Ion xpress plus fragment library kit

The 100 mg of fresh plant tissue was used to extract genomic DNA using the Genomic Mini AX Plant Spin kit (A&A Biotechnology, Gdańsk, Poland). The quality and concentration of the extracted DNA were verified using gel electrophoresis and a NanoDrop spectrophotometer (Thermo Fisher Scientific, Carlsbad, CA, United States). The genomic libraries were constructed with protocol: Ion Xpress™ Plus gDNA Fragment Library Preparation, using Ion Xpress Plus Fragment Library Kit (Pub. No. MAN0009847) (ThermoFisher Scientific, Waltham, MA, USA). Next-generation sequencing was performed on the GeneStudio™ S5 System (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions using the protocol: Ion 540 ™ Kit—OT2 User Guide (Cat. No. A27753, Publication No. MAN0010850, Revision D). The reads generated from next-generation sequencing were assembled and annotated using the Geneious Prime 2020.2.5 package. Reads were mapped to MT735327 sequence from genbank using Genious mapper with default settings and minimum mapping quality 30%. Sequences that were mapped were subsequently assembled de novo using Geneious algorithm on default settings and annotated based on MT735327 sequence. The obtained 22 complete ITS2 sequences were submitted to GenBank. Each sequence was assigned an accession number (Table S2).

To construct libraries of DNA, we used 1 μg of each of the bacterial extracts with the Ion Xpress Plus Fragment Library Kit (Thermo Fisher Scientific, Waltham, MA, United States). After adding barcode adaptors using the Ion Xpress Barcode Adaptors 1–16 Kit (Thermo Fisher Scientific), we purified the barcoded DNA using Agencourt AMPure XP (Beckman Coulter, Brea, CA, United States) and then combined 10 to 11 fragments. Next, we constructed a template DNA library on Ion Chef using the Ion 530 kit and 530 chip (Thermo Fisher Scientific), where we conducted sequencing by the Ion GeneStudio S5 sequencer (Thermo Fisher Scientific), according to the manufacturer’s protocols.

MeDIP-SC-seq was carried out as described previously [29 (link)]. In brief 100 ng of DNA library for each sample was prepared using the Ion XpressPlus Fragment Library Kit (Thermo Fisher Scientific, Paisley, UK). The DNA was end repaired, purified and ligated to ion-compatible barcoded adapters (Ion Xpress™ Barcode Adapters 1–96; Thermo Fisher Scientific, Paisley, UK) followed by nick-repair to complete the linkage between adapters and DNA inserts. The adapter-ligated library was then amplified (ten cycles) and size-selected using two rounds of AMPure XP bead (Beckman Coulter) capture to size-select fragments approximately 100–250 bp in length. Samples were then pooled at a 1:1 ratio and sequenced on an Ion Proton P1 microwell chip (Thermo Fisher Scientific, Paisley, UK). Samples were sequenced to between 24 and 31 M reads. Sperm sRNAs and ChIP DNA were sent for next-generation sequencing at Source Biosciences (Nottingham, UK). Single-end 50-bp sequencing was performed on a HiSeq 2500 machine. We obtained 32.5–62.5 million ChIP-Seq reads and 8.6–20.9 million sRNA-Seq reads. All sequencing data can be accessed through the European Nucleotide Archive, accession number PRJEB14719 [72 ].

DNA was extracted from the ASFV-989 strain with the High Pure PCR Template Preparation Kit (Roche Diagnostics, Meylan, France). For library preparation, the Ion Xpress Plus Fragment Library Kit and the Ion Xpress Barcode Adapters 1–96 Kit (Thermo Fisher Scientific, Frederick, MD, USA) were used, and barcoded DNA fragments between 250 bp and 290 bp were size-selected with magnetic beads from the Agencourt AMPure XP Kit (Beckman Coulter, Villepinte, France). All samples were sequenced with the Proton Ion Torrent technology (Thermo Fisher Scientific, Frederick, MD, USA). The raw reads were cleaned with the Trimmomatic [38 (link)] 0.36 software (ILLUMINACLIP: oligos.fasta: 2:30:5:1: true; LEADING: 3; TRAILING: 3; MAXINFO: 40:0.2; MINLEN: 36). Then, a bwa [39 (link)] (version 2.2.5) alignment was performed with cleaned reads versus NCBI reference FR682468.2. The consensus sequence was created with SAMtools [40 (link)] (version 1.8) and seqtk (version 1.2) (https://github.com/lh3/seqtk accessed on 17 July 2016). The obtained sequence was annotated with Prokka (Galaxy version 1.13), and then compared to the Georgia 2007/1 strain sequence [NC_044959.1] using Seaview (version 5.0) and the seg.sites {ape} function in R 3.6.1.
Data availability: all sequence data were uploaded to the NCBI under the study accession number PRJNA784367.

Barcoded libraries with 200-bp read lengths were prepared using Ion Xpress plus Fragment Library Kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Each library was prepared using 200 ng PCR2 product. For LSR junction sequencing equal amounts of each PCR2 product (using hs3, hs12 and h4 primers) were mixed together and diluted to 100 pM to prepare one barcoded library. Libraries were run on an Ion PI v3 chip on the Ion Proton sequencer (Life Technologies). Data analysis was performed using CSReport [21 (link)].

Samples 1 through 186 were sequenced using an Ion Torrent PGM instrument (ThermoFisher Scientific). In this case, pooled DNA amplicons were sheared, and Ion Torrent-compatible barcoded adaptors were ligated to the sheared DNA using Ion Xpress Plus Fragment Library Kit (ThermoFisher Scientific) to generate 400-bp libraries.
Samples 187 through 495 were sequenced using Illumina MiSeq instrument (2×300 bp PE, Illumina). All deep-sequencing analyses were carried out using Illumina MiSeq data. In this case, DNA amplicons were pooled and subjected to adaptor ligation and library construction (as detailed in manufacturer’s instructions, Illumina).