Single-cell omics sequencing technologies: the long-read generation.

Over the past decade, single-cell omics sequencing technologies have revolutionized biological and medical research and deepened our knowledge of cellular heterogeneities in life activities at the genomic, epigenomic, and transcriptomic levels. Concurrently, single-molecule long-read sequencing (SMS) technologies have also made amazingly rapid progress. In recent years, the convergence of these two exciting fields has injected new vitality into the generation of novel insights in genomics (repetitive elements, structural variations), epigenomics (allele-specific epigenetic modifications), and transcriptomics (alternative splicing) at the single-cell level, providing powerful new tools and opening new opportunities for biomedical fields.

Multi-omics analysis reveals single-cell meiotic hotspot dynamics and epigenomic regulations in female mammals.

Meiotic recombination initiates via DNA double-strand breaks (DSBs) at specialized hotspots, while the regulation of meiotic recombination hotspots in females remain elusive due to the scarcity of embryonic stage germ cells (EGCs). Here, we mapped genome-wide active recombination hotspots and estimated their activities in female EGCs at single-cell resolution, revealing the high variability in hotspot usage frequency among individual germ cells. Further investigation of nucleosome positioning and histone modifications at recombination hotspots revealed that PRDM9-mediated open chromatin and flanking H3K4me3 established earlier at high-frequency hotspots compared with less frequently used ones.

Systematic characterization of full-length RNA isoforms in human colorectal cancer at single-cell resolution.

Dysregulated RNA splicing is a well-recognized characteristic of colorectal cancer (CRC); however, its intricacies remain obscure, partly due to challenges in profiling full-length transcript variants at single-cell level. Here, we employ high-depth long-read scRNA-seq to define the full-length transcriptome of colorectal epithelial cells in 12 CRC patients, revealing extensive isoform diversities and splicing alterations. Cancer cells exhibited increased transcript complexity, with widespread 3'-UTR shortening and reduced intron retention.

EGR1 Promotes Craniofacial Bone Regeneration via Activation of ALPL⁺PDGFD⁺ Periosteal Stem Cells.

Oral and craniofacial bone regeneration remains challenging due to unique anatomical and functional demands. Rodent models have limited translational value because of significant structural differences from humans. The study reveals high similarity in calvarial periosteal cell composition between miniature pigs and humans at single-cell resolution.

Single-cell long-read Hi-C, scNanoHi-C2, details 3D genome reorganization in embryonic-stage germ cells.

During mouse development, embryonic-stage germ cells (EGCs) make crucial fate decisions, with female EGCs embarking on meiosis whereas male EGCs enter mitotic arrest until birth. Despite increasing understanding of the reprogramming of epigenetic modifications, the dynamics of three-dimensional (3D) genome structures within individual EGCs remains elusive. Here we present a single-cell input, long-read Hi-C method, termed scNanoHi-C2.

scExtract: leveraging large language models for fully automated single-cell RNA-seq data annotation and prior-informed multi-dataset integration.

Single-cell RNA sequencing has revolutionized cellular heterogeneity research, but analyzing the abundance of unannotated public datasets remains challenging. We present scExtract, a framework leveraging large language models to automate scRNA-seq data analysis from preprocessing to annotation and integration. scExtract extracts information from research articles to guide data processing, outperforming existing reference transfer methods in benchmarks.

Single-cell 5-hydroxymethylcytosine landscapes of mouse early embryos at single-base resolution.

DNA methylation and hydroxymethylation are extensively reprogrammed during mammalian early embryogenesis, and studying their regulatory functions requires comprehensive DNA hydroxymethylation maps at base resolution. Here, we develop single-cell 5-hydroxymethylcytosine (5hmC) chemical-assisted C-to-T conversion-enabled sequencing (schmC-CATCH), a method leveraging selective 5hmC labeling for a quantitative, base-resolution, genome-wide landscape of the DNA hydroxymethylome in mouse gametes and preimplantation embryos spanning from the zygote to blastocyst stage. We revealed that, in addition to late zygotic stages, onset of ten-eleven translocation (TET)-mediated DNA hydroxymethylation initiates immediately after fertilization and is characterized by the distinct 5hmC patterns on the parental genomes shaped by TET3 demethylase.

A computational DNA methylation method to remove contaminated DNA from spent embryo culture medium for noninvasive preimplantation genetic testing.

Background: In the last fifty years, assisted reproductive technology (ART) has achieved remarkable breakthroughs, culminating in the birth of 12 million infants. At the heart of ART success is preimplantation genetic testing (PGT), which enables the detection of chromosomal anomalies, single-gene disorders, and structural rearrangements, enhancing embryo selection and mitigating genetic risk. However, current PGT methods, including trophectoderm (TE) biopsy, face limitations such as challenges related to convenience and potential impacts on embryonic health.

Chromatin accessibility landscape of mouse early embryos revealed by single-cell NanoATAC-seq2.

In mammals, fertilized eggs undergo genome-wide epigenetic reprogramming to generate the organism. However, our understanding of epigenetic dynamics during preimplantation development at single-cell resolution remains incomplete. Here, we developed scNanoATAC-seq2, a single-cell assay for transposase-accessible chromatin using long-read sequencing for scarce samples.

SETD1B-mediated broad H3K4me3 controls proper temporal patterns of gene expression critical for spermatid development.

Epigenetic programming governs cell fate determination during development through intricately controlling sequential gene activation and repression. Although H3K4me3 is widely recognized as a hallmark of gene activation, its role in modulating transcription output and timing within a continuously developing system remains poorly understood. In this study, we provide a detailed characterization of the epigenomic landscapes in developing male germ cells.

Simultaneous de novo calling and phasing of genetic variants at chromosome-scale using NanoStrand-seq.

The successful accomplishment of the first telomere-to-telomere human genome assembly, T2T-CHM13, marked a milestone in achieving completeness of the human reference genome. The upcoming era of genome study will focus on fully phased diploid genome assembly, with an emphasis on genetic differences between individual haplotypes. Most existing sequencing approaches only achieved localized haplotype phasing and relied on additional pedigree information for further whole-chromosome scale phasing.

Unveiling gene regulatory networks during cellular state transitions without linkage across time points.

Time-stamped cross-sectional data, which lack linkage across time points, are commonly generated in single-cell transcriptional profiling. Many previous methods for inferring gene regulatory networks (GRNs) driving cell-state transitions relied on constructing single-cell temporal ordering. Introducing COSLIR (COvariance restricted Sparse LInear Regression), we presented a direct approach to reconstructing GRNs that govern cell-state transitions, utilizing only the first and second moments of samples between two consecutive time points.

DEAD-box helicase 17 (DDX17) protects cardiac function by promoting mitochondrial homeostasis in heart failure.

DEAD-box helicase 17 (DDX17) is a typical member of the DEAD-box family with transcriptional cofactor activity. Although DDX17 is abundantly expressed in the myocardium, its role in heart is not fully understood. We generated cardiomyocyte-specific Ddx17-knockout mice (Ddx17-cKO), cardiomyocyte-specific Ddx17 transgenic mice (Ddx17-Tg), and various models of cardiomyocyte injury and heart failure (HF).

Integrative analysis of transcriptome, DNA methylome, and chromatin accessibility reveals candidate therapeutic targets in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease and is characterized by primary left ventricular hypertrophy usually caused by mutations in sarcomere genes. The mechanism underlying cardiac remodeling in HCM remains incompletely understood. An investigation of HCM through integrative analysis at multi-omics levels will be helpful for treating HCM.

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.

A single-cell characterised signature integrating heterogeneity and microenvironment of lung adenocarcinoma for prognostic stratification.

Background: The high heterogeneity of tumour and the complexity of tumour microenvironment (TME) greatly impacted the tumour development and the prognosis of cancer in the era of immunotherapy. In this study, we aimed to portray the single cell-characterised landscape of lung adenocarcinoma (LUAD), and develop an integrated signature incorporating both tumour heterogeneity and TME for prognosis stratification.

Methods: Single-cell tagged reverse transcription sequencing (STRT-seq) was performed on tumour tissues and matched normal tissues from 14 patients with LUAD for immune landscape depiction and candidate key genes selection for signature construction.

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

Unlabelled: 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.

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).

Single-cell analysis reveals insights into epithelial abnormalities in ovarian endometriosis.

Ovarian endometriosis is characterized by the growth of endometrial tissue within the ovary, causing infertility and chronic pain. However, its pathophysiology remains unclear. Utilizing high-precision single-cell RNA sequencing, we profile the normal, eutopic, and ectopic endometrium from 34 individuals across proliferative and secretory phases.