads (2.5mm) to each tube and vortexing vigorously, prior to Nucleic Acid (NA) extraction protocols: -NA extraction for DNA barcoding: 300µl of homogenized samples were pipetted out in Eppendorf tubes, centrifuged for 5min at 1000g at room temperature, then 200µl of supernatants were transferred to new Eppendorf tubes, mixed with 200µl lysis buffer and 50µl proteinase K, well vortexed, incubated overnight at 56ºC, mitochondrial DNA was extracted later on using Gene JET Viral DNA/RNA Purification Kit (Thermo scientific), and following the manufacturer protocol. – NA extraction for library preparation: 200µl of homogenized samples were filtered using Sartorius filter tubes by centrifugation at 10000rpm for 10 min at 4ºC, then immediately an enzymatic enrichment protocol was accomplished, after that and following the manufacturer protocol the nucleic acid was extracted using Gene JET Viral DNA/RNA Purification Kit (Thermo scientific).3-Bat DNA barcoding:PCR amplification of Cox1 gene, the reaction mix and cycling conditions were conducted according to GoTaq® G2 Flexi DNA Polymerase (Promega) kit protocol, the final volume was 25µl, using 3µl DNA template, per reaction, primers used SSF145f, SSF351r as described previously (Walker et all,2016), a 202 bp bands were visualised on 2% agarose gel stained with ethidium bromide after a gel excision and a gel DNA extraction using Gel/PCR DNA Fragments Kit (Geneaid ) ,then the samples were sequenced by Sanger method.4-Library preparation and metagenomic analysis Prior to the library preparation two PCRs are necessaire for RNA viruses, a reverse transcription step using a random hexamer primer (MTG) plus AMV-RT enzyme (Promega) in order to synthetize cDNA from RNAs, and a random amplification step using a complimentary primer (MTG comb) and Dream Taq polymerase, then using NEBNext® Fast DNA Library Prep Set for Ion Torrent kit and following the manufacturer protocol metagenomic libraries have been prepared.After sequencing, the data have been analysed using Megan 6 ,1179 reads were obtained for Picornavirus, after a multiple alignment of the consensus sequence with the reference sequences using muscle alignment, a 3′ race and primer walking method were used to fill the gaps, then using NGS we were able to get almost a full genome sequence.5-Phylogenetic and co-phylogenetic analysis:5.1 Source and selection of virus and host sequences:all bat picornaviruses representing either partial or complete coding sequence including 3Dpol gene were retrieved from GenBank

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