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Cancer is a disease largely caused by genomic aberrations. Utilizing many rapidly emerging sequencing technologies, researchers have studied cancer genomes to understand the molecular statuses of cancer cells and to reveal their vulnerabilities, such as driver mutations or gene expression. Long-read technologies enable us to identify and characterize novel types of cancerous mutations, including complicated structural variants in haplotype resolution. In this review, we introduce three representative platforms for long-read sequencing and research trends of cancer genomics with long-read data. Further, we describe that aberrant transcriptome and epigenome statuses, namely, fusion transcripts, as well as aberrant transcript isoforms and the phase information of DNA methylation, are able to be elucidated by long-read sequencers. Long-read sequencing may shed light on novel types of aberrations in cancer genomics that are being missed by conventional short-read sequencing analyses.

Here, we review single-cell sequencing techniques for individual and multiomics profiling in single cells. We mainly describe single-cell genomic, epigenomic, and transcriptomic methods, and examples of their applications. For the integration of multilayered data sets, such as the transcriptome data derived from single-cell RNA sequencing and chromatin accessibility data derived from single-cell ATAC-seq, there are several computational integration methods. We also describe single-cell experimental methods for the simultaneous measurement of two or more omics layers. We can achieve a detailed understanding of the basic molecular profiles and those associated with disease in each cell by utilizing a large number of single-cell sequencing techniques and the accumulated data sets.Single-cell sequencing: Greater insight through integrated dataCombining data from different single-cell sequencing techniques could greatly improve understanding of the molecular profiles associated with disease. Sequencing studies provide valuable insights into diseased and healthy states at a single-cell level, for example the evolutionary paths of brain tumors and cancerous mutations. Ayako Suzuki at the University of Tokyo in Chiba, Japan, and co-workers examined the challenges of integrating data from various experimental and computational single-cell sequencing methods. These methods usually determine the genomic, epigenomic (DNA modifications) or transcriptomic (messenger RNAs) state of a cell, and can be combined to create a detailed picture. Other ‘multiomics’ techniques provide multilayered information from the same cell. The researchers recommend detailed analysis of individual data layers prior to integration, and highlight emerging techniques that analyze larger tissue sections, thus retaining the temporal and spatial information around a cell.

The mechanism underlying the development of tumors, particularly at early stages, still remains mostly elusive. Here, we report whole-genome long and short read sequencing analysis of 76 lung cancers, focusing on very early-stage lung adenocarcinomas such as adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma. The obtained data is further integrated with bulk and spatial transcriptomic data and epigenomic data. These analyses reveal key events in lung carcinogenesis. Minimal somatic mutations in pivotal driver mutations and essential proliferative factors are the only detectable somatic mutations in the very early-stage of AIS. These initial events are followed by copy number changes and global DNA hypomethylation. Particularly, drastic changes are initiated at the later AIS stage, i.e., in Noguchi type B tumors, wherein cancer cells are exposed to the surrounding microenvironment. This study sheds light on the pathogenesis of lung adenocarcinoma from integrated pathological and molecular viewpoints. Current sequencing technologies can shed light on the stepwise progression of lung adenocarcinoma. Here, the authors characterize tumor progression in lung adenocarcinomas from an early stage using short and long read whole-genome sequencing, bulk and spatial transcriptomics, and epigenomics.

Background Genome graph is an emerging approach for representing structural variants on genomes with branches. For example, representing structural variants of cancer genomes as a genome graph is more natural than representing such genomes as differences from the linear reference genome. While more and more structural variants are being identified by long-read sequencing, many of them are difficult to visualize using existing structural variants visualization tools. To this end, visualization method for large genome graphs such as human cancer genome graphs is demanded. Results We developed MOdular Multi-scale Integrated Genome graph browser, MoMI-G, a web-based genome graph browser that can visualize genome graphs with structural variants and supporting evidences such as read alignments, read depth, and annotations. This browser allows more intuitive recognition of large, nested, and potentially more complex structural variations. MoMI-G has view modules for different scales, which allow users to view the whole genome down to nucleotide-level alignments of long reads. Alignments spanning reference alleles and those spanning alternative alleles are shown in the same view. Users can customize the view, if they are not satisfied with the preset views. In addition, MoMI-G has Interval Card Deck, a feature for rapid manual inspection of hundreds of structural variants. Herein, we describe the utility of MoMI-G by using representative examples of large and nested structural variations found in two cell lines, LC-2/ad and CHM1. Conclusions Users can inspect complex and large structural variations found by long-read analysis in large genomes such as human genomes more smoothly and more intuitively. In addition, users can easily filter out false positives by manually inspecting hundreds of identified structural variants with supporting long-read alignments and annotations in a short time. Software availability MoMI-G is freely available at https://github.com/MoMI-G/MoMI-G under the MIT license.

Chromosomal backgrounds of cancerous mutations still remain elusive. Here, we conduct the phasing analysis of non-small cell lung cancer specimens of 20 Japanese patients. By the combinatory use of short and long read sequencing data, we obtain long phased blocks of 834 kb in N50 length with >99% concordance rate. By analyzing the obtained phasing information, we reveal that several cancer genomes harbor regions in which mutations are unevenly distributed to either of two haplotypes. Large-scale chromosomal rearrangement events, which resemble chromothripsis events but have smaller scales, occur on only one chromosome, and these events account for the observed biased distributions. Interestingly, the events are characteristic of EGFR mutation-positive lung adenocarcinomas. Further integration of long read epigenomic and transcriptomic data reveal that haploid chromosomes are not always at equivalent transcriptomic/epigenomic conditions. Distinct chromosomal backgrounds are responsible for later cancerous aberrations in a haplotype-specific manner. Long-read sequencing technologies are useful for the multifaceted task of characterising somatic mutations, including structural variants, in cancers. Here, the authors combine short and long read sequencing for the phasing analysis, which enables them to resolve the chromosomal backgrounds of somatic mutations in Japanese non-small cell lung cancers.

A liver cyst is hepatic fluid-filled cavities often detected in clinical surveillances such as a health examination. Although the liver cyst is usually asymptomatic and observed without any therapeutic intervention, it can be symptomatic and needs treatment due to its enlargement, hemorrhage, and infection. A 74-year-old woman presented with upper abdominal pain and a huge liver cyst in the left lobe. Several examinations including image findings revealed that the symptom could be derived from the liver cyst. Although there is no definite guideline of treatment for symptomatic liver cysts, percutaneous ultrasound-guided drainage with sclerotherapy or surgery is often selected. Because of anatomical accessibility to the liver cyst and the patient’s wish, we performed endoscopic transgastric drainage with insertion of both an internal stent and an external nasocystic tube. Sclerotherapy with minocycline hydrochloride was performed through the nasocystic tube, and the liver cyst shrunk completely without any complications. This is the first reported method of administering minocycline hydrochloride through a nasocystic tube, which can be a therapeutic option for patients with symptomatic liver cysts.

The functional relevancy of mutations occurring in the regulatory regions in cancers remains mostly elusive. Here, we identified and analyzed regulatory mutations having transcriptional consequences in lung adenocarcinoma-derived cell lines. We phased the mutations in the regulatory regions to the downstream heterozygous SNPs in the coding regions and examined whether the ChIP-Seq variant tags of the regulatory SNVs and the RNA-Seq variant tags of their target transcripts showed biased frequency between the mutant and reference alleles. We identified 137 potential regulatory mutations affecting the transcriptional regulation of 146 RefSeq transcripts with at least 84 SNVs that create and/or disrupt potential transcription factor binding sites. For example, in the regulatory region of NFATC1 gene, a novel and active binding site for the ETS transcription factor family was created. Further examination revealed that 31 of these disruptions were presented in clinical lung adenocarcinoma samples and were associated with prognosis of patients.

Tubulocystic carcinoma of the bile duct is extremely rare and has not been reported in the literature. We reported a case of cystic neoplasm of the liver with distinct histopathological features that could not be clearly classified as of either mucinous or intraductal papillary neoplasm. A 68-year-old Japanese patient had a multicystic biliary tumor within the liver. This tumor was detected on follow-up of polymyalgia rheumatica. The exophytic, multicystic, 35 × 50 mm mass was composed of complex tubulocystic structures. We initially suspected cystadenocarcinoma of the liver and performed radical operation. However, pathology ultimately showed it to be very rare tubulocystic carcinoma that derived from the bile duct. We reviewed the literature and describe the process of our differential diagnosis.