Nextera xt reagents

RNA was extracted using Roche High Pure viral RNA kits. Water only controls were extracted, amplified and sequenced in parallel with each set of samples. Capsid coding regions were amplified in duplicate by one-step RT-PCR using a SuperScript III HiFi kit and primers P1F (5’-GCGAGTTGGATTGGCCATCCAGTG -3’) and P1R (5’-TGGAAGGTGGGTCCCACAAACGAC-3’). Products were purified using AMPure XP magnetic beads (Beckman Coulter), quantified using Qubit High Sensitivity dsDNA assay (Life Technologies), analysed on an Agilent High Sensitivity DNA chip (Agilent) and diluted to 0.2 ng/μl in molecular grade 10mM Tris–EDTA, pH8.0.
Sequencing libraries were prepared using Nextera XT reagents (Illumina) and the manufacturer's protocol, and sequenced on a MiSeq using a 2 × 251 paired-end v2 Flow Cell (Illumina). Quality trimming and assembly were carried out as in Mee at al. [26 (link)]. Reads were then mapped to parental reference sequences using Geneious R7 (Biomatters) software and SNPs present at ≥ 0.5% identified. Only those SNPs present in both replica amplicons were retained.

F. filiformis strains collected in the study are shown in Table 1 and Table 2. In total, 232 strains of F. filiformis strains were collected in this study, including 157 cultivars and 75 wild strains. To obtain a comprehensive understanding of the genetic variation present within the F. filiformis, we selected cultivars from various countries, although a significant proportion of the strains were sourced from China. The mycelia were grown in solid Potato dextrose agar (PDA) medium at 25 °C until they reached full growth. Genomic DNA was extracted from mycelia using the CTAB method [27 ]. A Nanodrop and 1.0% agarose gel electrophoresis were used to assess the concentration and integrity of the DNA solution. Whole-genome sequencing libraries were prepared using NexteraXT reagents (Illumina). The Illumina Novoseq platform from Novogene was then used for sequencing the DNA samples. Briefly, approximately 2 μg of DNA from each sample was used for fragmentation by Biorupter (high power: (15 s, on/90 s, off), six cycles) and end preparation by NEXT flex TM End-Repair. After PCR amplification (10 cycles), the library was purified using AMPure beads. Qubit was used to evaluate the quality and quantity of each library. The sequencing statistics of the samples are summarized in Table 1.

RNA was extracted from samples of clarified infected cell lysate or purified virus using TRIzol and prepared for next-generation sequencing as described previously (22 (link)). Briefly, reverse transcription used both random hexamers and FMDV-specific primers. Subsequent DNase treatment and cleanup was followed by second-strand synthesis before library preparation using Nextera XT reagents (Illumina) and sequencing on the MiSeq v2 (Illumina). Although originally described as a consensus-level sequencing methodology, depth of coverage was such that deep sequencing analysis also could be carried out. Bioinformatics analysis of the data was completed using the pipeline previously described (22 (link)). The windowed adaptive quality trimming tool Sickle was used to trim reads using Q30 as a cutoff. A de novo reference sequence was created with Velvet (using Velvet optimizer), and reads subsequently were aligned to this reference using Bowtie2. Shannon's entropy was calculated using R.

The nearly complete genome of EV-D94 was determined in a different project characterizing EVs present in infected cell cultures from clinical stool samples from AFP surveillance. As it is expected that such cell culture samples contain high concentration of virus, we opted for sequencing PCR products generated by random primers as opposed to specific primers required to amplify low concentrations of viral RNA present in environmental samples. PCR fragments for sequencing were generated by sequence-independent single-primer amplification (SISPA) of purified RNAs from infected cells and NGS analysis was performed as described before [30 (link),31 (link)]. RT-PCR templates were generated in a random RT-PCR reaction using primers RA01-N8 (5’-GCC GGA GCT CTG CAG ATA TCN NNN NNN N-3′) and RA01 (5´-GCC GGA GCT CTG CAG ATA TC-3´). Sequencing libraries were prepared using Nextera XT reagents and sequenced on a MiSeq using a 2 × 301-mer paired-end v3 Flow Cell and manufacturer’s protocols (both Illumina, San Diego, CA, USA). Quality trimming of NGS reads was performed as described in Section 2.3. Host contaminating human genome sequences were filtered out using the hg38 database 27. Filtered reads were then assembled de novo using stringent assembly conditions as described in Section 2.4 and the resulting contigs were analyzed by BLAST analysis.

To ensure that the donors represented a diverse set of HLAs four-digit HLA typing (S8 Table) was performed at the La Jolla Institute (LJI) as described previously [25 (link)]. Here, genomic DNA was isolated from PBMC using standard techniques (REPLI-g; Qiagen). Amplicons for HLA class I and class II genes were generated using PCR and locus-specific primers. Amplicons of the correct size were purified using Zymo DNA Clean-up Kit, according to the manufacturer’s instructions. Sequencing libraries were prepared using Nextera XT reagents (Illumina), according to manufacturer’s instructions. The libraries were purified using AMPure XP (Beckman Coulter) with a ratio of 0.5:1 beads to DNA (vol/vol). The libraries were pooled in equimolar amounts and loaded at 5.4pM on one MiSeq flowcell with 1% phiX spiked in (MiSeq Reagent Kit v3). Paired-end sequencing was performed with 300 cycles in each direction. HLA typing calls were made using HLATyphon (https://github.com/LJI-Bioinformatics/HLATyphon).