Insights into the mechanisms and structure of breakage-fusion-bridge cycles in cervical cancer using long-read sequencing.

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and our study applies long-read sequencing to this important chromosomal rearrangement type. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes, and only one BFB breakpoint showed chromothripsis. Five cell lines have a chromosomal region 11q BFB event, with YAP1-BIRC3-BIRC2 amplification. Indeed, YAP1 amplification is associated with a 10-year-earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that individuals with cervical cancer and YAP1-BIRC3-BIRC2 amplification, especially those of African ancestry, might benefit from targeted therapy. In summary, we uncovered valuable insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

Insights into the Mechanisms and Structure of Breakage-Fusion-Bridge Cycles in Cervical Cancer using Long-Read Sequencing.

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and this is one of the first analyses of these events using long-read sequencing. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes and only one BFB breakpoint showed chromothripsis. Five cell lines have a Chr11q BFB event, with YAP1/BIRC2/BIRC3 gene amplification. Indeed, YAP1 amplification is associated with a 10-year earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that cervical cancer patients with YAP1/BIRC2/BIRC3-amplification, especially those of African American ancestry, might benefit from targeted therapy. In summary, we uncovered new insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

Extrachromosomal Amplification of Human Papillomavirus Episomes Is a Mechanism of Cervical Carcinogenesis.

SIGNIFICANCE: Multimers of the HPV genome are generated in cervical tumors replicating as extrachromosomal episomes, which is associated with deletion and rearrangement of the HPV genome and provides a mechanism for oncogenesis without integration.

HPV16 E7 Nucleotide Variants Found in Cancer-Free Subjects Affect E7 Protein Expression and Transformation.

The human papillomavirus (HPV) type 16 E7 oncogene is critical to carcinogenesis and highly conserved. Previous studies identified a preponderance of non-synonymous E7 variants amongst HPV16-positive cancer-free controls compared to those with cervical cancer. To investigate the function of E7 variants, we constructed full-length HPV16 E7 genes and tested variants at positions H9R, D21N, N29S, E33K, T56I, D62N, S63F, S63P, T64M, E80K, D81N, P92L, and P92S (found only in controls); D14E, N29H cervical intraepithelial neoplasia (CIN2), and P6L, H51N, R77S (CIN3). We determined the steady-state level of cytoplasmic and nuclear HPV16 E7 protein. All variants from controls showed a reduced level of E7 protein, with 7/13 variants having lower protein levels. In contrast, 2/3 variants from the CIN3 precancer group had near-wild type E7 levels. We assayed the activity of representative variants in stably transfected NIH3T3 cells. The H9R, E33K, P92L, and P92S variants found in control subjects had lower transforming activity than D14E and N29H variants (CIN2), and the R77S (CIN3) had activity only slightly reduced from wild-type E7. In addition, R77S and WT E7 caused increased migration of NIH3T3 cells in a wound-healing assay compared with H9R, E33K, P92L, and P92S (controls) and D14E (CIN2). These data provide evidence that the E7 variants found in HPV16-positive cancer-free women are partially defective for transformation and cell migration, further demonstrating the importance of fully active E7 in cancer development.

Phylogenomic Analysis of Human Papillomavirus Type 31 and Cervical Carcinogenesis: A Study of 2093 Viral Genomes.

Human papillomavirus (HPV) type 31 (HPV31) is closely related to the most carcinogenic type, HPV16, but only accounts for 4% of cervical cancer cases worldwide. Viral genetic and epigenetic variations have been associated with carcinogenesis for other high-risk HPV types, but little is known about HPV31. We sequenced 2093 HPV31 viral whole genomes from two large studies, one from the U.S. and one international. In addition, we investigated CpG methylation in a subset of 175 samples. We evaluated the association of HPV31 lineages/sublineages, single nucleotide polymorphisms (SNPs) and viral methylation with cervical carcinogenesis. HPV31 A/B clade was >1.8-fold more associated with cervical intraepithelial neoplasia grade 3 and cancer (CIN3+) compared to the most common C lineage. Lineage/sublineage distribution varied by race/ethnicity and geographic region. A viral genome-wide association analysis identified SNPs within the A/B clade associated with CIN3+, including H23Y (C626T) (odds ratio = 1.60, confidence intervals = 1.17-2.19) located in the pRb CR2 binding-site within the E7 oncogene. Viral CpG methylation was higher in lineage B, compared to the other lineages, and was most elevated in CIN3+. In conclusion, these data support the increased oncogenicity of the A/B lineages and suggest variation of E7 as a contributing risk factor.

APOBEC Mutagenesis Is Concordant between Tumor and Viral Genomes in HPV-Positive Head and Neck Squamous Cell Carcinoma.

APOBEC is a mutagenic source in human papillomavirus (HPV)-mediated malignancies, including HPV+ oropharyngeal squamous cell carcinoma (HPV + OPSCC), and in HPV genomes. It is unknown why APOBEC mutations predominate in HPV + OPSCC, or if the APOBEC-induced mutations observed in both human cancers and HPV genomes are directly linked. We performed sequencing of host somatic exomes, transcriptomes, and HPV16 genomes from 79 HPV + OPSCC samples, quantifying APOBEC mutational burden and activity in both host and virus. APOBEC was the dominant mutational signature in somatic exomes. In viral genomes, there was a mean of five (range 0-29) mutations per genome. The mean of APOBEC mutations in viral genomes was one (range 0-5). Viral APOBEC mutations, compared to non-APOBEC mutations, were more likely to be low-variant allele fraction mutations, suggesting that APOBEC mutagenesis actively occurrs in viral genomes during infection. HPV16 APOBEC-induced mutation patterns in OPSCC were similar to those previously observed in cervical samples. Paired host and viral analyses revealed that APOBEC-enriched tumor samples had higher viral APOBEC mutation rates (p = 0.028), and APOBEC-associated RNA editing (p = 0.008), supporting the concept that APOBEC mutagenesis in host and viral genomes is directly linked and occurrs during infection. Using paired sequencing of host somatic exomes, transcriptomes, and viral genomes, we demonstrated for the first-time definitive evidence of concordance between tumor and viral APOBEC mutagenesis. This finding provides a missing link connecting APOBEC mutagenesis in host and virus and supports a common mechanism driving APOBEC dysregulation.

Oropharyngeal Squamous Cell Carcinoma Morphology and Subtypes by Human Papillomavirus Type and by 16 Lineages and Sublineages.

Oropharyngeal squamous cell carcinoma (SCC) is increasing in incidence and, in Western countries, strongly associated with transcriptionally-active high-risk human papillomavirus (HPV). Within HPV-positive tumors, there is wide morphologic diversity with numerous histologic subtypes of SCC. There are also variable degrees of keratinization, anaplasia, stromal fibrosis, and maturing squamous differentiation. Unlike in the uterine cervix, where associations between HPV types and lineages/sublineages within types have been investigated with some clear correlations identified, little to no data exists for oropharyngeal SCC. In this study, for a large cohort of oropharyngeal SCC patients, we performed RTPCR for high-risk HPV. For the HPV positive patients, we sequenced the DNA of the entire HPV16 genome and determined lineages and sublineages, correlating HPV status, genotype, and HPV16 lineages/sublineages with SCC subtype and various histologic features. Of the 259 patients, 224 (86.5%) were high-risk HPV positive, of which 210/224 (93.8%) were HPV type 16 and 6/224 (2.7%) HPV type 33. Of the four HPV16 lineages, A was the most frequent (192/214 or 89.8%) and of the HPV16 A sublineages, A1 was the most frequent (112/210 or 53.3%). Patients with HPV negative tumors were more often keratinizing vs other types (23/35 or 65.7%) and thus more likely to have more maturing squamous differentiation and stromal desmoplasia. There was no significant correlation between HPV type (16 versus other), between HPV16 lineage (A versus others), or HPV16 A sublineages (A1 or A2 versus others) and morphologic type of SCC nor the various morphologic features of anaplasia/multinucleation, degree of keratinization, nor amount of stromal desmoplasia. In summary, in our cohort, there was no correlation between the type of HPV, the HPV 16 lineage or sublineage, and any of the histologic features or morphologic SCC subtypes.

Radiation-related genomic profile of papillary thyroid carcinoma after the Chernobyl accident.

The 1986 Chernobyl nuclear power plant accident increased papillary thyroid carcinoma (PTC) incidence in surrounding regions, particularly for radioactive iodine (131I)-exposed children. We analyzed genomic, transcriptomic, and epigenomic characteristics of 440 PTCs from Ukraine (from 359 individuals with estimated childhood 131I exposure and 81 unexposed children born after 1986). PTCs displayed radiation dose-dependent enrichment of fusion drivers, nearly all in the mitogen-activated protein kinase pathway, and increases in small deletions and simple/balanced structural variants that were clonal and bore hallmarks of nonhomologous end-joining repair. Radiation-related genomic alterations were more pronounced for individuals who were younger at exposure. Transcriptomic and epigenomic features were strongly associated with driver events but not radiation dose. Our results point to DNA double-strand breaks as early carcinogenic events that subsequently enable PTC growth after environmental radiation exposure.

HPV+ oropharyngeal squamous cell carcinomas from patients with two tumors display synchrony of viral genomes yet discordant mutational profiles and signatures.

Human papillomavirus (HPV) positive oropharyngeal squamous cell carcinoma (HPV + OPSCC) is increasing in prevalence in the USA, as are cases of patients with multiple HPV + OPSCCs (mHPV + OPSCC). mHPV + OPSCCs present a unique opportunity to examine HPV + OPSCC mutation acquisition and evolution. We performed sequencing of the viral genome, somatic exome and somatic transcriptome from 8 patients each with 2 spatially distinct HPV + OPSCCs, and 37 'traditional' HPV + OPSCCs to first address if paired tumors are caused by the same viral isolate and next, if acquired alterations, and the underlying processes driving mutagenesis, are shared within pairs. All tumor pairs contained viral genomes from the same HPV type 16 sublineage and differed by 0-2 clonal single nucleotide polymorphisms (SNPs), suggesting infection with the same viral isolate. Despite this, there was significant discordance in expression profiles, mutational burden and mutational profiles between tumors in a pair, with only two pairs sharing any overlapping mutations (3/3343 variants). Within tumor pairs there was a striking discrepancy of mutational signatures, exemplified by no paired tumors sharing high APOBEC mutational burden. Here, leveraging mHPV + OPSCCs as a model system to study mutation acquisition in virally mediated tumors, in which the germline, environmental exposures, immune surveillance and tissue/organ type were internally controlled, we demonstrate that despite infection by the same viral isolate, paired mHPV + OPSCCs develop drastically different somatic alterations and even more strikingly, appear to be driven by disparate underlying mutational processes. Thus, despite a common starting point, HPV + OPSCCs evolve through variable mutational processes with resultant stochastic mutational profiles.

An optimized protocol for retina single-cell RNA sequencing.

Purpose: Single-cell RNA sequencing (scRNA-seq) is a powerful technique used to explore gene expression at the single cell level. However, appropriate preparation of samples is essential to obtain the most information out of this transformative technology. Generating high-quality single-cell suspensions from the retina is critical to preserve the native expression profile that will ensure meaningful transcriptome data analysis. Methods: We modified the conditions for rapid and optimal dissociation of retina sample preparation. We also included additional filtering steps in data analysis for retinal scRNA-seq. Results: We report a gentle method for dissociation of the mouse retina that minimizes cell death and preserves cell morphology. This protocol also results in detection of higher transcriptional complexity. In addition, the modified computational pipeline leads to better-quality single-cell RNA-sequencing data in retina samples. We also demonstrate the advantages and limitations of using fresh versus frozen retinas to prepare cell or nuclei suspensions for scRNA-seq. Conclusions: We provide a simple yet robust and reproducible protocol for retinal scRNA-seq analysis, especially for comparative studies.

The D2 and D3 Sublineages of Human Papilloma Virus 16-Positive Cervical Cancer in Guatemala Differ in Integration Rate and Age of Diagnosis.

Human papillomavirus (HPV) 16 displays substantial sequence variation; four HPV16 lineages (A, B, C, and D) have been described as well as multiple sublineages. To identify molecular events associated with HPV16 carcinogenesis, we evaluated viral variation, the integration of HPV16, and somatic mutation in 96 cervical cancer samples from Guatemala. A total of 65% (62/96) of the samples had integrated HPV16 sequences and integration was associated with an earlier age of diagnosis and premenopausal disease. HPV16 integration sites were broadly distributed in the genome, but in one tumor, HPV16 integrated into the promoter of the IFN regulatory factor 4 (IRF4) gene, which plays an important role in the regulation of the IFN response to viral infection. The HPV16 D2 and D3 sublineages were found in 23% and 30% of the tumors, respectively, and were significantly associated with adenocarcinoma. D2-positive tumors had a higher rate of integration, earlier age of diagnosis, and a lower rate of somatic mutation, whereas D3-positive tumors were less likely to integrate, had later age of diagnosis, and exhibited a higher rate of somatic mutation. In conclusion, Guatemalan cervical tumors have a high frequency of very high-risk HPV16 D2 and D3 sublineages harboring distinct histology, which may help guide future therapeutic strategies to target the tumor and reduce recurrence. SIGNIFICANCE: This study details the biological and molecular properties of the most pathogenic forms of HPV16, the cause of the majority of cervical cancers.

Association of HPV35 with cervical carcinogenesis among women of African ancestry: Evidence of viral-host interaction with implications for disease intervention.

HPV35 has been found in only ∼2% of invasive cervical cancers (ICC) worldwide but up to 10% in Sub-Saharan Africa, warranting further investigation and consideration of impact on preventive strategies. We studied HPV35 and ethnicity, in relation to the known steps in cervical carcinogenesis, using multiple large epidemiologic studies in the U.S. and internationally. Combining five U.S. studies, we measured HPV35 positivity and, in Northern California, observed HPV35 type-specific population prevalence and estimated 5-year risk of developing precancer when HPV35-positive. HPV35 genetic variation was examined for differences in carcinogenicity in 1053 HPV35+ cervical specimens from a U.S. cohort and an international collection. African-American women had more HPV35 (12.1% vs 5.1%, P < .001) and more HPV35-associated precancers (7.4% vs 2.1%, P < .001) compared to other ethnicities. Precancer risks after HPV35 infection did not vary by ethnicity (global P = .52). The HPV35 A2 sublineage showed an increased association with precancer/cancer in African-Americans (OR = 5.6 vs A1, 95% CI = 1.3-24.8) and A2 was more prevalent among ICC in Africa than other world regions (41.9% vs 10.4%, P < .01). Our analyses support a strong link between HPV35 and cervical carcinogenesis in women of African ancestry. Current HPV vaccines cover the majority of cervical precancer/cancer across all ethnic groups; additional analyses are required to determine whether the addition of HPV35 to the already highly effective nine-valent HPV vaccine would provide better protection for women in Africa or of African ancestry.

Correction to: Low-cost HPV testing and the prevalence of cervical infection in asymptomatic populations in Guatemala.

Following publication of the original article [1], an error was reported in the tagging of Joël Fokom Domgue in the author group. The tagging in this correction article has been fixed.

Mutations in the HPV16 genome induced by APOBEC3 are associated with viral clearance.

HPV16 causes half of cervical cancers worldwide; for unknown reasons, most infections resolve within two years. Here, we analyze the viral genomes of 5,328 HPV16-positive case-control samples to investigate mutational signatures and the role of human APOBEC3-induced mutations in viral clearance and cervical carcinogenesis. We identify four de novo mutational signatures, one of which matches the COSMIC APOBEC-associated signature 2. The viral genomes of the precancer/cancer cases are less likely to contain within-host somatic HPV16 APOBEC3-induced mutations (Fisher's exact test, P = 6.2 x 10-14), and have a 30% lower nonsynonymous APOBEC3 mutation burden compared to controls. We replicate the low prevalence of HPV16 APOBEC3-induced mutations in 1,749 additional cases. APOBEC3 mutations also historically contribute to the evolution of HPV16 lineages. We demonstrate that cervical infections with a greater burden of somatic HPV16 APOBEC3-induced mutations are more likely to be benign or subsequently clear, suggesting they may reduce persistence, and thus progression, within the host.

Human papillomavirus 16 sub-lineage dispersal and cervical cancer risk worldwide: Whole viral genome sequences from 7116 HPV16-positive women.

BACKGROUND: Human papillomavirus (HPV)16 can be separated into genetic sub-lineages (A1-4, B1-4, C1-4, D1-4) which may have differential cervical cancer risk. METHODS: A next-generation sequencing assay was used to whole-genome sequence 7116 HPV16-positive cervical samples from well-characterised international epidemiological studies, including 2076 controls, 1878 squamous cell carcinoma (SCC) and 186 adenocarcinoma/adenosquamous cell carcinoma (ADC), and to assign HPV16 sub-lineage. Logistic regression was used to estimate region-stratified country-adjusted odds ratios (OR) and 95%CI. RESULTS: A1 was the most globally widespread sub-lineage, with others showing stronger regional specificity (A3 and A4 for East Asia, B1-4 and C1-4 for Africa, D2 for the Americas, B4, C4 and D4 for North Africa). Increased cancer risks versus A1 were seen for A3, A4 and D (sub)lineages in regions where they were common: A3 in East Asia (OR=2.2, 95%CI:1.0-4.7); A4 in East Asia (6.6, 3.1-14.1) and North America (3.8, 1.7-8.3); and D in North (6.2, 4.1-9.3) and South/Central America (2.2, 0.8-5.7), where D lineages were also more frequent in ADC than SCC (3.2, 1.5-6.5; 12.1, 5.7-25.6, respectively). CONCLUSIONS: HPV16 genetic variation can strongly influence cervical cancer risk. However, burden of cervical cancer attributable to different sub-lineages worldwide is largely driven by historical HPV16 sub-lineage dispersal.

Low-cost HPV testing and the prevalence of cervical infection in asymptomatic populations in Guatemala.

BACKGROUND: A low cost and accurate method for detecting high-risk (HR) human papillomavirus (HPV) is important to permit HPV testing for cervical cancer prevention. We used a commercially available HPV method (H13, Hybribio) which was documented to function accurately in a reduced volume of cervical specimen to determine the most prevalent HPV types and the distribution of HPV infections in over 1795 cancer-free women in Guatemala undergoing primary screening for cervical cancer by cytology. METHODS: HR-HPV detection was attempted in cervical samples from 1795 cancer-free women receiving Pap smears using the Hybribio™ real-time PCR assay of 13 HR types. The test includes a globin gene internal control. HPV positive samples were sequenced to determine viral type. Age-specific prevalence of HPV was also assessed in the study population. RESULTS: A total of 13% (226/1717) of women tested HPV+, with 78 samples (4.3%) failing to amplify the internal control. The highest prevalence was found in younger women (< 30 years, 22%) and older ones (≥60 years, 15%). The six most common HR-HPV types among the 148 HPV+ typed were HPV16 (22%), HPV18 (11%), HPV39 (11%), HPV58 (10%), HPV52 (8%), and HPV45 (8%). CONCLUSIONS: In this sample of cancer free women in Guatemala, HPV16 was the most prevalent HR type in Guatemala and the age-specific prevalence curve peaked in younger ages. Women in the 30-59-year age groups had a prevalence of HR-HPV of 8%, however, larger studies to better describe the epidemiology of HPV in Guatemala are needed.

The Intersection of HPV Epidemiology, Genomics and Mechanistic Studies of HPV-Mediated Carcinogenesis.

Of the ~60 human papillomavirus (HPV) genotypes that infect the cervicovaginal epithelium, only 12-13 "high-risk" types are well-established as causing cervical cancer, with HPV16 accounting for over half of all cases worldwide. While HPV16 is the most important carcinogenic type, variants of HPV16 can differ in their carcinogenicity by 10-fold or more in epidemiologic studies. Strong genotype-phenotype associations embedded in the small 8-kb HPV16 genome motivate molecular studies to understand the underlying molecular mechanisms. Understanding the mechanisms of HPV genomic findings is complicated by the linkage of HPV genome variants. A panel of experts in various disciplines gathered on 21 November 2016 to discuss the interdisciplinary science of HPV oncogenesis. Here, we summarize the discussion of the complexity of the viral-host interaction and highlight important next steps for selected applied basic laboratory studies guided by epidemiological genomic findings.

HPV16 E7 Genetic Conservation Is Critical to Carcinogenesis.

Although most cervical human papillomavirus type 16 (HPV16) infections become undetectable within 1-2 years, persistent HPV16 causes half of all cervical cancers. We used a novel HPV whole-genome sequencing technique to evaluate an exceptionally large collection of 5,570 HPV16-infected case-control samples to determine whether viral genetic variation influences risk of cervical precancer and cancer. We observed thousands of unique HPV16 genomes; very few women shared the identical HPV16 sequence, which should stimulate a careful re-evaluation of the clinical implications of HPV mutation rates, transmission, clearance, and persistence. In case-control analyses, HPV16 in the controls had significantly more amino acid changing variants throughout the genome. Strikingly, E7 was devoid of variants in precancers/cancers compared to higher levels in the controls; we confirmed this in cancers from around the world. Strict conservation of the 98 amino acids of E7, which disrupts Rb function, is critical for HPV16 carcinogenesis, presenting a highly specific target for etiologic and therapeutic research.

Novel and known ribosomal causes of Diamond-Blackfan anaemia identified through comprehensive genomic characterisation.

BACKGROUND: Diamond-Blackfan anaemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterised by erythroid hypoplasia. It is associated with congenital anomalies and a high risk of developing specific cancers. DBA is caused predominantly by autosomal dominant pathogenic variants in at least 15 genes affecting ribosomal biogenesis and function. Two X-linked recessive genes have been identified. OBJECTIVES: We aim to identify the genetic aetiology of DBA. METHODS: Of 87 families with DBA enrolled in an institutional review board-approved cohort study (ClinicalTrials.gov Identifier:NCT00027274), 61 had genetic testing information available. Thirty-five families did not have a known genetic cause and thus underwent comprehensive genomic evaluation with whole exome sequencing, deletion and CNV analyses to identify their disease-associated pathogenic variant. Controls for functional studies were healthy mutation-negative individuals enrolled in the same study. RESULTS: Our analyses uncovered heterozygous pathogenic variants in two previously undescribed genes in two families. One family had a non-synonymous variant (p.K77N) in RPL35; the second family had a non-synonymous variant (p. L51S) in RPL18. Both of these variants result in pre-rRNA processing defects. We identified heterozygous pathogenic variants in previously known DBA genes in 16 of 35 families. Seventeen families who underwent genetic analyses are yet to have a genetic cause of disease identified. CONCLUSIONS: Overall, heterozygous pathogenic variants in ribosomal genes were identified in 44 of the 61 families (72%). De novo pathogenic variants were observed in 57% of patients with DBA. Ongoing studies of DBA genomics will be important to understand this complex disorder.

Genomic diversity and phylogenetic relationships of human papillomavirus 16 (HPV16) in Nepal.

OBJECTIVES/BACKGROUND: Sequence variants in HPV16 confer differences in oncogenic potential; however, to date there have not been any HPV sequence studies performed in Nepal. The objective of this study was to characterize HPV16 viral genome sequences from Nepal compared to a reference sequence in order to determine their lineages. Additionally, we sought to determine if five High-grade Squamous Intraepithelial Lesion (HSIL) subjects were genetically distinct from the non-HSIL subjects. METHODS: DNA was isolated from exfoliated cervical cells from 17 individuals in Nepal who were previously identified to be HPV16-positive. A custom HPV16 Ion Ampliseq panel of multiplexed degenerate primers was designed that generated 47 overlapping amplicons and covered 99% of the viral genome for all known HPV16 variant lineages. All sequence data were processed through a custom quality control and analysis pipeline of sequence comparisons and phylogenetic analysis. RESULTS: There were high similarities across the genomes, with two major indels observed in the non-coding region between E5 and L2. Compared to the PAVE reference HPV16 genome, there were up to 9, 4, 38, 27, 8, 7, 52, and 32 nucleotide variants in the E6, E7, E1, E2, E4, E5, L2, and L1 genes in the Nepalese samples, respectively. Based on sequence variation, HPV16 from Nepal falls across the A, C, and D lineages in this study. We found no evidence of genetic distinctness between HSIL and non-HSIL subjects. CONCLUSIONS: The evolutionary and pathological characteristics of the representative HPV16 genomes from Nepal seem similar to results from other parts of the world and provide the basis for further studies.