Clc genomics workbench v 10

Operational taxonomic unit (OTU) clustering and taxonomic analyses were performed using CLC Genomics Workbench V.10.1.1 and CLC Microbial Genomics Module V.2.5 (Qiagen). Sequences were first trimmed to remove 13 bases at the 5′ terminal position and merged considering the alignment scores as follows: mismatch cost of 2, gap cost of 2, zero maximum unaligned end mismatches and minimum score of 30.
After merging, sequences were clustered into OTUs at 97% sequence similarity level. The most abundant sequences were selected as representative of each cluster and then assigned to a taxonomy level using default values and the Greengenes Database 2013 release. Low depth samples (less than 2000 sequences per sample) were removed from the analysis. Alpha diversity indexes (Simpson, Shannon and total OTU number) were calculated. Bray-Curtis and unweighted UniFrac metric were used to calculate intersample diversity (beta diversity).
Samples were rarefied using QIIME 1.9, and multiple comparisons and statistical analyses were performed using CLC Genomics Workbench V.10.1.1 and CLC Microbial Genomics Module V.2.5 (Qiagen). A Negative Binomial GLM model was used to obtain maximum likelihood estimates for an OTU’s log-fold change between two conditions, and the Wald test was used to determine significance. False discovery rate (FDR) was performed to correct p values.

For full genome sequencing, viral RNA from the DF-1 culture supernatant was used. Next-generation sequencing (NGS) was conducted by the Tri-I Biotech Company (Taipei, Taiwan). Briefly, a cDNA library was constructed using the NuGEN Trio RNA-Seq kit (Tecan, CA), and sequencing was conducted using the Illumina NovaSeq Sequencing System (2 × 151 pair-ends) (Illumina, San Diego, CA). The obtained reads were assembled by reference mapping using CLC Genomics Workbench v10 (Qiagen) and de novo assembly using SPAdes v3.14.1 (Bankevich et al., 2012 (link)).