PRKDC-Mediated NHEJ May Play a Crucial Role in Aneuploidy of Chromosome 8-Driven Progression of Ovarian Cancer.

High malignancy is a prominent characteristic of epithelial ovarian cancer (EOC), emphasizing the necessity for further elucidation of the potential mechanisms underlying cancer progression. Aneuploidy and copy number variation (CNV) partially contribute to the heightened malignancy observed in EOC; however, the precise features of aneuploidy and their underlying molecular patterns, as well as the relationship between CNV and aneuploidy in EOC, remain unclear. In this study, we employed single-cell sequencing data along with The Cancer Genome Atlas (TCGA) to investigate aneuploidy and CNV in EOC. The technique of fluorescence in situ hybridization (FISH) was employed using specific probes. The copy number variation within the genomic region of chromosome 8 (42754568-47889815) was assessed and utilized as a representative measure for the ploidy status of individual cells in chromosome 8. Differential expression analysis was performed between different subgroups based on chromosome 8 ploidy. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI), and hub-gene analyses were subsequently utilized to identify crucial genes involved. By classifying enriched tumor cells into distinct subtypes based on chromosome 8 ploidy combined with TCGA data integration, we identified key genes driving chromosome 8 aneuploidy in EOC, revealing that PRKDC gene involvement through the mediated non-homologous end-joining pathway may play a pivotal role in disease progression. Further validation through analysis of the GEO and TCGA database and survival assessment, considering both mRNA expression levels and CNV status of PRKDC, has confirmed its involvement in the progression of EOC. Further functional analysis revealed an upregulation of PRKDC in both ovarian EOC cells and tissues, with its expression showing a significant correlation with the extent of copy number variation (CNV) on chromosome 8. Taken together, CNV amplification and aneuploidy of chromosome 8 are important characteristics of EOC. PRKDC and the mediated NHEJ pathway may play a crucial role in driving aneuploidy on chromosome 8 during the progression of EOC.

Single-Cell Chromatin Accessibility Analysis Reveals the Epigenetic Basis and Signature Transcription Factors for the Molecular Subtypes of Colorectal Cancers.

Colorectal cancer is a highly heterogeneous disease, with well-characterized subtypes based on genome, DNA methylome, and transcriptome signatures. To chart the epigenetic landscape of colorectal cancers, we generated a high-quality single-cell chromatin accessibility atlas of epithelial cells for 29 patients. Abnormal chromatin states acquired in adenomas were largely retained in colorectal cancers, which were tightly accompanied by opposite changes of DNA methylation. Unsupervised analysis on malignant cells revealed two epigenetic subtypes, exactly matching the iCMS classification, and key iCMS-specific transcription factors (TFs) were identified, including HNF4A and PPARA for iCMS2 tumors and FOXA3 and MAFK for iCMS3 tumors. Notably, subtype-specific TFs bind to distinct target gene sets and contribute to both interpatient similarities and diversities for both chromatin accessibilities and RNA expressions. Moreover, we identified CpG-island methylator phenotypes and pinpointed chromatin state signatures and TF regulators for the CIMP-high subtype. Our work systematically revealed the epigenetic basis of the well-known iCMS and CIMP classifications of colorectal cancers. SIGNIFICANCE: Our work revealed the epigenetic basis of the well-known iCMS and CIMP classifications of colorectal cancers. Moreover, interpatient minor similarities and major diversities of chromatin accessibility signatures of TF target genes can faithfully explain the corresponding interpatient minor similarities and major diversities of RNA expression signatures of colorectal cancers, respectively. This article is featured in Selected Articles from This Issue, p. 897.

Mapping crossover events of mouse meiotic recombination by restriction fragment ligation-based Refresh-seq.

Single-cell whole-genome sequencing methods have undergone great improvements over the past decade. However, allele dropout, which means the inability to detect both alleles simultaneously in an individual diploid cell, largely restricts the application of these methods particularly for medical applications. Here, we develop a new single-cell whole-genome sequencing method based on third-generation sequencing (TGS) platform named Refresh-seq (restriction fragment ligation-based genome amplification and TGS). It is based on restriction endonuclease cutting and ligation strategy in which two alleles in an individual cell can be cut into equal fragments and tend to be amplified simultaneously. As a new single-cell long-read genome sequencing method, Refresh-seq features much lower allele dropout rate compared with SMOOTH-seq. Furthermore, we apply Refresh-seq to 688 sperm cells and 272 female haploid cells (secondary polar bodies and parthenogenetic oocytes) from F1 hybrid mice. We acquire high-resolution genetic map of mouse meiosis recombination at low sequencing depth and reveal the sexual dimorphism in meiotic crossovers. We also phase the structure variations (deletions and insertions) in sperm cells and female haploid cells with high precision. Refresh-seq shows great performance in screening aneuploid sperm cells and oocytes due to the low allele dropout rate and has great potential for medical applications such as preimplantation genetic diagnosis.

Long-read-based single sperm genome sequencing for chromosome-wide haplotype phasing of both SNPs and SVs.

Although localized haploid phasing can be achieved using long read genome sequencing without parental data, reliable chromosome-scale phasing remains a great challenge. Given that sperm is a natural haploid cell, single-sperm genome sequencing can provide a chromosome-wide phase signal. Due to the limitation of read length, current short-read-based single-sperm genome sequencing methods can only achieve SNP haplotyping and come with difficulties in detecting and haplotyping structural variations (SVs) in complex genomic regions. To overcome these limitations, we developed a long-read-based single-sperm genome sequencing method and a corresponding data analysis pipeline that can accurately identify crossover events and chromosomal level aneuploidies in single sperm and efficiently detect SVs within individual sperm cells. Importantly, without parental genome information, our method can accurately conduct de novo phasing of heterozygous SVs as well as SNPs from male individuals at the whole chromosome scale. The accuracy for phasing of SVs was as high as 98.59% using 100 single sperm cells, and the accuracy for phasing of SNPs was as high as 99.95%. Additionally, our method reliably enabled deduction of the repeat expansions of haplotype-resolved STRs/VNTRs in single sperm cells. Our method provides a new opportunity for studying haplotype-related genetics in mammals.

Study on the correlation between B vitamins and breast cancer.

BACKGROUND: Relevant studies suggest that serum vitamin level is related to the risk of breast cancer, and dietary pattern and drug supplementation can significantly affect the level of vitamin in the body. Therefore, intervention of vitamin level in the body is expected to be a potential strategy to reduce the risk of breast cancer. However, the current epidemiological findings of serum vitamin levels and breast cancer risk are inconsistent, and the relationship between serum vitamin and breast cancer is still controversial. In this study, we compared the serum vitamin expression levels of healthy people, benign breast patients, and breast cancer patients, and evaluated the relationship between B vitamin levels and breast cancer risk. METHODS: The study used liquid chromatography-tandem mass spectrometry to determine the serum vitamin levels of 520 people who attended Yunnan Cancer Hospital from September 2020 to December 2020. After screening by exclusion criteria, 38 patients with benign breast diseases, 87 patients with breast cancer and 91 healthy controls were finally included. The kruskal-wallis H test was used to compare the differences in serum vitamin levels of subjects. Χ2 test was used to evaluate the relationship between B vitamin level and age,BMI,TNM staging,Ki-67,Her-2,surgery and chemotherapy, and other baseline characteristics and through binary logistic regression analysis, calculating odds ratio and 95% confidence interval (CI) to evaluate the relationship between B vitamins and breast cancer risk. CONCLUSION: The levels of VitB1 and VitB5 in the serum of breast cancer patients and patients with benign breast diseases were higher than those in the healthy control group, while the expression levels of VitB3 in breast cancer patients were lower than those in the healthy control group and the breast benign disease groups. The level of VitB1 was positively correlated with breast cancer risk. The VitB3 level was negatively correlated with breast cancer risk. The VitB5 level is not significantly related to the risk of breast cancer.

Single-Cell Dissection of the Multiomic Landscape of High-Grade Serous Ovarian Cancer.

High-grade serous cancer (HGSC) is the most common subtype of ovarian cancer. HGSC is highly aggressive with poor patient outcomes, and a deeper understanding of HGSC tumorigenesis could help guide future treatment development. To systematically characterize the underlying pathologic mechanisms and intratumoral heterogeneity in human HGSC, we used an optimized single-cell multiomics sequencing technology to simultaneously analyze somatic copy-number alterations (SCNA), DNA methylation, chromatin accessibility, and transcriptome in individual cancer cells. Genes associated with interferon signaling, metallothioneins, and metabolism were commonly upregulated in ovarian cancer cells. Integrated multiomics analyses revealed that upregulation of interferon signaling and metallothioneins was influenced by both demethylation of their promoters and hypomethylation of satellites and LINE1, and potential key transcription factors regulating glycolysis using chromatin accessibility data were uncovered. In addition, gene expression and DNA methylation displayed similar patterns in matched primary and abdominal metastatic tumor cells of the same genetic lineage, suggesting that metastatic cells potentially preexist in the subclones of primary tumors. Finally, the lineages of cancer cells with higher residual DNA methylation levels and upregulated expression of CCN1 and HSP90AA1 presented greater metastatic potential. This study characterizes the critical genetic, epigenetic, and transcriptomic features and their mutual regulatory relationships in ovarian cancer, providing valuable resources for identifying new molecular mechanisms and potential therapeutic targets for HGSC. SIGNIFICANCE: Integrated analysis of multiomic changes and epigenetic regulation in high-grade serous ovarian cancer provides insights into the molecular characteristics of this disease, which could help improve diagnosis and treatment.

De novo assembly of human genome at single-cell levels.

Genome assembly has been benefited from long-read sequencing technologies with higher accuracy and higher continuity. However, most human genome assembly require large amount of DNAs from homogeneous cell lines without keeping cell heterogeneities, since cell heterogeneity could profoundly affect haplotype assembly results. Herein, using single-cell genome long-read sequencing technology (SMOOTH-seq), we have sequenced K562 and HG002 cells on PacBio HiFi and Oxford Nanopore Technologies (ONT) platforms and conducted de novo genome assembly. For the first time, we have completed the human genome assembly with high continuity (with NG50 of ∼2 Mb using 95 individual K562 cells) at single-cell levels, and explored the impact of different assemblers and sequencing strategies on genome assembly. With sequencing data from 30 diploid individual HG002 cells of relatively high genome coverage (average coverage ∼41.7%) on ONT platform, the NG50 can reach over 1.3 Mb. Furthermore, with the assembled genome from K562 single-cell dataset, more complete and accurate set of insertion events and complex structural variations could be identified. This study opened a new chapter on the practice of single-cell genome de novo assembly.

Integrating single-cell datasets with ambiguous batch information by incorporating molecular network features.

With the rapid development of single-cell sequencing techniques, several large-scale cell atlas projects have been launched across the world. However, it is still challenging to integrate single-cell RNA-seq (scRNA-seq) datasets with diverse tissue sources, developmental stages and/or few overlaps, due to the ambiguity in determining the batch information, which is particularly important for current batch-effect correction methods. Here, we present SCORE, a simple network-based integration methodology, which incorporates curated molecular network features to infer cellular states and generate a unified workflow for integrating scRNA-seq datasets. Validating on real single-cell datasets, we showed that regardless of batch information, SCORE outperforms existing methods in accuracy, robustness, scalability and data integration.

SMOOTH-seq: single-cell genome sequencing of human cells on a third-generation sequencing platform.

There is no effective way to detect structure variations (SVs) and extra-chromosomal circular DNAs (ecDNAs) at single-cell whole-genome level. Here, we develop a novel third-generation sequencing platform-based single-cell whole-genome sequencing (scWGS) method named SMOOTH-seq (single-molecule real-time sequencing of long fragments amplified through transposon insertion). We evaluate the method for detecting CNVs, SVs, and SNVs in human cancer cell lines and a colorectal cancer sample and show that SMOOTH-seq reliably and effectively detects SVs and ecDNAs in individual cells, but shows relatively limited accuracy in detection of CNVs and SNVs. SMOOTH-seq opens a new chapter in scWGS as it generates high fidelity reads of kilobases long.

LncADeep: an ab initio lncRNA identification and functional annotation tool based on deep learning.

Motivation: To characterize long non-coding RNAs (lncRNAs), both identifying and functionally annotating them are essential to be addressed. Moreover, a comprehensive construction for lncRNA annotation is desired to facilitate the research in the field. Results: We present LncADeep, a novel lncRNA identification and functional annotation tool. For lncRNA identification, LncADeep integrates intrinsic and homology features into a deep belief network and constructs models targeting both full- and partial-length transcripts. For functional annotation, LncADeep predicts a lncRNA's interacting proteins based on deep neural networks, using both sequence and structure information. Furthermore, LncADeep integrates KEGG and Reactome pathway enrichment analysis and functional module detection with the predicted interacting proteins, and provides the enriched pathways and functional modules as functional annotations for lncRNAs. Test results show that LncADeep outperforms state-of-the-art tools, both for lncRNA identification and lncRNA-protein interaction prediction, and then presents a functional interpretation. We expect that LncADeep can contribute to identifying and annotating novel lncRNAs. Availability and implementation: LncADeep is freely available for academic use at http://cqb.pku.edu.cn/ZhuLab/lncadeep/ and https://github.com/cyang235/LncADeep/. Supplementary information: Supplementary data are available at Bioinformatics online.