Clc main workbench v 6

Sequencing data were processed and analysed using CLC MAIN WORKBENCH v. 6.9.2 (QIAGEN) and GENEIOUS v. 11.0.2 (Biomatters) computer software. Sequences were compared to all known badnaviruses on the NCBI database using BLAST algorithms available on the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The presence of putative ORFs was predicted using GENEIOUS v. 11.0.2 and SNAPGENE software (GLS Biotech). Virus sequences were further aligned and analysed with the CLUSTALW multiple alignment application using BIOEDIT v. 7.1.9 sequence alignment editor program (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Phylogenetic trees were constructed from CLUSTALW-aligned sequences on MEGA v. 7.0 (http://www.megasoftware.net/mega.php), using the maximum-likelihood method and a Kimura 2-parameter model with 1000 bootstrap replications. Pairwise sequence comparison (PASC) was carried out on aligned sequences using GENEIOUS v. 11.0.2 computer software. For taxonomic purposes, the 1.2 kb polymerase gene covering the RT/RNase H domains was used to compare sequences from the different genera in the family Caulimoviridae while the core 529 bp sequence of the RT/RNase H-coding region (excluding the BadnaFP/RP primer binding sites) was used to compare sequences from TaBV and TaBCHV isolates.

Sanger‐derived sequences were trimmed to remove primer‐binding sites and analysed using CLC Main Workbench v6.9.2 (Qiagen, USA) and Geneious v11.0.2 (Biomatters, New Zealand). For RNAseq data, adapter sequences were removed using the fastx_clipper and reads were further trimmed to attain optimum quality using the DynamicTrim function of SolexaQA++ v.3.1.3 software (Cox, Peterson, & Biggs, 2010 (link)) and fastx‐trimmer module of FASTX‐Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). De novo assembly of reads from each sample was performed using Trinity v.2.0.3 (Grabherr et al., 2011 ) and virus contigs were identified by BLASTn analysis against the NCBI‐derived local virus database (ftp://ftp.ncbi.nih.gov/genomes/Viruses/) using a blast command line analysis (Altschul, Gish, Miller, Myers, & Lipman, 1990 (link)). Reads were subsequently mapped onto reference sequences using CLC Genomics Workbench v.7.5.1 (https://www.qiagenbioinformatics.com/) and Geneious Prime 2000 (Biomatters) with default parameters. ORFs were predicted and annotated using Geneious Prime 2000 and sequences were designated ‘complete’ based on comparison with the reference sequence used for mapping. Processed Sanger and NGS data were compared to sequences on the NCBI database using BLAST algorithms available on the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Two PCR fragments representative of each Eimeria species detected were sequenced to confirm amplicon identity and validate PCR detection, resulting in 14 sequences from 31 positive reactions (45 %). Amplicons were purified using a Qiagen PCR purification kit, cloned using pGEM-T Easy (Promega, Madison, USA) in XL1-Blue MRF Escherichia coli (Stratagene, La Jolla, USA), miniprepped (Qiagen) and sequenced (GATC Biotech, Konstanz, Germany) as described by the respective manufacturers. Sequence assembly, annotation and interrogation were undertaken using CLC Main Workbench v6.0.2 (CLC Bio, Katrinebjerg, Denmark) and sequences were identified using BLASTn against the GenBank non-redundant database with default parameters. The sequences have been submitted to GenBank under the accession numbers LT549029-LT549042.