Background: Human papillomavirus (HPV) testing has been employed by several European countries to augment cytology-based cervical screening programs. A number of research groups have demonstrated potential utility of next-generation sequencing (NGS) for HPV genotyping, with comparable performance and broader detection spectrum than current gold standards. Nevertheless, most of these NGS platforms may not be the best choice for medium sample throughput and laboratories with less resources and space. In light of this, we developed a Nanopore sequencing assay for HPV genotyping and compared its performance with cobas HPV Test and Roche Linear Array HPV Genotyping Test (LA). Methods: Two hundred and one cervicovaginal swabs were routinely tested for Papanicolaou smear, cobas HPV Test and LA. Residual DNA was used for Nanopore protocol after routine testing. Briefly, HPV L1 region was amplified using PGMY and MGP primers, and PCR-positive specimens were sequenced on MinION flow cells (R9.4.1). Data generated in first 2 h were aligned with reference sequences from Papillomavirus Episteme database for genotyping. Results: Nanopore detected 96 HPV-positive (47.76%) and 95 HPV-negative (47.26%) specimens, with 10 lacking β-globin band and not further analyzed (4.98%). Substantial agreement was achieved with cobas HPV Test and LA (κ: 0.83–0.93). In particular, Nanopore appeared to be more sensitive than cobas HPV Test for HPV 52 (n = 7). For LA, Nanopore revealed higher concordance for high-risk (κ: 0.93) than non-high risk types (κ: 0.83), and with similar high-risk positivity in each cytology grading. Nanopore also provided better resolution for HPV 52 in 3 specimens co-infected with HPV 33 or 58, and for HPV 87 which was identified as HPV 84 by LA. Interestingly, Nanopore identified 5 additional HPV types, with an unexpected high incidence of HPV 90 (n = 12) which was reported in North America and Belgium but not in Hong Kong. Conclusions: We developed a Nanopore workflow for HPV genotyping which was economical (about USD 50.77 per patient specimen for 24-plex runs), and with comparable or better performance than 2 reference methods in the market. Future prospective study with larger sample size is warranted to further evaluate test performance and streamline the protocol.
Keywords: Cervical cancer; HPV; Nanopore; NGS
Human papillomavirus (HPV) is generally accepted as the causative agent of cervical cancer (CC) [[
Two hundred and one cervicovaginal swabs were collected from March to July, 2019 in Hong Kong Sanatorium & Hospital. The swabs were preserved in SurePath preservative fluid (Becton, Dickson and Company, Sparks, MD, USA) and routinely tested for Papanicolaou smear (Pap smear, following The Bethesda System for reporting), cobas HPV Test and LA (Roche Diagnostics, Mannheim, Germany). Routine test results are shown in Table 1.
Results of Pap smear, cobas HPV Test, Roche Linear Array HPV Genotyping Test, and Nanopore sequencing
Patient Pap smear Roche Linear Array Cobas HPV Nanopore (PGMY) Nanopore (MGP) Total HPV reads HR Non-HR HR Non-HR HR Non-HR 1 AGUS Neg Neg Neg Neg Neg Neg Neg ND 2 ASCH 52, 59 62 Other HR 59 Neg 59 90 4956 3 ASCUS 52 55 Neg 52 55 Neg Neg 4262 4 ASCUS Neg Neg Neg Neg Neg Neg Neg ND 5 ASCUS 31, 33 54 Other HR 31, 33, 52 Neg Neg 90 8973 6 ASCUS Neg Neg Neg Neg Neg Neg Neg ND 7 ASCUS 31 Neg Other HR Neg Neg 31 Neg 1430 8 ASCUS Neg Neg Neg Neg Neg Neg Neg ND 9 ASCUS Neg 81 Neg Neg 81 Neg 81 48,477 10 ASCUS 18 Neg 18 18 Neg 18 Neg 16,206 11 ASCUS Neg Neg Neg Neg Neg Neg Neg ND 12 ASCUS Neg Neg Neg Neg Neg Neg Neg ND 13 ASCUS 52 53, 54 Other HR 52 44, 53, 74 52 74, 90 15,419 14 ASCUS Neg Neg Neg Neg Neg Neg Neg ND 15 ASCUS Neg Neg Neg Neg Neg Neg Neg ND 16 ASCUS 52 81 Neg 52 81 Neg 81 8873 17 ASCUS 52 54 Other HR 52 54 52 54 36,258 18 ASCUS 52, 59 11 Other HR 52, 59 11 52, 59 11 44,702 19 ASCUS Neg Neg Neg PCR inhibition 20 ASCUS Neg Neg Neg Neg Neg Neg Neg 7 21 ASCUS Neg Neg Neg Neg Neg Neg Neg ND 22 ASCUS Neg Neg Neg Neg Neg Neg Neg ND 23 ASCUS 39 61, 72 Other HR 39 61, 72 39 87 1624 24 ASCUS 66 Neg Other HR 66 Neg 66 Neg 10,383 25 ASCUS 68 61 Other HR Neg 61 Neg 61 10,644 26 ASCUS Neg Neg Neg Neg Neg Neg 90 541 27 ASCUS 52 Neg Neg 52 Neg Neg 87 3614 28 ASCUS Neg 62 Neg Neg 62 Neg 62 45 29 ASCUS Neg Neg Neg Neg Neg Neg Neg ND 30 ASCUS 35 Neg Other HR 35 Neg 35 Neg 1641 31 ASCUS Neg Neg Neg Neg Neg Neg Neg ND 32 ASCUS 52 Neg Other HR 52 Neg 52 Neg 399 33 ASCUS Neg Neg Neg Neg Neg Neg Neg ND 34 ASCUS 51 84 Other HR 51 Neg Neg Neg 1853 35 ASCUS Neg Neg Neg Neg 74 Neg 74 11,499 36 ASCUS Neg Neg Neg Neg Neg Neg Neg 93 37 ASCUS 51 Neg Other HR 51 Neg 51 Neg 2897 38 ASCUS Neg 40, 55, 83 Neg Neg 40, 55, 83 Neg 40, 55, 83 47,736 39 ASCUS Neg Neg Neg Neg Neg Neg Neg ND 40 ASCUS 58 53, 55, 62 Other HR 52, 58 53, 55, 62, 74 52 53, 62, 74 42,106 41 ASCUS 52 42, 73 Other HR 52 42, 73 52 42, 73 15,778 42 ASCUS Neg Neg Neg Neg Neg Neg Neg 116 43 HSIL 16 Neg 16 16 Neg 16 Neg 15,918 44 HSIL 16 Neg 16 16 Neg 16 Neg 34,654 45 HSIL 59 Neg Other HR 59 Neg 59 Neg 15,381 46 HSIL 31, 58 Neg Other HR 31, 58 Neg 31, 58 Neg 3367 47 LSIL 52, 68 84 Other HR 52, 68 84 52, 68 84, 90 24,366 48 LSIL 66 84 Other HR 66 44, 84 66 44 57,206 49 LSIL 52 Neg Neg 52 Neg 52 Neg 14,516 50 LSIL Neg 40, 53 Neg Neg 40, 53 Neg 40, 53 9265 51 LSIL 52 11, 81 Other HR 52 11, 81 52 11, 43, 81 29,748 52 LSIL 66 Neg Other HR 66 Neg 66 Neg 40,328 53 LSIL 51 Neg Other HR 51 Neg 51 43, 90 4454 54 LSIL 16, 51, 56 54, 62, 81 16, other HR 16, 51, 56 54, 62, 81 16, 51 40, 62, 81 20,455 55 LSIL 56 53 Other HR 56 53 56 53 28,377 56 LSIL Neg Neg Neg Neg Neg Neg Neg ND 57 LSIL 66 54, 55, 81 Other HR 66 54, 55, 81 66 55, 81, 90 25,606 58 LSIL 52 Neg Neg 52 42 52 90 15,103 59 LSIL 59 Neg Other HR 59 Neg Neg Neg 11,235 60 LSIL 59 89 Neg 59 89 Neg 89 67,220 61 LSIL 56 82 Other HR 56 82 56 43, 82 42,160 62 LSIL 52 Neg Other HR 52 Neg 52 Neg 39,323 63 LSIL 33, 51 Neg Other HR 33, 51 44 51 44 19,704 64 LSIL+ ASCH 51 Neg Other HR 51 Neg 51 Neg 4621 65 NIL 16 Neg 16 16 Neg 16 Neg 1958 66 NIL Neg Neg Neg Neg Neg Neg Neg ND 67 NIL Neg Neg Neg Neg Neg Neg Neg ND 68 NIL Neg Neg Neg 59 Neg 59 Neg 2455 69 NIL Neg Neg Neg Neg 87 Neg 87 8775 70 NIL Neg Neg Neg Neg Neg Neg Neg ND 71 NIL Neg Neg Neg Neg Neg Neg Neg ND 72 NIL Neg Neg Neg Neg Neg Neg Neg ND 73 NIL Neg Neg Neg Neg Neg Neg Neg ND 74 NIL 58 Neg Other HR 58 Neg 52, 58 62 8619 75 NIL 58 Neg Other HR 58 Neg 58 Neg 13,149 76 NIL Neg Neg Neg Neg Neg Neg Neg ND 77 NIL Neg Neg Neg Neg Neg Neg Neg ND 78 NIL Neg Neg Neg Neg Neg Neg 90 2289 79 NIL 56 70 Other HR Neg 44, 70 56 44, 70 7855 80 NIL Neg Neg Neg PCR inhibition 81 NIL Neg Neg Neg Neg Neg Neg Neg 74 82 NIL Neg 42 Neg Neg Neg Neg 42 1406 83 NIL Neg Neg Neg Neg 74 Neg 74 7441 84 NIL Neg Neg Neg Neg Neg Neg Neg ND 85 NIL Neg 82 Neg Neg 82 Neg 82 1162 86 NIL Neg 62 Neg Neg 62 Neg 62 65,368 87 NIL Neg Neg Neg Neg Neg Neg Neg ND 88 NIL Neg Neg Neg Neg Neg Neg Neg ND 89 NIL Neg Neg Neg Neg Neg Neg Neg ND 90 NIL Neg Neg Neg Neg Neg Neg Neg 142 91 NIL 39, 52 Neg Other HR 52 Neg 52 90 15,703 92 NIL 68 Neg Other HR 68 42 68 Neg 19,777 93 NIL Neg Neg Neg Neg Neg Neg Neg ND 94 NIL Neg Neg Neg Neg Neg Neg Neg ND 95 NIL Neg Neg Neg Neg Neg Neg Neg ND 96 NIL 52 Neg Neg 52 Neg 52 Neg 5242 97 NIL Neg Neg Neg Neg Neg Neg Neg ND 98 NIL Neg Neg Neg Neg Neg Neg Neg ND 99 NIL Neg Neg Neg Neg Neg Neg Neg 41 100 NIL 52 Neg Other HR 52 Neg 52 Neg 24,478 101 NIL Neg 61 Neg PCR inhibition 102 NIL Neg Neg Neg Neg Neg Neg Neg 72 103 NIL 39 Neg Neg Neg Neg Neg Neg ND 104 NIL Neg 62, 84 Neg Neg 62 Neg 62 3589 105 NIL Neg 71 Neg Neg Neg Neg Neg ND 106 NIL Neg Neg Neg Neg Neg Neg Neg ND 107 NIL 52 62 Other HR 52 44, 53, 62 52 44 18,086 108 NIL Neg Neg Neg Neg Neg Neg Neg ND 109 NIL Neg Neg Neg Neg Neg Neg Neg ND 110 NIL Neg Neg Neg Neg Neg Neg Neg ND 111 NIL Neg 84 Neg Neg Neg Neg Neg ND 112 NIL 16, 52 Neg 16 16, 52 Neg 16 Neg 72,357 113 NIL Neg Neg Neg Neg Neg Neg Neg ND 114 NIL Neg 55, 89 Neg Neg 26, 55, 89 59 26, 55, 62, 89 8926 115 NIL Neg Neg Neg Neg Neg Neg 74 1586 116 NIL Neg 81 Neg Neg Neg Neg Neg ND 117 NIL Neg Neg Neg Neg Neg Neg Neg ND 118 NIL Neg 6, 62 Neg Neg 6, 62 Neg 6, 62 9414 119 NIL Neg Neg Neg Neg Neg Neg Neg ND 120 NIL Neg 54 Neg Neg Neg Neg Neg ND 121 NIL Neg Neg Neg PCR inhibition 122 NIL Neg Neg Neg Neg Neg Neg Neg 8 123 NIL 68 Neg Other HR Neg Neg Neg Neg ND 124 NIL Neg 81 Neg Neg 81 Neg 81 8735 125 NIL Neg 84 Neg Neg Neg Neg 87 1025 126 NIL Neg Neg Neg Neg Neg Neg 90 1719 127 NIL Neg Neg Neg Neg Neg Neg Neg ND 128 NIL Neg Neg Neg Neg Neg Neg Neg ND 129 NIL Neg Neg Neg Neg Neg Neg Neg 10 130 NIL Neg Neg Neg Neg Neg Neg Neg ND 131 NIL Neg 84 Neg Neg Neg Neg Neg ND 132 NIL Neg Neg Neg Neg Neg Neg Neg ND 133 NIL 59 62, 71 Other HR Neg Neg Neg Neg 30 134 NIL Neg Neg Neg Neg Neg Neg Neg ND 135 NIL Neg Neg Neg Neg Neg Neg Neg 522 136 NIL Neg Neg Neg Neg Neg Neg Neg ND 137 NIL 51 84 Other HR PCR inhibition 138 NIL 39 Neg Other HR 39 Neg 39 Neg 19,305 139 NIL Neg Neg Neg Neg Neg Neg Neg 195 140 NIL Neg Neg Neg Neg Neg Neg Neg ND 141 NIL Neg Neg Neg Neg Neg Neg Neg 23 142 NIL Neg Neg Neg Neg Neg Neg Neg ND 143 NIL Neg 42, 81 Neg Neg 40, 74, 81 Neg 40, 74, 81, 87 19,118 144 NIL Neg Neg Neg Neg Neg Neg Neg ND 145 NIL Neg Neg Neg Neg Neg Neg Neg ND 146 NIL Neg Neg Neg Neg Neg Neg Neg ND 147 NIL Neg Neg Neg Neg Neg Neg Neg 40 148 NIL 59 Neg Neg 59 Neg Neg Neg 12,681 149 NIL Neg Neg Neg Neg Neg Neg Neg 14 150 NIL Neg Neg Neg PCR inhibition 151 NIL Neg Neg Neg Neg Neg Neg Neg 79 152 NIL Neg 62 Neg Neg 62 Neg 62 14,353 153 NIL Neg Neg Neg Neg Neg Neg Neg ND 154 NIL Neg Neg Neg Neg Neg Neg Neg ND 155 NIL Neg Neg Neg Neg Neg Neg Neg ND 156 NIL 52 54 Neg 52 54 52 54 18,397 157 NIL 39, 52 53, 61 Other HR 39 53, 61 39 53, 61 20,332 158 NIL Neg Neg Neg Neg Neg Neg Neg ND 159 NIL Neg Neg Neg Neg Neg Neg Neg 60 160 NIL Neg Neg Neg PCR inhibition 161 NIL Neg 62 Neg Neg 62 Neg 62 13,545 162 NIL Neg Neg Neg Neg 74 Neg 74 4514 163 NIL Neg 62 Neg Neg 62 Neg 62 11,894 164 NIL Neg Neg Neg Neg Neg Neg Neg ND 165 NIL 59 Neg Neg PCR inhibition 166 NIL Neg Neg Neg Neg Neg Neg Neg ND 167 NIL 39 Neg Other HR 39 Neg 39 Neg 52,831 168 NIL Neg Neg Neg Neg Neg Neg Neg ND 169 NIL Neg Neg Neg Neg Neg Neg Neg ND 170 NIL 66 Neg Other HR 66 Neg 66 Neg 54,943 171 NIL Neg Neg Neg Neg Neg Neg Neg ND 172 NIL Neg Neg Neg Neg Neg Neg Neg ND 173 NIL Neg Neg Neg Neg Neg Neg Neg ND 174 NIL 66 Neg Other HR 66 Neg 66 Neg 57,791 175 NIL Neg 54 Neg Neg 54 Neg 54 23,583 176 NIL Neg Neg Neg PCR inhibition 177 NIL 16 62 16 Neg 53, 62 16 62 28,181 178 NIL Neg Neg Neg Neg Neg Neg Neg 206 179 NIL Neg Neg Neg Neg Neg Neg Neg ND 180 NIL Neg Neg Neg Neg Neg Neg Neg ND 181 NIL 51, 66 Neg Other HR 51, 66, 68 Neg 51, 66, 68 Neg 6952 182 NIL 16, 51, 58 61 Other HR 58 61 Neg 61 5737 183 NIL Neg Neg Neg Neg Neg Neg Neg ND 184 NIL 58 Neg Other HR 58 Neg 58 Neg 43,034 185 NIL 58 70, 89 Other HR 58 70, 89 58 89 33,842 186 ND Neg Neg Neg Neg Neg Neg Neg 414 187 ND Neg Neg Neg Neg Neg Neg Neg ND 188 ND 16 Neg 16 16 Neg 16 Neg 96,549 189 ND Neg Neg Neg Neg Neg Neg Neg ND 190 ND Neg Neg Neg Neg Neg Neg Neg ND 191 ND 56 Neg Other HR 56 Neg 56 Neg 18,782 192 ND 51 Neg Other HR 51 Neg 51 Neg 6020 193 ND Neg 62 Neg Neg 62 Neg 62 20,373 194 ND Neg Neg Neg Neg Neg Neg Neg ND 195 ND 52, 59 Neg Other HR 52, 59 Neg 59 Neg 11,926 196 ND 59 Neg Other HR 59 Neg 59 Neg 24,045 197 ND 52, 59 54, 70 Other HR 52, 59 70 52, 59 70, 90 46,523 198 ND 56, 66 53, 61, 84 Other HR 66 32, 53, 61, 84 56 32, 53, 61, 84 62,600 199 ND Neg 62 Neg Neg Neg Neg Neg ND 200 ND Neg 53, 54, 81, 83 Neg Neg 53, 54, 83 Neg 53, 81, 83 32,868 201 ND Neg Neg Neg PCR inhibition
AGUS Atypical glandular cells of undetermined significance, ASCH Atypical squamous cells – cannot exclude HSIL, ASCUS Atypical squamous cells of undetermined significance, HR High-risk, HSIL High-grade squamous intraepithelial lesion, LSIL Low-grade squamous intraepithelial lesion, ND Pap smear/ MinION sequencing not done, Neg Negative, NIL normal cytology
DNA extraction and cobas HPV Test were performed using cobas 4800 system (Roche Diagnostics, Rotkreuz, Switzerland). Briefly, 500 μL of cervicovaginal specimen was added to 500 μL of sample preparation buffer and heated at 120 °C for 20 min. The mixture was brought to ambient temperature for 10 min and processed on cobas × 480 using 'high-risk HPV DNA PCR' protocol. Real-time polymerase chain reaction (PCR) was performed on cobas z 480. Fifty microliter of DNA extract was used for LA according to manufacturer's recommendations. Residual DNA was used for Nanopore protocol after routine testing.
For each specimen, L1 region of HPV genome was amplified in 2 separate PCRs using PGMY and MGP primer sets [[
Primer sequences
Primer 5′ to 3′ sequence References PGMY11-A GCA CAG GGA CAT AAC AAT GG [ PGMY11-B GCG CAG GGC CAC AAT AAT GG PGMY11-C GCA CAG GGA CAT AAT AAT GG PGMY11-D GCC CAG GGC CAC AAC AAT GG PGMY11-E GCT CAG GGT TTA AAC AAT GG PGMY09-F CGT CCC AAA GGA AAC TGA TC PGMY09-G CGA CCT AAA GGA AAC TGA TC PGMY09-H CGT CCA AAA GGA AAC TGA TC PGMY09-I G CCA AGG GGA AAC TGA TC PGMY09-J CGT CCC AAA GGA TAC TGA TC PGMY09-K CGT CCA AGG GGA TAC TGA TC PGMY09-L CGA CCT AAA GGG AAT TGA TC PGMY09-M CGA CCT AGT GGA AAT TGA TC PGMY09-N CGA CCA AGG GGA TAT TGA TC PGMY09-P G CCC AAC GGA AAC TGA TC PGMY09-Q CGA CCC AAG GGA AAC TGG TC PGMY09-R CGT CCT AAA GGA AAC TGG TC HMB01 GCG ACC CAA TGC AAA TTG GT Human β-globin forward GAAGAGCCAAGGACAGGTAC [ Human β-globin reverse GGAAAATAGACCAATAGGCAG MGPA ACGTTGGATGTTTGTTACTGTGGTGGATACTAC [ MGPB ACGTTGGATGTTTGTTACCGTTGTTGATACTAC MGPC ACGTTGGATGTTTGTTACTAAGGTAGATACCACTC MGPD ACGTTGGATGTTTGTTACTGTTGTGGATACAAC MGP31 ACGTTGGATGTTTGTTACTATGGTAGATACCACAC MGPG ACGTTGGATGGAAAAATAAACTGTAAATCATATTCCT MGPH ACGTTGGATGGAAAAATAAATTGTAAATCATACTC MGPI ACGTTGGATGGAAATATAAATTGTAAATCAAATTC MGPJ ACGTTGGATGGAAAAATAAACTGTAAATCATATTC MGP18 ACGTTGGATGGAAAAATAAACTGCAAATCATATTC
Master mix constituents and PCR conditions
PGMY PCR 10X PCR buffer II (Applied Biosystems) 5 25 mM MgCl2 (Applied Biosystems) 3 PGMY primer mix (10 μM) 1 Human β-globin primer mix (5 μM) 1 10 mM dNTPs (Roche) 1 5 M betaine (Sigma) 10 AmpliTaq Gold DNA Polymerase (Applied Biosystems) 0.25 Molecular grade water (Sigma) 23.75 DNA 5 95 9 min 1 95 1 min 40 (50% ramp) 55 1 min 72 1 min 72 5 min 1 15 Hold / MGP PCR 10X PCR buffer II (Applied Biosystems) 2.5 25 mM MgCl2 (Applied Biosystems) 1.5 MGP primer mix (10 μM) 0.5 10 mM dNTPs (Roche) 0.5 AmpliTaq Gold DNA Polymerase (Applied Biosystems) 0.1 Molecular grade water (Sigma) 14.9 DNA 5 95 10 min 1 95 30 s 5 42 30 s 72 30 s 95 30 s 45 64 30 s 72 30 s 72 5 min 1 15 Hold /
PGMY and MGP PCR amplicons of each positive specimen were pooled and purified using AMPure XP beads (Beckman-Coulter, Brea, CA, USA). Nanopore sequencing libraries were prepared from purified amplicons using Ligation Sequencing Kit 1D (SQK-LSK109) and PCR-free Native Barcoding Expansion Kit (EXP-NBD104/114) (Oxford Nanopore Technologies, Oxford, England). The barcoded libraries were loaded and sequenced on MinION flow cells (FLO-MIN106D R9.4.1, Oxford Nanopore Technologies, Oxford, England) after quality control runs.
Data from first 2 h of sequencing runs was analyzed. FASTQ files generated by live basecalling (MinKNOW version 2.0) were demultiplexed using 'FASTQ Barcoding' workflow on EPI2ME (Oxford Nanopore Technologies, Oxford, England) with default minimum qscore of 7, 'auto' and 'split by barcode' options. FASTQ files of each specimen were concatenated into a single file and analyzed using a 2-step custom workflow on Galaxy bioinformatics platform. Briefly, FASTQ files were converted into FASTA format, followed by aligning sequences against HPV reference genomes from PaVE database using NCBI BLAST+ blastn (Galaxy version 1.1.1). PGMY and MGP reads were sorted based on sequence length and analyzed individually. Threshold of each run was derived from average number of background reads plus 10 standard deviations, which were calculated using interquartile rule, excluding first and last quartiles. A positive HPV call was based on either (
As HPV 66 is categorized as 'other high-risk' by cobas HPV Test, all calculations were based on this grouping, albeit HPV 66 was found as a single infection in cancers with extreme rarity and re-classified as possible carcinogen (Group 2B) by IARC Monographs Working Group [[
The results are summarized in Table 1. PCR was successful for 191 specimens (191/201, 95.02%), with 10 specimens (10/201, 4.98%) lacking β-globin band and therefore regarded as inappropriate for further analysis. Seventy-six specimens (76/201, 37.81%) were negative for both PGMY and MGP PCRs, and 115 (115/201, 57.21%) were positive for either of the two. PCR-positive specimens were sequenced on 10 MinION flow cells with 145–890 active pores, generating 31,748–525,880 HPV reads in first 2 h (Table 4). For the 115 specimens sequenced, 19 were negative (7–522 reads, 113 in average) and 96 were positive (45–96,549 reads, 20,158 in average) for HPV. Taken together, there were 95 HPV-negative (95/201, 47.26%) and 96 HPV-positive (96/201, 47.76%) specimens by Nanopore workflow.
Details of Nanopore sequencing runs
Run No. of active pores Elapsed sequencing time No. of HPV reads 1 611 2 h 11 min 60,976 2 458 1 h 59 min 246,521 3 690 2 h 1 min 279,520 4 467 2 h 5 min 111,885 5 462 2 h 5 min 31,748 6 247 2 h 3 min 113,521 7 330 2 h 5 min 111,702 8 753 2 h 1 min 478,711 9 145 1 h 59 min 207,094 10 890 1 h 59 min 525,880
Table 5 shows concordance of Nanopore workflow with cobas HPV Test and LA, which was based on the 37 HPV types detectable by LA. For cobas HPV Test, our workflow achieved 93.19, 93.19 and 81.94% for perfect, total and positive agreement, respectively, with Cohen's kappa of 0.85. For LA, Nanopore achieved a perfect agreement of 83.77% for both high-risk and non-high risk HPVs. For high-risk types, total and positive agreement were 96.86 and 91.78%, respectively, with Cohen's kappa of 0.93. For non-high risk types, total and positive agreement were 93.19 and 77.59%, respectively, with Cohen's kappa of 0.83.
Agreement between cobas HPV Test, Roche Linear Array HPV Genotyping Test (LA) and Nanopore
Nanopore Perfect agreement Total agreement Positive agreement Cohen's κ + – 59 2 93.19% 93.19% 81.94% 0.85 11 119 67 4 83.77% 96.86% 91.78% 0.93 2 118 45 10 93.19% 77.59% 0.83 3 133
Table 6 shows per-type concordance of Nanopore and LA. A total of 13 high-risk and 19 non-high risk HPV types were evaluated. Positive agreement for HPV 16 (n = 8) and 18 (n = 1) were 87.5 and 100%, respectively. Positive agreement was 75–100% for high-risk HPV 31, 33, 35, 39, 51, 52, 56, 58, 59 and 66, and 20% for HPV 68 (n = 5). For non-high risk HPVs, positive agreement was 37.5–100% for HPV 6, 11, 40, 42, 53, 54, 55, 61, 62, 70, 72, 73, 81, 82, 83, 84 and 89. There were 2 non-high risk types with 0% positive agreement (HPV 26 and 71). HPV 26 (n = 1) was only detected by Nanopore workflow, whereas HPV 71 (n = 2) was only detected by LA.
Per HPV type positive agreement between Roche Linear Array Genotyping Test (LA) and Nanopore
HPV Genotypes Number of specimens Positive agreement Nanopore−/LA−/LA- Nanopore +/LA- Nanopore−/LA+ Nanopore+/LA+ Total 183 0 1 7 191 87.5% 190 0 0 1 191 100% 188 0 0 3 191 100% 189 0 0 2 191 100% 190 0 0 1 191 100% 185 0 1 5 191 83.33% 182 0 1 8 191 88.89% 165 3 2 21 191 80.77% 185 0 0 6 191 100% 184 0 0 7 191 100% 179 2 1 9 191 75% 182 1 0 8 191 88.89% 186 2 2 1 191 20% 190 0 0 1 191 100% 189 0 0 2 191 100% 190 1 0 0 191 0% 187 2 0 2 191 50% 186 2 1 2 191 40% 181 3 0 7 191 70% 181 0 4 6 191 60% 186 0 0 5 191 100% 186 0 0 5 191 100% 174 2 2 13 191 76.47% 188 0 0 3 191 100% 189 0 2 0 191 0% 190 0 0 1 191 100% 190 0 0 1 191 100% 182 0 1 8 191 88.89% 189 0 0 2 191 100% 189 0 0 2 191 100% 183 0 5 3 191 37.5% 188 0 0 3 191 100%
Table 7 reveals the percentage of identity of Nanopore consensus sequences to HPV reference genomes. In general, Nanopore consensus sequences showed an average identity of 98% to the best matches, with an average difference of 15% from second BLAST hits.
Percentage of identity of Nanopore consensus sequences to HPV reference genomes
Patient Nanopore results Best BLAST hit Second BLAST hit Difference HPV type % identity HPV type % identity 59 99% 18 77% 22% 90 97% 106 84% 15% 52 99% 58 80% 19% 55 100% 44 93% 7% 31 98% 35 80% 18% 33 99% 58 86% 13% 52 99% 58 80% 19% 90 97% 106 85% 12% 31 95% 35 79% 16% 81 99% 62 85% 14% 18 99% 45 85% 14% 44 99% 55 92% 7% 52 99% 58 80% 19% 53 99% 30 85% 14% 74 99% 55 83% 16% 90 97% 106 85% 12% 52 99% 58 81% 18% 81 99% 62 85% 14% 52 99% 58 80% 19% 54 99% 45 74% 25% 11 99% 6 87% 12% 52 99% 58 80% 19% 59 99% 18 77% 22% 39 99% 70 81% 18% 61 99% mEV06c12b 83% 16% 72 92% mEV06c12b 89% 3% 87 98% 86 85% 13% 66 98% 56 84% 14% 61 99% mEV06c12b 83% 16% 90 97% 106 85% 12% 52 99% 58 80% 19% 87 98% 86 84% 14% 62 99% 81 84% 15% 35 98% 31 80% 18% 52 99% 58 81% 18% 51 99% 82 85% 14% 74 99% 55 84% 15% 51 99% 82 85% 14% 40 99% 7 88% 11% 55 99% 44 93% 6% 83 99% 102 84% 15% 52 99% 58 80% 19% 53 98% 30 85% 13% 55 100% 44 93% 7% 58 99% 33 86% 13% 62 99% 81 85% 14% 74 98% 55 84% 14% 42 98% 32 83% 15% 52 100% 58 81% 19% 73 99% 34 85% 14% 16 100% 35 78% 22% 16 99% 35 78% 21% 59 99% 18 76% 23% 31 98% 35 80% 18% 58 99% 33 86% 13% 52 98% 58 80% 18% 68 93% 39 81% 12% 84 98% 87 84% 14% 90 97% 106 85% 12% 44 99% 55 93% 6% 66 98% 56 84% 14% 84 99% 87 84% 15% 52 99% 58 80% 19% 40 98% 7 87% 11% 53 98% 30 85% 13% 11 100% 6 87% 13% 43 95% 45 77% 18% 52 99% 58 80% 19% 81 99% 62 84% 15% 66 98% 56 83% 15% 43 95% 45 78% 17% 51 99% 82 84% 15% 90 97% 106 85% 12% 16 100% 35 78% 22% 40 93% 7 85% 8% 51 99% 82 84% 15% 54 99% 45 73% 26% 56 90% 66 76% 14% 62 99% 81 84% 15% 81 99% 62 85% 14% 53 99% 56 79% 20% 56 99% 66 84% 15% 54 87% 31 74% 13% 55 100% 44 93% 7% 66 98% 56 84% 14% 81 99% 62 84% 15% 90 97% 106 85% 12% 42 99% 32 84% 15% 52 98% 58 80% 18% 90 97% 106 85% 12% 59 99% 18 77% 22% 59 99% 18 76% 23% 89 99% 81 78% 21% 43 96% 45 79% 17% 56 97% 66 83% 14% 82 99% 51 84% 15% 52 99% 58 80% 19% 33 99% 58 86% 13% 44 99% 55 93% 6% 51 99% 82 83% 16% 51 99% 82 84% 15% 16 100% 35 78% 22% 59 99% 18 77% 22% 87 99% 86 86% 13% 52 99% 58 81% 18% 58 99% 33 86% 13% 62 99% 81 85% 14% 58 99% 33 85% 14% 90 97% 106 85% 12% 44 99% 55 92% 7% 56 96% 66 84% 12% 70 99% 39 81% 18% 74 93% 55 81% 12% 42 95% 32 83% 12% 74 97% 55 83% 14% 82 99% 51 84% 15% 62 99% 81 85% 14% 52 99% 58 80% 19% 90 97% 106 84% 13% 42 93% 32 78% 15% 68 92% 39 80% 12% 52 99% 58 80% 19% 52 99% 58 80% 19% 62 98% 81 85% 13% 44 99% 55 93% 6% 52 99% 58 81% 18% 53 100% 30 86% 14% 62 99% 81 85% 14% 16 98% 58 78% 20% 52 99% 58 81% 18% 26 100% 69 83% 17% 55 100% 44 93% 7% 59 99% 18 77% 22% 62 99% 81 85% 14% 89 99% 81 77% 22% 74 95% 55 83% 12% 6 99% 11 87% 12% 62 99% 81 84% 15% 81 99% 62 85% 14% 87 98% 86 85% 13% 90 97% 106 85% 12% 39 99% 68 81% 18% 40 99% 7 88% 11% 74 98% 55 84% 14% 81 99% 62 84% 15% 87 97% 86 84% 13% 59 99% 18 77% 22% 62 98% 81 85% 13% 52 99% 58 81% 18% 54 95% 6 74% 21% 39 94% 70 81% 13% 53 96% 30 84% 12% 61 99% mEV06c12b 83% 16% 62 98% 81 83% 15% 74 94% 55 85% 9% 62 99% 81 85% 14% 39 99% 70 81% 18% 66 98% 56 83% 15% 66 98% 56 83% 15% 54 99% 45 73% 26% 16 99% 35 80% 19% 53 99% 30 84% 15% 62 99% 81 85% 14% 51 99% 82 85% 14% 66 98% 56 83% 15% 68 98% 39 81% 17% 58 98% 33 87% 11% 61 100% mEV06c12b 83% 17% 58 99% 33 85% 14% 58 99% 33 85% 14% 70 99% 39 81% 18% 89 99% 81 78% 21% 16 100% 35 78% 22% 56 99% 66 83% 16% 51 98% 82 84% 14% 62 99% 81 85% 14% 52 99% 58 81% 18% 59 99% 18 76% 23% 59 99% 18 77% 22% 52 100% 58 81% 19% 59 99% 18 76% 23% 70 99% 39 81% 18% 90 97% 106 85% 12% 32 99% 42 84% 15% 53 99% 30 86% 13% 56 99% 66 84% 15% 61 100% mEV06c12b 83% 17% 66 98% 56 83% 15% 84 99% 87 84% 15% 53 98% 30 85% 13% 54 99% 45 74% 25% 81 99% 62 84% 15% 83 95% 102 82% 13%
Table 8 summarizes HPV status of each cytology grading. For high-grade and low-grade squamous intraepithelial lesion (HSIL and LSIL), nearly all specimens were positive for high-risk HPV (HSIL: 4/4, 100%; LSIL: 16/18, 88.89%). For atypical squamous/ glandular cells, about half of the specimens were positive for high-risk HPV (by LA: 19/41, 46.34%; by Nanopore: 18/41, 43.90%). For cases without observable abnormalities, 22.12% (25/113) and 21.24% (24/113) were positive for high-risk HPV by LA and Nanopore, respectively.
Results of Pap smear, LA and Nanopore workflow. The calculations were based 176 quality control-valid specimens with Pap smear results available
Pap smear interpretation HPV status No. of specimens LA Nanopore HSIL ( HR/ HR + non-HR 4 4 Non-HR only 0 0 Negative 0 0 LSIL/ LSIL + ASCH ( HR/ HR + non-HR 16 16 Non-HR only 1 1 Negative 1 1 AGUS/ ASCH/ ASCUS ( HR/ HR + non-HR 19 18 Non-HR only 3 6 Negative 19 17 NIL ( HR/ HR + non-HR 25 24 Non-HR only 18 18 Negative 70 71
Hong Kong has been one of the Asian regions with the lowest incidence and mortality rate of CC [[
Cytology and HPV testing have their own value for CC screening. High quality cytology has high specificity for CC, but with lower sensitivity ranging from 50% suggested by cross-sectional studies to 75% estimated longitudinally [[
Compared with HPV assays in the market, HPV genotyping by NGS offers a broader detection spectrum which, despite minimal benefit of non-high risk HPV information for CC screening, may provide important etiologic clues for other HPV-associated infections and a more complete picture of HPV epidemiology. For the latter, Nanopore identified more HPV types per sample (Fig. 1) and 5 extra HPV types (HPV 43, 44, 74, 87 and 90, n = 34) not detectable by LA (Fig. 2), with an unexpected high incidence of HPV 90 (n = 12) which was reported in North America and Belgium but not in Hong Kong [[
Graph: Fig. 1 Number of HPV types detected per sample by Nanopore workflow and LA
Graph: Fig. 2 Diversity of HPV types detected by Nanopore workflow and LA
In general, Nanopore had substantial agreement with cobas HPV Test and LA. Compared with cobas HPV Test, Nanopore appeared to be more sensitive for HPV 52 (n = 7) and 59 (n = 4), with 81.82% (9/11) of these discrepant results matched with LA. Compared with LA, concordance for high-risk HPV was higher than non-high risk types. Among the 37 discrepant results, 22 were false negatives by Nanopore and 15 were not detected by LA.
For the false negatives by Nanopore, more than half (12/22, 54.55%) were mixed infections, and similar finding was reported by other research groups using HPV consensus primers for NGS-based genotyping [[
For the 15 HPV types missed by LA, the average identity of Nanopore consensus sequences was 98.27% with an average difference of 16% from second BLAST hits (Table 7). As distinct HPV types generally have more than 10% difference in L1 sequence [[
The Nanopore method and LA revealed very similar high-risk HPV positivity in each cytology grading. The goal of combined cytology-HPV testing approach is to enhance cost effectiveness of CC screening. While minimizing unnecessary referral for colposcopy, HPV genotyping may identify high-risk individuals before observable cytological abnormalities, for instance, the 4 HPV 16-positive patients without abnormal cytology findings in this study. This may facilitate an early detection approach for cancer prevention.
Our study had several limitations. First, the sample size of certain HPV types, for example, HPV 18 (n = 1), was less satisfactory for evaluating type-specific performance. Second, as residual DNA was used after routine testing, DNA input for PGMY and MGP PCRs was constrained which might lower the sensitivity. In addition, as flow cells with suboptimal number of active pores were used, sequencing time and depth might be further improved if new flow cells were used.
We developed a Nanopore workflow for HPV genotyping, with performance comparable to or better than 2 reference methods in the market. Our method was economical, with a reagent cost of about USD 50.77 per patient specimen for 24-plex runs, which was competitive when compared to an average price of USD 106.14 (from 4 randomly-selected laboratories) for HPV genotyping referral service in our region (Table 9). The protocol was also straightforward with reasonable turnaround time of about 12 h from samples to answers. The small size and portability of MinION sequencers may well suit remote or resource-limited laboratories with constraints in space. Future prospective study with larger sample size is warranted to further evaluate test performance and streamline the protocol. As LA was discontinued in Hong Kong, the Nanopore workflow described here may provide an economical option for broad-range HPV genotyping.
Comparison of estimated reagent cost of Nanopore workflow (24-plex) and randomly-selected prices of HPV genotyping referral service in Hong Kong
Procedure Number of specimens Cost DNA extraction and PCRs 201 patients +20 controls = 221 USD 20.02 × 221 reactions = USD 4424.42 Nanopore sequencing 115 patients / 24 = at least 5 runs N = 120 for 1 positive control per run USD 1155.94 × 5 runs = USD 5779.70 (4424.42 + 5779.70) / 201 = Lab A USD 77.19 Lab B USD 124.79 Lab C USD 101.63 Lab D USD 120.93
Not applicable.
We thank the colleagues of Department of Pathology, Hong Kong Sanatorium & Hospital for their dedicated and professional work on routine laboratory diagnostics.
BSFT, TLC and WSC conceived and designed the study. BSFT, ESKM, MKMC, TLC, CPL, CHA, MYT, MKN, SML and WSC were involved in data collection and analysis. WSC wrote the first draft. All authors critically reviewed and approved the manuscript.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
This study was approved by Research Ethics Committee (REC) of Hong Kong Sanatorium & Hospital under the reference number RC-2019-18. No patient-identifying data was collected throughout the whole study.
Not applicable.
The authors declare that they have no competing interests.
• CC
- Cervical cancer
• HPV
- Human papillomavirus
• HSIL
- High-grade squamous intraepithelial lesion
• IARC
- International Agency for Research on Cancer (IARC)
• LA
- Roche Linear Array HPV Genotyping Test
• LSIL
- Low-grade squamous intraepithelial lesion
• NGS
- Next-generation sequencing
- Pap smear
- Papanicolaou smear
• PCR
- Polymerase chain reaction
• WHO
- World Health Organization
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By Wai Sing Chan; Tsun Leung Chan; Chun Hang Au; Chin Pang Leung; Man Yan To; Man Kin Ng; Sau Man Leung; May Kwok Mei Chan; Edmond Shiu Kwan Ma and Bone Siu Fai Tang
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