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.

Assessing methylation detection for primary human tissue using Nanopore sequencing.

DNA methylation most commonly occurs as 5-methylcytosine (5-mC) in the human genome and has been associated with human diseases. Recent developments in single-molecule sequencing technologies (Oxford Nanopore Technologies (ONT) and Pacific Biosciences) have enabled readouts of long, native DNA molecules, including cytosine methylation. ONT recently upgraded their Nanopore sequencing chemistry and kits from R9 to the R10 version, which yielded increased accuracy and sequencing throughput. However the effects on methylation detection have not yet been documented. Here we performed a series of computational analyses to characterize differences in Nanopore-based 5mC detection between the ONT R9 and R10 chemistries. We compared 5mC calls in R9 and R10 for three human genome datasets: a cell line, a frontal cortex brain sample, and a blood sample. We performed an in-depth analysis on CpG islands and homopolymer regions, and documented high concordance for methylation detection among sequencing technologies. The strongest correlation was observed between Nanopore R10 and Illumina bisulfite technologies for cell line-derived datasets. Subtle differences in methylation datasets between technologies can impact analysis tools such as differential methylation calling software. Our findings show that comparisons can be drawn between methylation data from different Nanopore chemistries using guided hypotheses. This work will facilitate comparison among Nanopore data cohorts derived using different chemistries from large scale sequencing efforts, such as the NIH CARD Long Read Initiative.

The Utility of Long-Read Sequencing in Diagnosing Genetic Autosomal Recessive Parkinson's Disease: a genetic screening study.

Background: Mutations within the genes PRKN and PINK1 are the leading cause of early onset autosomal recessive Parkinson's disease (PD). However, the genetic cause of most early-onset PD (EOPD) cases still remains unresolved. Long-read sequencing has successfully identified many pathogenic structural variants that cause disease, but this technology has not been widely applied to PD. We recently identified the genetic cause of EOPD in a pair of monozygotic twins by uncovering a complex structural variant that spans over 7 Mb, utilizing Oxford Nanopore Technologies (ONT) long-read sequencing. In this study, we aimed to expand on this and assess whether a second variant could be detected with ONT long-read sequencing in other unresolved EOPD cases reported to carry one heterozygous variant in PRKN or PINK1. Methods: ONT long-read sequencing was performed on patients with one reported PRKN/PINK1 pathogenic variant. EOPD patients with an age at onset younger than 50 were included in this study. As a positive control, we also included EOPD patients who had already been identified to carry two known PRKN pathogenic variants. Initial genetic testing was performed using either short-read targeted panel sequencing for single nucleotide variants and multiplex ligation-dependent probe amplification (MLPA) for copy number variants. Results: 48 patients were included in this study (PRKN "one-variant" n = 24, PINK1 "one-variant" n = 12, PRKN "two-variants" n = 12). Using ONT long-read sequencing, we detected a second pathogenic variant in six PRKN "one-variant" patients (26%, 6/23) but none in the PINK1 "one-variant" patients (0%, 0/12). Long-read sequencing identified one case with a complex inversion, two instances of structural variant overlap, and three cases of duplication. In addition, in the positive control PRKN "two-variants" group, we were able to identify both pathogenic variants in PRKN in all the patients (100%, 12/12). Conclusions: This data highlights that ONT long-read sequencing is a powerful tool to identify a pathogenic structural variant at the PRKN locus that is often missed by conventional methods. Therefore, for cases where conventional methods fail to detect a second variant for EOPD, long-read sequencing should be considered as an alternative and complementary approach.

Profiling complex repeat expansions in RFC1 in Parkinson's disease.

A biallelic (AAGGG) expansion in the poly(A) tail of an AluSx3 transposable element within the gene RFC1 is a frequent cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS), and more recently, has been reported as a rare cause of Parkinson's disease (PD) in the Finnish population. Here, we investigate the prevalence of RFC1 (AAGGG) expansions in PD patients of non-Finnish European ancestry in 1609 individuals from the Parkinson's Progression Markers Initiative study. We identified four PD patients carrying the biallelic RFC1 (AAGGG) expansion and did not identify any carriers in controls.

Characterizing a complex CT-rich haplotype in intron 4 of SNCA using large-scale targeted amplicon long-read sequencing.

Parkinson's disease (PD) is a common neurodegenerative disorder with a significant risk proportion driven by genetics. While much progress has been made, most of the heritability remains unknown. This is in-part because previous genetic studies have focused on the contribution of single nucleotide variants. More complex forms of variation, such as structural variants and tandem repeats, are already associated with several synucleinopathies. However, because more sophisticated sequencing methods are usually required to detect these regions, little is understood regarding their contribution to PD. One example is a polymorphic CT-rich region in intron 4 of the SNCA gene. This haplotype has been suggested to be associated with risk of Lewy Body (LB) pathology in Alzheimer's Disease and SNCA gene expression, but is yet to be investigated in PD. Here, we attempt to resolve this CT-rich haplotype and investigate its role in PD. We performed targeted PacBio HiFi sequencing of the region in 1375 PD cases and 959 controls. We replicate the previously reported associations and a novel association between two PD risk SNVs (rs356182 and rs5019538) and haplotype 4, the largest haplotype. Through quantitative trait locus analyzes we identify a significant haplotype 4 association with alternative CAGE transcriptional start site usage, not leading to significant differential SNCA gene expression in post-mortem frontal cortex brain tissue. Therefore, disease association in this locus might not be biologically driven by this CT-rich repeat region. Our data demonstrates the complexity of this SNCA region and highlights that further follow up functional studies are warranted.