Exosap it kit

We vortexed the samples (clinical and fecal swabs) to homogenously distribute cells in the Trizol suspension. We performed RNA extraction using the Direct-zol RNA MiniPrep kit (Zymo Research, USA). We carried out cDNA synthesis from RNA elute using Invitrogen Superscript III (ThermoFisher, USA) and stored in -80 °C freezer until downstream processing.
We performed PCR screening for the CDV phosphoprotein (P) gene using a Morbillivirus specific primer set that amplifies the ~ 390 bp region of the gene [19 (link)]. PCR amplicons were visualized on 1.5% agarose gels, and the amplified products were purified using ExoSAP-IT™ kit (Thermofisher, USA), and the sequencing reactions were performed in an MJ Research PTC-225 Peltier Thermal Cycler using the ABI PRISM® BigDye™ Terminator V3.1 Cycle Sequencing Kits (Applied Biosystems, USA) following the manufacturer’s protocols. Finally, the sequencing products were resolved on ABI 310 Genetic Analyzer (Applied Biosystems, USA), and the P gene sequences were used to confirm the presence of CDV using blast tool against Genbank database.

The genomic DNA of the pure fungal isolate SPH2 was extracted using DNeasy Plant mini kit (Qiagen GmbH, Hilden, Germany, Cat. No 69104) by following the manufacturer’s instructions. The extracted DNA was used for PCR amplification by primer ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) according to Kumar et al. (2011) [16 (link)]. Genomic DNA (100–200 ng) was amplified on a PTC-200 Thermal Cycler (MJ Research, San Diego, CA, USA) in a 25 µL final volume with the AmpONE Taq DNA polymerase PCR kit (GeneAll, Seoul, Korea) for 35 cycles (95 °C, 1 min; 50 °C, 20 s; 72 °C, 1.5 min) after an initial denaturation (95 °C, 2 min) and followed by a final extension (72 °C, 7 min). Amplicons were checked by agarose gel (1%) electrophoresis, purified using the EXO-SAP-IT kit (Affimetrix-USB; Thermo Fisher Scientific, Waltham, MS, USA), and sequenced on an AB 3500 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MS, USA) at the University of La Laguna (La Laguna, Spain) genomic service. A BLASTN search of the sequence against the NCBI nucleotide identified strain SPH2 as Aspergillus sp., similar to these in the group Circumdati (A. ochraceus, GENBANK accession number KX901282.1 and A. westerdijkiae, GENBANK accession number KY608057.1).

PCR primers were designed on the basis of the potential viral integrations that we identified running ViR on WGS data from Ae. albopictus (Additional file 5). These primes were used on DNA extracted from different mosquitoes than those used as source of WGS data. Aedes albopictus mosquitoes are reared at the insectary of the University of Pavia as previously described [23 (link)]. Genomic DNA was extracted from individual mosquitoes using the Promega Wizard® Genomic DNA Purification Kit, according to manufacturer’s protocol. PCR reactions were carried out with the DreamTaq Green PCR Master Mix (ThermoFisher) using 1 μl of genomic DNA. Amplified bands were purified with ExoSAP-IT kit (ThermoFisher) and send to Macrogen (Madrid, Spain) for Sanger sequencing. Sequences were analyzed with Bioedit [44 ].

For Sanger sequencing, PCR were done using the HotStar Taq DNA polymerase kit (Qiagen GmbH, Hilden, Germany). PCR products have been controlled on agarose gel and then purified with the ExoSAP-IT kit (Thermo Fisher Scientific Inc, Waltham, MA, USA). Sequence reactions were prepared and purified using BigDye Terminator v1.1 and BigDye XTerminator kits (Thermo Fisher Scientific Inc, Waltham, MA, USA) following standard protocol. The sequencing products were then analyzed by capillary electrophoresis on an ABI PRISM 3130 × l genetic analyzer (Thermo Fisher Scientific Inc, Waltham, MA, USA). Results were analyzed using the FinchTV software (Digital World Biology LLC, Seattle WA, USA).

Bacterial DNA was extracted from the collected samples (fecal, soil and water) using Bacterial DNA extraction kit (Zymo Research, USA) using manufacturer’s protocol. Campylobacter was detected by PCR amplifying ~ 800 bp fragment of the 16S rRNA gene using Campylobacter genus specific PCR primer sets C412F and C1288R [29 (link)]. PCR amplification was done in 25μl volume containing reaction buffer, 0.2nmol primers, Taq polymerase and 2 μl template. The cycling condition for the PCR- initial denaturation at 95˚C for 4 minutes, 35 cycles of denaturation at 95˚C for 30 sec/cycle, annealing temperature of 55˚C for 30 sec and extension at 72˚C for 30 sec, and a final extension at 72˚C for 10 min. The PCR products were separated on 1.5% agarose gel electrophoresis.
8μl amplified PCR product was cleaned using 2μl of ExoSAP-IT™ kit (Thermofisher, Catalog No. 78200.200.UL). The reaction mixture was then incubated for 30 minutes at 55˚C to get rid of excess PCR primers, followed by 85˚C for 10 minutes for reaction deactivation. The purified PCR products were then sequenced on an ABI thermocycler using BigDye^TM Terminator V3.1 Cycle Sequencing Kit (Catalog No. 4337455). Excess salts and dye terminators were removed using BigDye® XTerminator™ Purification Kit (Catalog No.4376486). The samples were then analyzed on ABI 310 Genetic Analyzer.