Changes in genome architecture and transcriptional dynamics progress independently of sensory experience during post-natal brain development.

Both transcription and three-dimensional (3D) architecture of the mammalian genome play critical roles in neurodevelopment and its disorders. However, 3D genome structures of single brain cells have not been solved; little is known about the dynamics of single-cell transcriptome and 3D genome after birth. Here, we generated a transcriptome (3,517 cells) and 3D genome (3,646 cells) atlas of the developing mouse cortex and hippocampus by using our high-resolution multiple annealing and looping-based amplification cycles for digital transcriptomics (MALBAC-DT) and diploid chromatin conformation capture (Dip-C) methods and developing multi-omic analysis pipelines. In adults, 3D genome "structure types" delineate all major cell types, with high correlation between chromatin A/B compartments and gene expression. During development, both transcriptome and 3D genome are extensively transformed in the first post-natal month. In neurons, 3D genome is rewired across scales, correlated with gene expression modules, and independent of sensory experience. Finally, we examine allele-specific structure of imprinted genes, revealing local and chromosome (chr)-wide differences. These findings uncover an unknown dimension of neurodevelopment.

Structurally Resolved SARS-CoV-2 Antibody Shows High Efficacy in Severely Infected Hamsters and Provides a Potent Cocktail Pairing Strategy.

Understanding how potent neutralizing antibodies (NAbs) inhibit SARS-CoV-2 is critical for effective therapeutic development. We previously described BD-368-2, a SARS-CoV-2 NAb with high potency; however, its neutralization mechanism is largely unknown. Here, we report the 3.5-Å cryo-EM structure of BD-368-2/trimeric-spike complex, revealing that BD-368-2 fully blocks ACE2 recognition by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their "up" or "down" conformations. Also, BD-368-2 treats infected adult hamsters at low dosages and at various administering windows, in contrast to placebo hamsters that manifested severe interstitial pneumonia. Moreover, BD-368-2's epitope completely avoids the common binding site of VH3-53/VH3-66 recurrent NAbs, evidenced by tripartite co-crystal structures with RBDs. Pairing BD-368-2 with a potent recurrent NAb neutralizes SARS-CoV-2 pseudovirus at pM level and rescues mutation-induced neutralization escapes. Together, our results rationalized a new RBD epitope that leads to high neutralization potency and demonstrated BD-368-2's therapeutic potential in treating COVID-19.

Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients' B Cells.

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 Å cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.

Mechanism of transcriptional bursting in bacteria.

Transcription of highly expressed genes has been shown to occur in stochastic bursts. But the origin of such ubiquitous phenomenon has not been understood. Here, we present the mechanism in bacteria. We developed a high-throughput, in vitro, single-molecule assay to follow transcription on individual DNA templates in real time. We showed that positive supercoiling buildup on a DNA segment by transcription slows down transcription elongation and eventually stops transcription initiation. Transcription can be resumed upon gyrase binding to the DNA segment. Furthermore, using single-cell mRNA counting fluorescence in situ hybridization (FISH), we found that duty cycles of transcriptional bursting depend on the intracellular gyrase concentration. Together, these findings prove that transcriptional bursting of highly expressed genes in bacteria is primarily caused by reversible gyrase dissociation from and rebinding to a DNA segment, changing the supercoiling level of the segment.

Genome analyses of single human oocytes.

Single-cell genome analyses of human oocytes are important for meiosis research and preimplantation genomic screening. However, the nonuniformity of single-cell whole-genome amplification hindered its use. Here, we demonstrate genome analyses of single human oocytes using multiple annealing and looping-based amplification cycle (MALBAC)-based sequencing technology. By sequencing the triads of the first and second polar bodies (PB1 and PB2) and the oocyte pronuclei from same female egg donors, we phase the genomes of these donors with detected SNPs and determine the crossover maps of their oocytes. Our data exhibit an expected crossover interference and indicate a weak chromatid interference. Further, the genome of the oocyte pronucleus, including information regarding aneuploidy and SNPs in disease-associated alleles, can be accurately deduced from the genomes of PB1 and PB2. The MALBAC-based preimplantation genomic screening in in vitro fertilization (IVF) enables accurate and cost-effective selection of normal fertilized eggs for embryo transfer.

MINERVA: A Facile Strategy for SARS-CoV-2 Whole-Genome Deep Sequencing of Clinical Samples.

Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from clinical samples is crucial for understanding viral spread and evolution as well as for vaccine development. Existing RNA sequencing methods are demanding on user technique and time and, thus, not ideal for time-sensitive clinical samples; these methods are also not optimized for high performance on viral genomes. We developed a facile, practical, and robust approach for metagenomic and deep viral sequencing from clinical samples. We demonstrate the utility of our approach on pharyngeal, sputum, and stool samples collected from coronavirus disease 2019 (COVID-19) patients, successfully obtaining whole metatranscriptomes and complete high-depth, high-coverage SARS-CoV-2 genomes with high yield and robustness. With a shortened hands-on time from sample to virus-enriched sequencing-ready library, this rapid, versatile, and clinic-friendly approach will facilitate molecular epidemiology studies during current and future outbreaks.

Highly sensitive single-cell chromatin accessibility assay and transcriptome coassay with METATAC.

Recent advances in single-cell assay for transposase accessible chromatin using sequencing (scATAC-seq) and its coassays have transformed the field of single-cell epigenomics and transcriptomics. However, the low detection efficiency of current methods has limited our understanding of the true complexity of chromatin accessibility and its relationship with gene expression in single cells. Here, we report a high-sensitivity scATAC-seq method, termed multiplexed end-tagging amplification of transposase accessible chromatin (METATAC), which detects a large number of accessible sites per cell and is compatible with automation. Our high detectability and statistical framework allowed precise linking of enhancers to promoters without merging single cells. We systematically investigated allele-specific accessibility in the mouse cerebral cortex, revealing allele-specific accessibility of promotors of certain imprinted genes but biallelic accessibility of their enhancers. Finally, we combined METATAC with our high-sensitivity single-cell RNA sequencing (scRNA-seq) method, multiple annealing and looping based amplification cycles for digital transcriptomics (MALBAC-DT), to develop a joint ATAC-RNA assay, termed METATAC and MALBAC-DT coassay by sequencing (M2C-seq). M2C-seq achieved significant improvements for both ATAC and RNA compared with previous methods, with consistent performance across cell lines and early mouse embryos.

Protein and lipid mass concentration measurement in tissues by stimulated Raman scattering microscopy.

Cell mass and chemical composition are important aggregate cellular properties that are especially relevant to physiological processes, such as growth control and tissue homeostasis. Despite their importance, it has been difficult to measure these features quantitatively at the individual cell level in intact tissue. Here, we introduce normalized Raman imaging (NoRI), a stimulated Raman scattering (SRS) microscopy method that provides the local concentrations of protein, lipid, and water from live or fixed tissue samples with high spatial resolution. Using NoRI, we demonstrate that protein, lipid, and water concentrations at the single cell are maintained in a tight range in cells under the same physiological conditions and are altered in different physiological states, such as cell cycle stages, attachment to substrates of different stiffness, or by entering senescence. In animal tissues, protein and lipid concentration varies with cell types, yet an unexpected cell-to-cell heterogeneity was found in cerebellar Purkinje cells. The protein and lipid concentration profile provides means to quantitatively compare disease-related pathology, as demonstrated using models of Alzheimer's disease. This demonstration shows that NoRI is a broadly applicable technique for probing the biological regulation of protein mass, lipid mass, and water mass for studies of cellular and tissue growth, homeostasis, and disease.

Accurate SNV detection in single cells by transposon-based whole-genome amplification of complementary strands.

Single-nucleotide variants (SNVs), pertinent to aging and disease, occur sporadically in the human genome, hence necessitating single-cell measurements. However, detection of single-cell SNVs suffers from false positives (FPs) due to intracellular single-stranded DNA damage and the process of whole-genome amplification (WGA). Here, we report a single-cell WGA method termed multiplexed end-tagging amplification of complementary strands (META-CS), which eliminates nearly all FPs by virtue of DNA complementarity, and achieved the highest accuracy thus far. We validated META-CS by sequencing kindred cells and human sperm, and applied it to other human tissues. Investigation of mature single human neurons revealed increasing SNVs with age and potentially unrepaired strand-specific oxidative guanine damage. We determined SNV frequencies along the genome in differentiated single human blood cells, and identified cell type-dependent mutational patterns for major types of lymphocytes.

RNA sequencing by direct tagmentation of RNA/DNA hybrids.

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second-strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.

Single-cell sequencing deciphers a convergent evolution of copy number alterations from primary to circulating tumor cells.

Copy number alteration (CNA) is a major contributor to genome instability, a hallmark of cancer. Here, we studied genomic alterations in single primary tumor cells and circulating tumor cells (CTCs) from the same patient. Single-nucleotide variants (SNVs) in single cells from both samples occurred sporadically, whereas CNAs among primary tumor cells emerged accumulatively rather than abruptly, converging toward the CNA in CTCs. Focal CNAs affecting the MYC gene and the PTEN gene were observed only in a minor portion of primary tumor cells but were present in all CTCs, suggesting a strong selection toward metastasis. Single-cell structural variant (SV) analyses revealed a two-step mechanism, a complex rearrangement followed by gene amplification, for the simultaneous formation of anomalous CNAs in multiple chromosome regions. Integrative CNA analyses of 97 CTCs from 23 patients confirmed the convergence of CNAs and revealed single, concurrent, and mutually exclusive CNAs that could be the driving events in cancer metastasis.

Mapping allele with resolved carrier status of Robertsonian and reciprocal translocation in human preimplantation embryos.

Reciprocal translocations (RecT) and Robertsonian translocations (RobT) are among the most common chromosomal abnormalities that cause infertility and birth defects. Preimplantation genetic testing for aneuploidy using comprehensive chromosome screening for in vitro fertilization enables embryo selection with balanced chromosomal ploidy; however, it is normally unable to determine whether an embryo is a translocation carrier. Here we report a method named "Mapping Allele with Resolved Carrier Status" (MaReCs), which enables chromosomal ploidy screening and resolution of the translocation carrier status of the same embryo. We performed MaReCs on 108 embryos, of which 96 were from 13 RecT carriers and 12 were from three RobT carriers. Thirteen of the sixteen patients had at least one diploid embryo. We have confirmed the accuracy of our carrier status determination in amniotic fluid karyotyping of seven cases as well as in the live birth we have thus far. Therefore, MaReCs accurately enables the selection of translocation-free embryos from patients carrying chromosomal translocations. We expect MaReCs will help reduce the propagation of RecT/RobT in the human population.

Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization.

Preimplantation genetic screening (PGS) is widely used to select in vitro-fertilized embryos free of chromosomal abnormalities and to improve the clinical outcome of in vitro fertilization (IVF). A disadvantage of PGS is that it requires biopsy of the preimplantation human embryo, which can limit the clinical applicability of PGS due to the invasiveness and complexity of the process. Here, we present and validate a noninvasive chromosome screening (NICS) method based on sequencing the genomic DNA secreted into the culture medium from the human blastocyst. By using multiple annealing and looping-based amplification cycles (MALBAC) for whole-genome amplification (WGA), we performed next-generation sequencing (NGS) on the spent culture medium used to culture human blastocysts (n = 42) and obtained the ploidy information of all 24 chromosomes. We validated these results by comparing each with their corresponding whole donated embryo and obtained a high correlation for identification of chromosomal abnormalities (sensitivity, 0.882, and specificity, 0.840). With this validated NICS method, we performed chromosome screening on IVF embryos from seven couples with balanced translocation, azoospermia, or recurrent pregnancy loss. Six of them achieved successful clinical pregnancies, and five have already achieved healthy live births thus far. The NICS method avoids the need for embryo biopsy and therefore substantially increases the safety of its use. The method has the potential of much wider chromosome screening applicability in clinical IVF, due to its high accuracy and noninvasiveness.

Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification.

Whole-genome amplification (WGA) for next-generation sequencing has seen wide applications in biology and medicine when characterization of the genome of a single cell is required. High uniformity and fidelity of WGA is needed to accurately determine genomic variations, such as copy number variations (CNVs) and single-nucleotide variations (SNVs). Prevailing WGA methods have been limited by fluctuation of the amplification yield along the genome, as well as false-positive and -negative errors for SNV identification. Here, we report emulsion WGA (eWGA) to overcome these problems. We divide single-cell genomic DNA into a large number (10(5)) of picoliter aqueous droplets in oil. Containing only a few DNA fragments, each droplet is led to reach saturation of DNA amplification before demulsification such that the differences in amplification gain among the fragments are minimized. We demonstrate the proof-of-principle of eWGA with multiple displacement amplification (MDA), a popular WGA method. This easy-to-operate approach enables simultaneous detection of CNVs and SNVs in an individual human cell, exhibiting significantly improved amplification evenness and accuracy.

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy.

Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time.

Live births after simultaneous avoidance of monogenic diseases and chromosome abnormality by next-generation sequencing with linkage analyses.

In vitro fertilization (IVF), preimplantation genetic diagnosis (PGD), and preimplantation genetic screening (PGS) help patients to select embryos free of monogenic diseases and aneuploidy (chromosome abnormality). Next-generation sequencing (NGS) methods, while experiencing a rapid cost reduction, have improved the precision of PGD/PGS. However, the precision of PGD has been limited by the false-positive and false-negative single-nucleotide variations (SNVs), which are not acceptable in IVF and can be circumvented by linkage analyses, such as short tandem repeats or karyomapping. It is noteworthy that existing methods of detecting SNV/copy number variation (CNV) and linkage analysis often require separate procedures for the same embryo. Here we report an NGS-based PGD/PGS procedure that can simultaneously detect a single-gene disorder and aneuploidy and is capable of linkage analysis in a cost-effective way. This method, called "mutated allele revealed by sequencing with aneuploidy and linkage analyses" (MARSALA), involves multiple annealing and looping-based amplification cycles (MALBAC) for single-cell whole-genome amplification. Aneuploidy is determined by CNVs, whereas SNVs associated with the monogenic diseases are detected by PCR amplification of the MALBAC product. The false-positive and -negative SNVs are avoided by an NGS-based linkage analysis. Two healthy babies, free of the monogenic diseases of their parents, were born after such embryo selection. The monogenic diseases originated from a single base mutation on the autosome and the X-chromosome of the disease-carrying father and mother, respectively.

Genotyping single-sperm cells by universal MARSALA enables the acquisition of linkage information for combined pre-implantation genetic diagnosis and genome screening.

PURPOSE: This paper aims to investigate the feasibility of performing pre-implantation genetic diagnosis (PGD) and pre-implantation genetic screening (PGS) simultaneously by a universal strategy without the requirement of genotyping relevant affected family members or lengthy preliminary work on linkage analysis. METHODS: By utilizing a universal Mutated Allele Revealed by Sequencing with Aneuploidy and Linkage Analyses (MARSALA) strategy based on low depth whole genome sequencing (~3x), not involving specific primers' design nor the enrichment of SNP markers for haplotype construction. Single-sperm cells and trephectoderm cells from in vitro fertilized embryos from a couple carrying HBB mutations were genotyped. Haplotypes of paternal alleles were constructed and investigated in embryos, and the chromosome copy number profiles were simultaneously analyzed. RESULTS: The universal MARSALA strategy allows the selection of a euploid embryo free of disease mutations for in uterus transfer and successful pregnancy. A follow-up amniocentesis was performed at 17 weeks of gestation to confirm the PGD/PGS results. CONCLUSION: We present the first successful PGD procedure based on genotyping multiple single-sperm cells to obtain SNP linkage information. Our improved PGD/PGS procedure does not require genotyping the proband or relevant family members and therefore can be applicable to a wider population of patients when conducting PGD for monogenic disorders.

Central dogma at the single-molecule level in living cells.

Gene expression originates from individual DNA molecules within living cells. Like many single-molecule processes, gene expression and regulation are stochastic, that is, sporadic in time. This leads to heterogeneity in the messenger-RNA and protein copy numbers in a population of cells with identical genomes. With advanced single-cell fluorescence microscopy, it is now possible to quantify transcriptomes and proteomes with single-molecule sensitivity. Dynamic processes such as transcription-factor binding, transcription and translation can be monitored in real time, providing quantitative descriptions of the central dogma of molecular biology and the demonstration that a stochastic single-molecule event can determine the phenotype of a cell.

Characterization and development of photoactivatable fluorescent proteins for single-molecule-based superresolution imaging.

Photoactivatable fluorescent proteins (PAFPs) have been widely used for superresolution imaging based on the switching and localization of single molecules. Several properties of PAFPs strongly influence the quality of the superresolution images. These properties include (i) the number of photons emitted per switching cycle, which affects the localization precision of individual molecules; (ii) the ratio of the on- and off-switching rate constants, which limits the achievable localization density; (iii) the dimerization tendency, which could cause undesired aggregation of target proteins; and (iv) the signaling efficiency, which determines the fraction of target-PAFP fusion proteins that is detectable in a cell. Here, we evaluated these properties for 12 commonly used PAFPs fused to both bacterial target proteins, H-NS, HU, and Tar, and mammalian target proteins, Zyxin and Vimentin. Notably, none of the existing PAFPs provided optimal performance in all four criteria, particularly in the signaling efficiency and dimerization tendency. The PAFPs with low dimerization tendencies exhibited low signaling efficiencies, whereas mMaple showed the highest signaling efficiency but also a high dimerization tendency. To address this limitation, we engineered two new PAFPs based on mMaple, which we termed mMaple2 and mMaple3. These proteins exhibited substantially reduced or undetectable dimerization tendencies compared with mMaple but maintained the high signaling efficiency of mMaple. In the meantime, these proteins provided photon numbers and on-off switching rate ratios that are comparable to the best achieved values among PAFPs.