Computational protocol: Metagenomic Survey of Viral Diversity Obtained from Feces of Subantarctic and South American Fur Seals

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Protocol publication

[…] Ion Torrent reads were trimmed using PRINSEQ (prinseq.sourceforge.net) and the quality of the sequences was analyzed with FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Trimmed reads were assembled de novo by using MetaVelvet v1.2.01 (metavelvet.dna.bio.keio.ac.jp) with a k-mer of 51. Illumina reads were trimmed for primers using Geneious 8.1.3 and de novo assembled with St. Petersburg genome assembler (SPAdes) 3.5.0 (bioinf.spbau.ru/spades). The resulting contigs (>100bp) were submitted to BLASTx search against the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov) non-redundant database (nr) and its viral database by using an E-value cutoff of 1e-05. The contigs were classified into eukaryotic viruses, bacteriophages, bacterium, eukaryotes and unknown based on lowest E-value. Contigs of eukaryotic viruses were used for sequence and phylogenetic analyses and bacteriophage sequences were not further analyzed. The GenBank accession numbers for the sequences derived in this study are: KR261062, KR261063, KR261065, KR816222, KR816223 (fur seal anelloviruses); KR261066-KR261068, KR261070-KR261075, KR261077-KR261079, KR816217, KR816218, KR816220, KR816221 (fur seal parvoviruses); KR106199-KR106202, KR816213, KR816215, KR337994 (fur seal picornaviruses); KR072975-KR072979, KR072981, KR072982, KR072984 (fur seal sakobuvirus); KR106194-KR106196, KR106198, KR816216 (fur seal picobirnavirus); KR072985-KR072990 (fur seal rotavirus), KR827461 (fur seal hepevirus); KR072992, KR072994, KR072995 (fur seal sapovirus). The sequence data obtained from this study is available at the NIH Sequence Read Archive (SRA) under the study accession number SRP070196. [...] Nucleotide or translated amino acid sequences from the contigs of anellovirus, parvovirus, picornavirus, picobirnavirus, rotavirus, sapovirus and hepevirus-like were aligned with MUSCLE (www.drive5.com/muscle) and phylogenetic trees were built using MEGA6 []. Trees were constructed by the neighbor-joining (NJ) method [] with a bootstrap of 1000 replicates, p-distance model, and gaps were treated as pairwise deletion. The contig sequences from this study were compared with other selected gene sequences available in the GenBank. […]

Pipeline specifications

Software tools PRINSEQ, FastQC, MetaVelvet, Geneious, BLASTX, MUSCLE, MEGA
Databases SRA
Applications Phylogenetics, Metagenomic sequencing analysis, Nucleotide sequence alignment
Organisms Zalophus californianus, Filamentous phage, Human poliovirus 1 Mahoney