Clc genomics workbench 8

Viral RNA was extracted from 100 μL of patient serum collected from day 3 post onset with the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany, Cat# 74134) without carrier RNA. A sequencing library was constructed and analyzed with the Ion Xpress Plus Fragment Library kit (Life Technologies, Carlsbad, CA, USA), then subsequently sequenced with the Ion Torrent PGM Hi-Q OT2 kit (Life Technologies) according to the manufacturer's instructions. The next-generation genomic sequencing data were analyzed with CLC Genomics Workbench 8.5.1 (CLC bio, Aarhus, Denmark). Reads shorter than 30 bp were removed and de novo assembly was performed using the default parameters in the CLC software. The phylogenic analysis of E gene was performed using MEGA6 MEGA7 program software.^{10 (link), 11 (link)} The phylogenic tree was constructed using the neighbor-joining method with the Poisson correction and a complete deletion of gaps according to the software instructions. The Atlas genome was constructed using BLAST Ring Image Generator software.^{12 (link)} The sequence was confirmed later with Vero cells and isolated YF virus from the same patient blood sample.

The total genomic DNA of two individuals was sequenced using an Illumina Hiseq4000 sequencer with the pair‐end strategy of 150 bp reads with an average 400 bp insert size. Approximately, 5.5 Gb raw reads were yielded for each species. The raw reads were trimmed with an error probability < .05 and by removing one nucleotide at both terminal ends, and then assembled on CLC Genomics Workbench 8.5.1 (CLC Bio) with the plastome of D. moniliforme (AB893950) as reference. The gaps and junctions between inverted repeat (IR) regions and single copy (SC) regions were confirmed by PCR amplification. Genes were annotated using DOGMA (Wyman, Jansen, & Boore, 2004) and tRNAscan‐SE 1.21 (Schattner, Brooks, & Lowe, 2005). The exact boundaries of annotated genes were manually checked by aligning them with homologous genes of other plastomes in the genus of Dendrobium.

Quality control of the reads, contamination screening, and assembly were performed with CLC Genomics Workbench 8.5.1 (CLC Bio). Initial quality control and adapter trimming of raw sequence data was based on the following parameters, quality filtering 0.05, trim first 15 bases, minimum read length 35 bases, and maximum read length 220 bases. For contamination screening, reads of the soil datasets were mapped to blank sample sequences, the human reference genome hg18 (GRCh38, UCSC Genome Browser), and an Escherichia coli reference genome (strain C43(DE3), GenBank accession number CP011938) to remove non-soil virus sequences. De novo assembly of each soil virome was performed with the assembly and mapping algorithm using default parameters for Ion Torrent data. Fourteen assembled viromes were uploaded to the MetaVir server (http://metavir-meb.univ-bpclermont.fr/, Project: Antarctic Soil) [32 (link), 33 (link)].

A schematic representation of the detailed methodology is presented in Figure 1. The amino acid sequences of structural proteins (VP4 and VP7) of Rotavirus group A were downloaded from the NCBI (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 2 March 2023). All the globally reported sequences of VP7 and VP4 genotypes were included in the study.
Multiple sequence alignments were performed separately for individual serotypes of VP4 and VP7. For each serotype, the consensus sequence was generated. All the consensus sequences were then subjected to alignment to generate the final consensus sequence of VP4 and VP7. MAAFT (https://www.ebi.ac.uk/Tools/msa/mafft/, accessed on 2 March 2023) [24 (link)] was used to perform multiple sequence alignment and the consensus sequence was generated through CLC Genomics Workbench 8.5.1 (CLC Bio, Aarhus, Denmark). The consensus sequences of VP4 and VP7 were further subjected for epitope identification.

Six-week-old female BALB/c mice purchased from Sankyo-Lab (Tsukuba, Japan) were housed under specific pathogen-free conditions at the National Institute of Advanced Industrial Science and Technology (AIST; Tsukuba, Japan). Fecal samples were obtained from five mice at 12 weeks of age and stored at −80 °C until analysis. For viral nucleic acid extraction and double-stranded cDNA synthesis from fecal samples, we followed the method of Sasaki et al.21 (link). The isolated double-stranded cDNA was used for metagenomics library preparation with the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA). Sequencing was performed on the Illumina MiSeq platform (Illumina). The reads from the mouse (Mus musculus) virome were compared against NCBI NT/NR database by using the DIAMOND, BLASTN and BLASTX programs with an expected value (e-value) ≤10⁻⁴. The results were then classified and summarized using MEGAN1 (link) with LCA minimum score, maximum expected and minimum support set to 25, 0.01 and 5, respectively. De novo assemblies were performed using CLC Genomics Workbench 8.5.1 (CLC bio, Aarhus, Denmark).
All experiments involving animals were performed in accordance with the ethical guidelines of the National Institute of Advanced Industrial Science and Technology (AIST). The protocols were approved by the Animal Welfare Committee of AIST.