Received Mar 29; Accepted May This article has been cited by other articles in PMC. Two-sample tests of proportions were used to examine differences in the diagnostic performance of the NGS assay to detect high vs.
Received Mar 29; Accepted May This article has been cited by other articles in PMC. Two-sample tests of proportions were used to examine differences in the diagnostic performance of the NGS assay to detect high vs. Results In total there were 94 type-specific infections detected in 10 samples with a median of 9. Using the LA as the gold standard, The NPV for all types was Conclusions The NGS assay accurately detects multiple HPV infections in individual clinical specimens with limited sample volume and has extended coverage compared to LA.
Electronic supplementary material The online version of this article doi: Human papillomavirus, Multiple HPVs, Next-generation sequencing, Ion-Torrent, Linear Array Background Identifying type-specific infections of human papillomavirus HPV is fundamental for understanding carcinogenic risk in epidemiologic studies, particularly in high burden areas such as sub-Saharan Africa.
Some studies have shown that these broad-spectrum primer-based PCR methods may be less sensitive at detecting multiple infections [ 11 , 12 ]. Our prior study could not test the performance of the NGS assay in samples with multiple infections because the head and neck squamous cell carcinomas and cervical cancer specimens tested were primarily associated with a single HPV type-specific infection, with the exception of one sample that had two type-specific infections.
High-risk HPV included 13 type specific infections: Samples without library product detection were included in the pool at equal volumes. Pooled samples were purified and then quantified for emulsion template preparation on the Qubit 2.
Sequencing was performed on the Ion Torrent PGM using the v2 sequencing chemistry and v2 chips. Data processing was performed by the ion torrent server, using Torrent Suite v4. Statistical analyses The median and interquartile range of the number of genotypes identified per sample were estimated for any HPV infection, high-risk HPV, and low-risk HPV infection. To compare the NGS-HPV genotyping assay with LA, the presence or absence of 33 type-specific infections that were detectable by both assays were included in the analysis 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 53, 54, 56, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, Bolding indicates high-risk.
There were 4 type-specific infections 52, 55, 64, and IS39 82 subtype that were not included in the comparison. If either 35 or 58 are present, then 52 may be underdiagnosed in the LA assay.
For the other three genotypes, genomic sequences were not present in the PaVE database and were not available for mapping and therefore could possibly be underdiagnosed in the NGS assay. In total there were type-specific infections that could be detected by both assays. Analyses were performed using Stata Statistical Software: Release 13 College Station, TX: Stata Corp LP. If we exclude these two types, the sensitivity of the NGS assay becomes
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