PCR testing for Human herpesvirus 3 (HHV) and Suid herpesvirus 1 (SuHV1).

<p>(A) PCR amplification was performed using a primer set designed from a single HHV3 read found in the dVIN sample 1 next-generation sequencing (NGS) data. The expected 163-bp product, identified as HHV3 by confirmatory Sanger sequencing, is only seen in an HHV3-positive cerebrospinal fluid (CSF) sample processed in parallel on the same NGS run, indicating that the single HHV3 read in the dVIN sample 1 NGS dataset was most likely due to cross-contamination. (B) PCR amplification was performed using a primer set designed from two SuHV1 ViroChip probes. Note that the dVIN sample 1 is negative for SuHV1. Abbreviations: L, DNA ladder; d1-d10, dVIN samples 1 through 10; N, normal skin vulvar biopsy; H, HHV3-positive CSF sample;; ntC, no template control.</p

Histology and p16^ink4a immunostaining patterns of high-grade dVIN and uVIN samples.

<p>(A) dVIN, showing characteristic elongation and anastomosis of rete ridges. The epithelium shows enlarged keratinoocytes with abundant eosinophilic cytoplasm. The inset displays prominent cytologic atypia localized to the lower 1/3 of the epithelium. (B) uVIN, warty subtype. A spiked surface epithelium with hyperkeratosis and hypergranulosis is visualized. The inset displays full-thickness cytologic atypia. (C) p16^ink4a immunostaining is negative in dVIN. (D) Full-thickness p16^ink4a immunopositivity is seen in high-grade uVIN. Abbreviations: dVIN, differentiated vulvar intraepithelial neoplasia; uVIN, usual-type vulvar intraepithelial neoplasia.</p

Schematic flowchart showing testing of FFPE vulvar tissues from 28 cases of high-grade differentiated VIN (dVIN) and usual-type VIN (uVIN).

<p>Histological (yellow boxes), p16^ink4a immunostaining (yellow boxes), and genomic (pink boxes) analyses were performed. Abbreviations: FFPE, formalin-fixed, paraffin-embedded; VIN, vulvar intraepithelial neoplasia; VSCC, vulvar squamous cell carcinoma; PCR, polymerase chain reaction.</p

Pan-viral analysis of high-grade dVIN samples.

<p>(A) Tissue samples (x-axis) were analyzed using the ViroChip, a pan-viral DNA detection microarray. The cluster heat map of HPV probes (y-axis) shows only one strong papillomavirus cluster corresponding to the HPV18 cervical cancer positive control (PC). Other scattered clusters corresponding to the dVIN samples (tissue samples 1–10) consisted of lower-intensity probes representing multiple papillomavirus subtypes, including HPV7, HPV34, HPV81, and HPV 83 (clusters I, II, and III). Confirmatory PCR testing from the HPV L1 region using conserved primers was negative for all of the dVIN samples (gel, left). PCR testing using primers specific for each HPV subtype was also negative (2 gels, right), with the exception of bands in dVIN sample 5 that were cloned and sequenced as <i>Pseudomonas aeruginosa</i> (asterisks), and attributed to bacterial surface contamination of the FFPE block. Thus, the dVIN samples were deemed negative for HPV infection. The red color bar denotes the normalized magnitude of hybridization intensity. (B) Mapping of 16 HPV18 probes out of the top 50 (by ranked Z-score analysis) to the HPV18 genome in the cervical cancer positive control. Abbreviations: HPV, human papillomavirus; VIN, vulvar intraepithelial neoplasia; N, normal skin vulvar biopsy; PC, positive control; ntC, no template control.</p

Viruses and bacteria identified in tissue samples by next-generation sequencing.

<p>Viruses and bacteria identified in tissue samples by next-generation sequencing.</p

Number of next-generation sequencing (NGS) reads at each step of the SURPI bioinformatics pipeline for pathogen identification.

<p>Number of next-generation sequencing (NGS) reads at each step of the SURPI bioinformatics pipeline for pathogen identification.</p

Ranked Z-score analysis of ViroChip microarrays corresponding to dVIN samples and controls for virus identification.

<p>*Criteria: ≥5 hits out of top 50 probes (10%) and probe hits mapped to ≥3 distinct locations on the viral genome.</p><p>**Distinct locations: mapped probe locations on the viral genome separated by at least 5% of the total genome length.</p><p>Ranked Z-score analysis of ViroChip microarrays corresponding to dVIN samples and controls for virus identification.</p

Additional file 2: Table S2. of Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis

MetaPORE performance according to system configuration and alignment mode. (XLSX 18 kb

Additional file 1: Table S1. of Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis

Optimization of word count for MegaBLAST alignment to the NT reference database. (XLSX 12 kb