Clc genomics workbench software

Sequencing reads were mapped to the corresponding reference genome sequences (Genome accession numbers described above for Streptomyces species and NC_008095 for M. xanthus) using CLC Genomics Workbench software (CLC Bio, Aarhus, Denmark) (Supplementary Tables 2 and 3). Mapped reads were then counted and normalized using the DESeq2 algorithm in R (Supplementary Data 1–2 and 4–11) [13 ]. Mapped read information was exported as a BAM file format, which was further converted to a GFF file format containing read counts for each genomic position. The GFF file was visualized on SignalMap (v2.0.0.5; Roche NimbleGen, Basel, Switzerland). The sequencing data were deposited in the European Nucleotide Archive (accession number: PRJEB25075).

The nucleotide sequences of the purified dioscorin genes were subsequently translated to protein sequence using bioinformatic resource tool from CLC Genomics Workbench software (CLC Bio Denmark). The nucleotide and translated protein sequences were further subjected to computer-based homology search with NCBI BLAST program. Phylogenetic analysis was carried out to compare the relationship of the major storage protein of D. bulbifera with the storage proteins of other Dioscorea spp.

Genomic DNA was isolated using a Genomic Tip 100G DNA kit (Qiagen) according to the manufacturer's instructions. Five resistant clones from the cycling experiment were sent to the Beijing Genome Institute (BGI; China) for whole-genome sequencing using the Illumina sequencing technology. CLC Genomics Workbench software (version 5.5.1; CLC bio, Aarhus, Denmark) was used to assemble the sequencing reads and detect regions with sequence variations (quality-based variant detection) and regions with no coverage compared to the sequence of the S. Typhimurium LT2 wild-type strain. The cutoff frequency was set to 75%.

The frequency of NS5A-Y93H was determined by ultra-deep sequencing using the Ion Torrent PGM (Life Technologies) according to previously described protocols [22 (link)] with some modifications. In brief, the fragment distributions of all amplicons were analyzed on a Bio-Analyzer 2100 (Agilent Technologies, Palo Alto, CA). The amplified fragments were modified by the Ion Xpress Plus Fragment Library Kit, and sequence analysis was performed using Ion PGM. Read mapping onto the HCV-KT9 reference sequence (GenBank accession no. AB435162) was performed using the CLC Genomics Workbench software (CLC bio, Aarhus, Denmark). This technique revealed an average coverage depth of over 10,000 reads per position in the unique regions of the genome. A wild type hepatitis C virus-expressing plasmid pHCV-KT9 [23 (link)] was used to estimate the minimum detection threshold of Y93H frequency. The sequence data have been deposited in the NCBI Short Read Archive under BioProject PRJNA275480.

Virus DNA was extracted from infected macrophage cultures presenting higher than 90% CPE. The nucleus and cytoplasmic fractions were separated using a nuclear extraction kit, with the viral DNA being isolated from the cytoplasmic fraction, following the manufacturer’s protocol (Active Motif cat# 40010). ASFV infected cells were treated with the hypotonic buffer on ice until the cell membrane was dissolved. The nucleus fraction was separated by centrifugation; the cytoplasmic fraction was collected and DNA extracted adding 10% (v/v) of 3M NaAc (Sigma-Aldrich 71196, St. Louis, MO, USA) and an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) with a pH of 6.5–6.9 (Sigma-Aldrich P3803-100ML). The preparation was centrifuged on maximum speed in a tabletop centrifuge, the aqueous layer was then ethanol precipitated using 2 volumes of 100% ethanol, washed with the same volume of 70% ethanol, and dried. The resulting DNA pellet was then reconstituted in sterile water. We then used this DNA library for NGS sequencing using Nextera XT kit in the NextSeq (Illumnia, San Diego, CA, USA), following the manufacturer’s protocol. Sequence analysis was performed using CLC Genomics Workbench software (CLCBio, Waltham, MA, USA).

RNA sequences were analyzed using CLC Genomics Workbench software (CLC bio, Inc.). After diminishing clone vector sequence contamination, the alignment of Salp15–like sequences was performed using the ClustalX 2.1 program. The nucleotide and amino acid similarities of salp15 among different Ixodes species were calculated using the Needle software package (v6.0.1) in WebLab [30] . The signal peptide cleavage sites in amino acid sequences were predicted by Signal P 4.0 [31] (link).