Ion torrent platform

Sample preparation and sequencing was done following protocols optimized for virome characterization using the Ion Torrent platform (Thermo Fisher Scientific, Waltham, MA, USA) (Vibin et al., 2018 (link); Conceição-Neto et al., 2015 (link)). Prior to library preparation, mucosal samples were purified and enriched to remove host cells and free nucleic acids as follows. Samples were first low-speed centrifuged at 5,200 g for 10 min to remove cell debris, then filtered using centrifugation through a 0.45 µm cellulose acetate filter (Spin-X Centrifuge tube filter, cat#8162; Corning Inc., Corning, NY, USA) at 13,000 g for 1 min. Subsequently, to remove free nucleic acids, the filtrate was treated with Pierce Universal Nuclease (cat#88702; Thermo Fisher Scientific, Waltham, MA, USA) at the rate of 10 units of enzyme per 1 ml of solution. Finally, samples were concentrated using a Pierce™ PES protein concentrator (cat#88503), allowing concentration of proteins at 100 K molecular weight cut-off (Thermo Fisher Scientific, Waltham, MA, USA) at 13,000 g to a final volume of 150 µl/sample.

The primer pair BactB-F and BactB-R [47 (link)] and EliZyme HS FAST MIX Red (Elisabeth Pharmacon, Czech Republic) master mix were used to amplify the V4-V5 hypervariable regions of 16 S rRNA genes with the DNA extracted using two methods used as a template. Thermal cycling conditions included an initial denaturation step for 5 min at 95 °C, followed by 25 cycles of 30 s at 95 °C, 30 s at 57 °C and 30 s at 72 °C, ending with a final elongation step for 5 min at 72 °C. The quality and size of obtained PCR amplicons were evaluated on 1.5% agarose gel electrophoresis and purified using Monarch PCR & DNA Cleanup Kit (New England BioLabs, USA). Obtained amplicons were subsequently used for library preparation using the NEBNext Fast DNA Library Prep Set kit (New England Biolabs, USA) according to Milani et al. [48 (link)]. The sequencing was then performed on an Ion Torrent platform (Thermo Fisher Scientific, USA) as it was described by Mekadim et al. [49 (link)].

The purified avian RT-PCR amplicons were sequenced on the IonTorrent platform (Thermo Fisher Scientific) as previously described [5 (link), 10 (link)]. Before library preparation for the respective platform, the samples were mechanically fragmented to a 500 bp size on a Covaris M220 Ultrasonicator (Covaris Ltd., Brighton, UK). The GeneRead DNA L Core Kit (Qiagen) was subsequently used for library preparation with Xpress Barcode Adapters (Qiagen). After a following size selection and clean-up step with AMPure XP Beads (Beckman Coulter), the final library was quality checked on an Agilent Bioanalyzer 2100 (Agilent Technologies, Böblingen, Germany) and quantized via qPCR with the KAPA Library Quantification Kit (Roche, Mannheim, Germany). Sequencing was conducted on the IonTorrent S5XL (Thermo Fisher Scientific) in combination with the Ion OneTouch 2 System (Thermo Fisher Scientific), encompassing twelve AIV samples per Ion 530 Chip (Thermo Fisher Scientific).

The BAC-end sequences for selected BAC-clones were obtained with the universal M13 Reverse (5′-CAGGAAACAGCTATGAC -3′) and T7 forward (5′-TAATACGACTCACTATAGGG-3′) primers using the BigDye3.1 Terminator kit (Applied Biosystems, USA). Each 20 μl reaction contained ~200 μg of BAC-DNA, 1.5 μl of BigDye 3.1, 0.25 pM of specific forward, or reverse primer, 4 μl of5X buffer and deionized water. After preliminary denaturation at 95 °C for 5 min, 80 cycles were run at 95 °C for 30s, 55 °C for 15 s, and 60 °C for 4 min. The reaction products were precipitated using ethanol, and separated in a 3730XL DNA Analyzer (Perkin Elmer Cetus, USA).
BAC clones marked by 5S rDNA were included in the pool of 134 BAC-clones belonging to different locations of chromosome 5BS, and were collectively sequenced as one sample on an IonTorrent platform (Thermo Fisher Scientific). The sequencing and assembly were described in Nesterov et al. [23 (link)].

Mutation analysis was performed by targeted sequencing using the Ion Torrent platform (Thermo Fisher Scientific, Waltham, MA, USA) and a custom-designed sequencing gene panel (Ion AmpliSeq, Thermo Fisher Scientific) encompassing 525 amplicons covering coding regions of 58 GC related genes. The multiplex PCR based Ion AmpliSeq targeted sequencing technology (Thermo Fisher Scientific) was used for DNA library preparation and amplification of target regions using the Ion AmpliSeq Library Kit v2.0, as well as the specific GC sequencing panel consisting of four primer pools as described in detail previously [19 , 20 (link)]. Automated template preparation of the final libraries as well as chip loading (Ion 520, 530, or 540) was performed on an Ion Chef instrument and sequenced using an Ion S5XL instrument (Thermo Fisher Scientific). Data analysis was performed referring to Pfarr et al. [20 (link)] and ANNOVAR was used to annotate the sequence variants [21 (link)].

The lower respiratory samples collected from severe CAP patients were used to construct the cDNA libraries and sequenced using an Ion Torrent platform with a type 318 chip (Thermo Fisher Scientific Inc, USA). The samples were treated with Turbo DNase (Life Technologies, USA) and RNAse One (Promega, USA) to decrease the host genome background according to the manufacturer’s instructions. Total nucleic acids were extracted and amplified using the Ovation RNA-Seq System (Nugen, California, USA). The libraries for deep sequencing were constructed using the NEBNext Fast DNA Library Prep set for the Ion Torrent (New England Biolabs). Alignments with mapping quality scores less than 30 and reads containing fewer than 50 nucleotides were excluded from the following analysis. The taxonomic assignment was performed using MEGAN software (version 5.2.3) with the parameters “Min Support 50, Min score 80, Top percent 10” 31 (link). All reads assigned to the Enterovirus genus were retrieved and aligned to the consensus sequence of the reference strain, HRV-A21_p1177_sR3307_2010 (GenBank accession number JN837693), using BWA (version 0.7.5, BWA-MEM algorithm) and GATK (version 2.5.2)32 (link)33 (link). Pileup files were generated by SAMtools, and the allele frequency was calculated as the proportion of reads presenting the allele amongst all reads mapped to this position34 (link).