Metagenomic next-generation sequencing of a dVIN (vulvar cancer) sample for pathogen detection and discovery
NGS libraries were constructed from total nucleic acid using a modified TruSeq protocol.  Library size and concentration were determined using the BioAnalyzer High-Sensitivity DNA kit (Agilent, Santa Clara, CA) and Kapa Universal qPCR kit (Kapa Biosystems, Woburn, MA), respectively.  Samples were sequenced on an Illumina MiSeq instrument using 251/135 base pair (bp) paired-end sequencing.  NGS data was analyzed using the SURPI bioinformatics pipeline for pathogen detection (Naccache, et al., 2014, Genome Research, 24(7):1180-1192).  The SURPI pipeline first identifies and computationally subtracts human host sequences using the nucleotide aligner SNAP, followed by SNAP identification of viruses, bacteria, fungi, and parasites by comprehensive mapping of all remaining reads to the National Center for Biotechnology Information (NCBI) nucleotide (nt) reference database (NCBI nt).  For discovery of novel viruses, remaining unmatched reads and de novo assembled contigs are then mapped to a viral protein database using the translated nucleotide aligner RAPSearch.  Viral hits by SURPI were further curated for accuracy by manual inspection and BLAST alignment to the NCBI nucleotide (nt) and amino acid (nr) reference databases.  The run includes metagenomic NGS of 3samples: a vulvar cancer sample (dVIN1), a normal sample, and an HHV3-positive CSF sample as a positive control.  In total, there are 6 paired-end files.  All FASTQ files are depleted of human reads by nucleotide alignment to the human genome prior to depositing into SRA.