Development and validation of a new HPV genotyping assay based on next-generation sequencing.
OBJECTIVE
We developed a new human papillomavirus (HPV) genotyping assay based on multiplex polymerase chain reaction and next-generation sequencing (NGS) methods for large-scale cervical cancer screening.


METHODS
We first trained the assay on 1,170 self-collected samples, balancing the cutoff points for high-risk types. Then using 4,262 separate self-collected specimens, we compared concordance, sensitivity, and specificity for cervical intraepithelial neoplasia type 2 (CIN2) or higher and CIN type 3 (CIN3) or higher of the HPV sequencing assay with that of Hybrid Capture 2 (HC2) direct samples and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay self-samples.


RESULTS
All assays had a good agreement. The sensitivity for CIN2 or higher and CIN3 or higher of the self-sampling specimens tested with the sequencing assay run on both MiSeq and Ion Torrent Personal Genome Machine sequencer was similar to that of direct-sampling specimens tested with HC2 (P > .05), but the specificity of the sequencing assay for CIN2 or higher and CIN3 or higher was significantly higher than that of HC2 (P < .01).


CONCLUSIONS
This population-based study has demonstrated the applicability of a new NGS high-risk HPV assay for primary cervical cancer screening based on self-collection.
From BGI Shenzhen, Shenzhen, China; BGI Tianjin, Tianjin, China;
Xin
X
Yi
From BGI Shenzhen, Shenzhen, China;
Jing
J
Zou
BGI Tianjin, Tianjin, China;
Jiajia
J
Xu
BGI Tianjin, Tianjin, China;
Tao
T
Liu
From BGI Shenzhen, Shenzhen, China;
Tengfei
T
Liu
From BGI Shenzhen, Shenzhen, China;
Sang
S
Hua
From BGI Shenzhen, Shenzhen, China;
Feng
F
Xi
BGI Tianjin, Tianjin, China;
Xifang
X
Nie
From BGI Shenzhen, Shenzhen, China;
Lili
L
Ye
From BGI Shenzhen, Shenzhen, China;
Yufen
Y
Luo
From BGI Shenzhen, Shenzhen, China;
Lin
L
Xu
Peking University Shenzhen Hospital, Shenzhen, China;
Hui
H
Du
Peking University Shenzhen Hospital, Shenzhen, China;
Ruifang
R
Wu
BGI Tianjin, Tianjin, China;
Ling
L
Yang
Shenzhen Second People's Hospital, Shenzhen, China; and.
Ren
R
Liu
Department of Anatomic Pathology, and.
Bin
B
Yang
From BGI Shenzhen, Shenzhen, China;
Jian
J
Wang
Preventive Oncology International and Women's Health Institute, Cleveland Clinic, Cleveland, OH. jlb@poiinc.org.
Jerome L
JL
Belinson
D052685:Alphapapillomavirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002294:Carcinoma, Squamous Cell / Q000175:diagnosis / Q000821:virology*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D055088:Early Detection of Cancer; D005260:Female; D060005:Genotyping Techniques / Q000592:standards*; D059014:High-Throughput Nucleotide Sequencing; D061809:Human Papillomavirus DNA Tests; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction; D030361:Papillomavirus Infections / Q000150:complications / Q000175:diagnosis / Q000821:virology*; D012372:ROC Curve; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods / Q000592:standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis / Q000821:virology*; D014626:Vaginal Smears; D002578:Uterine Cervical Dysplasia / Q000175:diagnosis / Q000821:virology*
D004279:DNA, Viral
Cervical cancer screening; HPV genotyping test; Ion Torrent PGM; MiSeq; Next-generation sequencing (NGS); Self-sampling