Samples
Genomic DNA was previously extracted from anal swab samples and genotyped on LA to determine the prevalence of HPV type-specific infections between HIV-positive and HIV-negative men who have sex with men in Abuja, Nigeria [
13]. In brief, DNA was extracted from 250 μL of Aptima Specimen Transport medium (Hologic, San Diego, CA) using the QIAmp MinElute Media Kit (Qiagen, Valencia, CA). DNA was resuspended in 100 μL of Buffer AVE, quantified by NanoDrop (ng/μL) and stored at -20
0 C. A 10-μL aliquot of the purified DNA was amplified using the PGMY 09/11 L1 consensus primer system which co-amplifies 37 HPV genotypes and a human β-globin internal control target. Both high- and low-risk HPV genotypes were detected using the LA (Roche Diagnostics, Indianapolis IN). High-risk HPV included 13 type specific infections: 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59, and 68 [
14]. For any HPV (both low and high-risk), the median number of infections was 4, (interquartile range [IQR]: 2–8, range: 0–15); 38% (59/154) of the specimens had 2–5 infections and 38% (58/154) had 6–15 infections. Approximately 49% (76/154) of the specimens had two or more high-risk HPV infections (median: 1, IQR:0–3, range 0–7).
Laboratory
For the present study, 10 samples of genomic DNA positive for multiple high-risk HPV infections were selected. Quantified genomic DNA (20 ng) were included in HPV library amplification as previously described [
1]. In brief, all samples were included in the sequencing pool at a standardized concentration of ~500pM, as determined by the BioAnalyzer. 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 2.0 Fluorometer and prepared using Ion personal genome machine (PGM) 200 kits on the OneTouch 2. Sequencing was performed on the Ion Torrent PGM using the 200 v2 sequencing chemistry and 316v2 chips.
Data processing was performed by the ion torrent server, using Torrent Suite v4.4.3, and mapped to the full genomic sequences of HPV downloaded from the Papillomavirus Episteme (PaVE) database with a minimum score of AQ17. Further filtering of only reads >100 bp was performed using NGSUtils. A sample contained >5000 reads to be included in the analysis and the reads for each type specific infection accounted for more than 0.05% of the total number of reads to be called positive.
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, 89. Bolding indicates high-risk). There were 4 type-specific infections (52, 55, 64, and IS39 (82 subtype) that were not included in the comparison. In the LA assay, HPV 52 cross reacts with 35 and 58. 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 330 type-specific infections that could be detected by both assays.
To estimate the ability of the NGS assay to detect the same types of infections detected on LA, we calculated the positive predictive value [(true positive/(true positive + false positive))*100] and the negative predictive value [(true negative/(true negative + false negative))*100] for any HPV, and separately for high-risk HPV and low-risk HPV with the associated 95% confidence intervals. To estimate the ability of the NGS assay to correctly identify those with and without HPV genotypes, we calculated the sensitivity [(true positive/(true positive + false negative))*100] and the specificity [(true negative/(true negative + false positive))*100] for any HPV, and separately for high-risk HPV and low-risk HPV with the associated 95% confidence intervals. Two-sample tests of proportions were used to compare the performance of the NGS-HPV assay for high and low risk HPV infections relative to the LA. Analyses were performed using Stata Statistical Software: Release 13 (College Station, TX: Stata Corp LP).