Rapid and signal crowdedness-robust in situ sequencing through hybrid block coding.

Spatial transcriptomics technology has revolutionized our understanding of cell types and tissue organization, opening possibilities for researchers to explore transcript distributions at subcellular levels. However, existing methods have limitations in resolution, sensitivity, or speed. To overcome these challenges, we introduce SPRINTseq (Spatially Resolved and signal-diluted Next-generation Targeted sequencing), an innovative in situ sequencing strategy that combines hybrid block coding and molecular dilution strategies. Our method enables fast and sensitive high-resolution data acquisition, as demonstrated by recovering over 142 million transcripts using a 108-gene panel from 453,843 cells from four mouse brain coronal slices in less than 2 d. Using this advanced technology, we uncover the cellular and subcellular molecular architecture of Alzheimer's disease, providing additional information into abnormal cellular behaviors and their subcellular mRNA distribution. This improved spatial transcriptomics technology holds great promise for exploring complex biological processes and disease mechanisms.

Low-frequency somatic copy number alterations in normal human lymphocytes revealed by large-scale single-cell whole-genome profiling.

Genomic-scale somatic copy number alterations in healthy humans are difficult to investigate because of low occurrence rates and the structural variations' stochastic natures. Using a Tn5-transposase-assisted single-cell whole-genome sequencing method, we sequenced over 20,000 single lymphocytes from 16 individuals. Then, with the scale increased to a few thousand single cells per individual, we found that about 7.5% of the cells had large-size copy number alterations. Trisomy 21 was the most prevalent aneuploid event among all autosomal copy number alterations, whereas monosomy X occurred most frequently in over-30-yr-old females. In the monosomy X single cells from individuals with phased genomes and identified X-inactivation ratios in bulk, the inactive X Chromosomes were lost more often than the active ones.

Rapid and sensitive single-cell RNA sequencing with SHERRY2.

BACKGROUND: Prevalent single-cell transcriptomic profiling (scRNA-seq) methods are mainly based on the synthesis and enrichment of full-length double-stranded complementary DNA. These approaches are challenging to generate accurate quantification of transcripts when their abundance is low or their full-length amplifications are difficult. RESULTS: Based on our previous finding that Tn5 transposase can directly cut-and-tag DNA/RNA hetero-duplexes, we present SHERRY2, a specifically optimized protocol for scRNA-seq without second-strand cDNA synthesis. SHERRY2 is free of pre-amplification and eliminates the sequence-dependent bias. In comparison with other widely used scRNA-seq methods, SHERRY2 exhibits significantly higher sensitivity and accuracy even for single nuclei. Besides, SHERRY2 is simple and robust and can be easily scaled up to high-throughput experiments. When testing single lymphocytes and neuron nuclei, SHERRY2 not only obtained accurate countings of transcription factors and long non-coding RNAs, but also provided bias-free results that enriched genes in specific cellular components or functions, which outperformed other protocols. With a few thousand cells sequenced by SHERRY2, we confirmed the expression and dynamics of Myc in different cell types of germinal centers, which were previously only revealed by gene-specific amplification methods. CONCLUSIONS: SHERRY2 is able to provide high sensitivity, high accuracy, and high throughput for those applications that require a high number of genes identified in each cell. It can reveal the subtle transcriptomic difference between cells and facilitate important biological discoveries.

Microfluidic Device for Studying Controllable Hydrodynamic Flow Induced Cellular Responses.

Hydrodynamic flow is an essential stimulus in many cellular functions, regulating many mechanical sensitive pathways and closely associating with human health status and diseases. The flow pattern of blood in vessels is the key factor in causing atherosclerosis. Hemodynamics has great effect on endothelial cells' gene expression and biological functions. There are various tools that can be used for studying flow-induced cellular responses but most of them are either bulky or lack precise controllability. We develop an integrated microfluidic device that can precisely generate different flow patterns to human endothelial cells cultured on-chip. We monitored cell morphology and used small-input RNA-seq technology to depict the transcriptome profiles of human umbilical vein endothelial cells under uni- or bidirectional flow. Such integrated and miniatured device has greatly facilitated our understanding of endothelial functions with shear stimulus, not only providing new data on the transcriptomic scale but also building the connection between cell phenotypic changes and expression alternations.

Microfluidic single-cell whole-transcriptome sequencing.

Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.

FAM46C-mediated tumor heterogeneity predicts extramedullary metastasis and poorer survival in multiple myeloma.

Cancers originate from a single cell according to Nowell's theory of clonal evolution. The enrichment of the most aggressive clones has been developed and the heterogeneity arises for genomic instability and environmental selection. Multiple myeloma (MM) is a multiple relapse plasma cell cancer generated from bone marrow. Although there were accumulating researches in multiple myeloma pathogenesis, the heterogeneity remains poorly understood. The participants enrolled in this study were 4 EMP+ (EMP, Extramedullary plasmacytoma) and 2 EMP- primarily untreated MM patients. Single cell RNA sequencing and analysis were conducted for the single cell suspension, which was sorted by flow cytometry from peripheral blood mononuclear cells or bone marrow cells. In our research, the results of single cell RNA sequencing show that FAM46C determines MM tumor heterogeneity predicting extramedullary metastasis by influencing RNA stability. Further, we integrated and analyzed 2280 multiple myeloma samples from 7 independent datasets, which uncover that FAM46C mediated tumor heterogeneity predicts poorer survival in multiple myeloma.

Ancient DNA reveals genetic admixture in China during tiger evolution.

The tiger (Panthera tigris) is a charismatic megafauna species that originated and diversified in Asia and probably experienced population contraction and expansion during the Pleistocene, resulting in low genetic diversity of modern tigers. However, little is known about patterns of genomic diversity in ancient populations. Here we generated whole-genome sequences from ancient or historical (100-10,000 yr old) specimens collected across mainland Asia, including a 10,600-yr-old Russian Far East specimen (RUSA21, 8× coverage) plus six ancient mitogenomes, 14 South China tigers (0.1-12×) and three Caspian tigers (4-8×). Admixture analysis showed that RUSA21 clustered within modern Northeast Asian phylogroups and partially derived from an extinct Late Pleistocene lineage. While some of the 8,000-10,000-yr-old Russian Far East mitogenomes are basal to all tigers, one 2,000-yr-old specimen resembles present Amur tigers. Phylogenomic analyses suggested that the Caspian tiger probably dispersed from an ancestral Northeast Asian population and experienced gene flow from southern Bengal tigers. Lastly, genome-wide monophyly supported the South China tiger as a distinct subspecies, albeit with mitochondrial paraphyly, hence resolving its longstanding taxonomic controversy. The distribution of mitochondrial haplogroups corroborated by biogeographical modelling suggested that Southwest China was a Late Pleistocene refugium for a relic basal lineage. As suitable habitat returned, admixture between divergent lineages of South China tigers took place in Eastern China, promoting the evolution of other northern subspecies. Altogether, our analysis of ancient genomes sheds light on the evolutionary history of tigers and supports the existence of nine modern subspecies.

Openly accessible microfluidic liquid handlers for automated high-throughput nanoliter cell culture.

Cell culture is typically performed in Petri dishes, with a few million cells growing together, or in microwell plates with thousands of cells in each compartment. When the throughput of each experiment, especially of screening based assays, is increased, even using microliter solution per well will cost a considerable amount of cells and reagents. We took a rational approach to reduce the volume of each cell culture chamber. We designed and fabricated a poly(dimethylsiloxane) based liquid pipet chip to deliver and transfer nanoliter (50-500 nL) samples and reagents with high accuracy and robustness. A few tens to a few hundreds of cells can be successfully seeded, transferred, passaged, transfected, and stimulated by drugs on a microwell chip using this pipet chip automatically. We have used this system to test the cell growth dynamically, observed the correlation between the culture conditions and cell viabilities, and quantitatively evaluated cell apoptosis induced by cis-diammineplatinum(II) dichloride (cisplatin). This system shows great potential to facilitate large-scale screening and high-throughput cell-array based bioassays with the volume of each individual cell colony at the nanoliter level.

Quantitative study of the dynamic tumor-endothelial cell interactions through an integrated microfluidic coculture system.

The interaction between tumor and endothelial cells is crucial to cancer metastasis and angiogenesis. We developed a novel microfluidic device to assess the cell-cell interaction quantitatively at the single cell resolution. This integrated chip offers 16 coculture experiments in parallel with controllable microenvironments to study interactions between cells dynamically. We applied this approach to model the tumor invasion using Hela cells and human umbilical vein endothelial cells (HUVECs) and monitored the migration of both. We observed the retreatment of HUVECs upon the approach of Hela cells during coculture, indicating that the interaction between two cells was mediated by soluble factors. This interaction was further analyzed through quantitatively processing the phase-contrast microscopic time-lapse images of each individual coculture chamber. We also confirmed this paracrine effect by varying the frequency of medium change. This microfluidic technique is highly controllable, contamination free, fully automatic, and inexpensive. This approach not only offers a unique way to quantitatively study the interaction between cells but also provides accurate spatial-temporal tunability of microenvironments for cell coculture. We believe this method, intrinsically high-throughput and quantitative, will greatly facilitate the study of cell-cell interactions and communications.

A chromosome-level genome of the human blood fluke Schistosoma japonicum identifies the genomic basis of host-switching.

The evolution and adaptation of S. japonicum, a zoonotic parasite that causes human schistosomiasis, remain unclear because of the lack of whole-genome data. We construct a chromosome-level S. japonicum genome and analyze it together with 72 samples representing six populations of the entire endemic region. We observe a Taiwan zoophilic lineage splitting from zoonotic populations ∼45,000 years ago, consistent with the divergent history of their intermediate hosts. Interestingly, we detect a severe population bottleneck in S. japonicum, largely coinciding with human history in Asia during the last glacial maximum. We identify several genomic regions underlying natural selection, including GATAD2A and Lmln, both showing remarkable differentiation among different areas. RNAi knockdown suggests association of GATAD2A with parasite development and infection in definitive hosts, while Lmln relates to the specificity of the intermediate hosts. Our study provides insights into the evolution of S. japonicum and serves as a resource for further studies.

A Late Pleistocene human genome from Southwest China.

Southern East Asia is the dispersal center regarding the prehistoric settlement and migrations of modern humans in Asia-Pacific regions. However, the settlement pattern and population structure of paleolithic humans in this region remain elusive, and ancient DNA can provide direct information. Here, we sequenced the genome of a Late Pleistocene hominin (MZR), dated ∼14.0 thousand years ago from Red Deer Cave located in Southwest China, which was previously reported possessing mosaic features of modern and archaic hominins. MZR is the first Late Pleistocene genome from southern East Asia. Our results indicate that MZR is a modern human who represents an early diversified lineage in East Asia. The mtDNA of MZR belongs to an extinct basal lineage of the M9 haplogroup, reflecting a rich matrilineal diversity in southern East Asia during the Late Pleistocene. Combined with the published data, we detected clear genetic stratification in ancient southern populations of East/Southeast Asia and some degree of south-versus-north divergency during the Late Pleistocene, and MZR was identified as a southern East Asian who exhibits genetic continuity to present day populations. Markedly, MZR is linked deeply to the East Asian ancestry that contributed to First Americans.

Deletion of Shp2 in the brain leads to defective proliferation and differentiation in neural stem cells and early postnatal lethality.

The intracellular signaling controlling neural stem/progenitor cell (NSC) self-renewal and neuronal/glial differentiation is not fully understood. We show here that Shp2, an introcellular tyrosine phosphatase with two SH2 domains, plays a critical role in NSC activities. Conditional deletion of Shp2 in neural progenitor cells mediated by Nestin-Cre resulted in early postnatal lethality, impaired corticogenesis, and reduced proliferation of progenitor cells in the ventricular zone. In vitro analyses suggest that Shp2 mediates basic fibroblast growth factor signals in stimulating self-renewing proliferation of NSCs, partly through control of Bmi-1 expression. Furthermore, Shp2 regulates cell fate decisions, by promoting neurogenesis while suppressing astrogliogenesis, through reciprocal regulation of the Erk and Stat3 signaling pathways. Together, these results identify Shp2 as a critical signaling molecule in coordinated regulation of progenitor cell proliferation and neuronal/astroglial cell differentiation.

Surfactant and oil formulations for monodisperse droplet emulsion PCR.

Emulsion PCR has become a popular and widely applied method in biological research and clinical diagnostics to provide evenly amplified products and perform highly quantitative counting of target sequences. However, there is still a lack of information to support further development of appropriate water-in-oil emulsion formulations, which need to be both thermally and mechanically stable for digital amplification reactions. Here, we present a systematic survey of the oil and surfactant components of stable monodisperse w/o emulsions suitable for use with our previously developed micro-capillary array (MiCA)-based centrifugal emulsion generation method. Our findings show that a binary formula consisting of isopropyl palmitate and a silicone copolymer demonstrated the best performance, and provided a general guideline for the development of emulsion systems for digital PCR and emulsion amplification applications.

A microfluidic approach for experimentally modelling the intercellular coupling system of a mammalian circadian clock at single-cell level.

In mammals, it is believed that the intercellular coupling mechanism between neurons in the suprachiasmatic nucleus (SCN) confers robustness and distinguishes the central clock from peripheral circadian oscillators. Current in vitro culturing methods used in Petri dishes to study intercellular coupling by exogenous factors invariably cause perturbations, such as simple media changes. Here, we design a microfluidic device to quantitatively study the intercellular coupling mechanism of circadian clock at the single cell level, and demonstrate that vasoactive intestinal peptide (VIP) induced coupling in clock mutant Cry1-/- mouse adult fibroblasts engineered to express the VIP receptor, VPAC2, is sufficient to synchronize and maintain robust circadian oscillations. Our study provides a proof-of-concept platform to reconstitute the intercellular coupling system of the central clock using uncoupled, single fibroblast cells in vitro, to mimic SCN slice cultures ex vivo and mouse behavior in vivo phenotypically. Such a versatile microfluidic platform may greatly facilitate the studies of intercellular regulation networks, and provide new insights into the coupling mechanisms of the circadian clock.

Long-read individual-molecule sequencing reveals CRISPR-induced genetic heterogeneity in human ESCs.

Quantifying the genetic heterogeneity of a cell population is essential to understanding of biological systems. We develop a universal method to label individual DNA molecules for single-base-resolution haplotype-resolved quantitative characterization of diverse types of rare variants, with frequency as low as 4 × 10-5, using both short- or long-read sequencing platforms. It provides the first quantitative evidence of persistent nonrandom large structural variants and an increase in single-nucleotide variants at the on-target locus following repair of double-strand breaks induced by CRISPR-Cas9 in human embryonic stem cells.

Single-cell RNA sequencing reveals chemokine self-feeding of myeloma cells promotes extramedullary metastasis.

In this study, we aimed to determine the mechanisms underlying the initial extramedullary translocation of myeloma cells from bone marrow into peripheral blood. We found that clonal circulating plasma cells (cPCs) are more frequently detected by flow cytometry in extramedullary plasmacytoma (EMP) patients and worsen their prognosis. It is technically much easier to collect single cPCs using FACS than it is to perform EMP biopsy. Therefore, combining EMP imaging with cPC detection may be a promising strategy for prognostic stratification. Here, using single-cell transcriptome analysis, we found that the chemokine CXCL12, a key molecule involved in CXCR4-dependent cell retention in the bone marrow, is abnormally upregulated in cPCs and might initially enable cPCs to evade bone marrow retention and translocate into the bloodstream.

Genomic Heterogeneity and Branched Evolution of Early Stage Primary Acral Melanoma Shown by Multiregional Microdissection Sequencing.

Acral melanoma (AM) is an extremely aggressive subtype of melanoma that is prevalent in eastern Asia. AM exhibits high intertumoral and intratumoral heterogeneities with poor prognosis. To associate the genomic heterogeneities with phenotypic traits and efficacy of treatments, a method is needed to recover genomic information from limited samples with high specificity and sensitivity from early stage AM specimens. We performed laser capture microdissection to isolate single micro-tumor nests, containing only dozens of cells, from stained tissue slices and then applied multiple annealing and looping-based amplification cycles, a highly efficient whole-genome amplification method originally developed for single cells, to amplify the whole genome of each tumor nest for sequencing. We were able to accurately profile the landscape of copy number alterations and single nucleotide variations of every single micro-tumor nest and to quantitatively characterize the heterogeneities at different levels, between tumor and nevi, among patients, among different phenotypes within a same tumor, and among adjacent tumor cell clusters with identical phenotypic appearance. We have found that genomic heterogeneity exists extensively and that branched evolution happens in the early stage of AM development. We are able to build the phylogenetic tree among these phenotypically addressable cell clusters.

Tagmentation on Microbeads: Restore Long-Range DNA Sequence Information Using Next Generation Sequencing with Library Prepared by Surface-Immobilized Transposomes.

The next generation sequencing (NGS) technologies have been rapidly evolved and applied to various research fields, but they often suffer from losing long-range information due to short library size and read length. Here, we develop a simple, cost-efficient, and versatile NGS library preparation method, called tagmentation on microbeads (TOM). This method is capable of recovering long-range information through tagmentation mediated by microbead-immobilized transposomes. Using transposomes with DNA barcodes to identically label adjacent sequences during tagmentation, we can restore inter-read connection of each fragment from original DNA molecule by fragment-barcode linkage after sequencing. In our proof-of-principle experiment, more than 4.5% of the reads are linked with their adjacent reads, and the longest linkage is over 1112 bp. We demonstrate TOM with eight barcodes, but the number of barcodes can be scaled up by an ultrahigh complexity construction. We also show this method has low amplification bias and effectively fits the applications to identify copy number variations.

Single-Cell-Based Platform for Copy Number Variation Profiling through Digital Counting of Amplified Genomic DNA Fragments.

We develop a novel single-cell-based platform through digital counting of amplified genomic DNA fragments, named multifraction amplification (mfA), to detect the copy number variations (CNVs) in a single cell. Amplification is required to acquire genomic information from a single cell, while introducing unavoidable bias. Unlike prevalent methods that directly infer CNV profiles from the pattern of sequencing depth, our mfA platform denatures and separates the DNA molecules from a single cell into multiple fractions of a reaction mix before amplification. By examining the sequencing result of each fraction for a specific fragment and applying a segment-merge maximum likelihood algorithm to the calculation of copy number, we digitize the sequencing-depth-based CNV identification and thus provide a method that is less sensitive to the amplification bias. In this paper, we demonstrate a mfA platform through multiple displacement amplification (MDA) chemistry. When performing the mfA platform, the noise of MDA is reduced; therefore, the resolution of single-cell CNV identification can be improved to 100 kb. We can also determine the genomic region free of allelic drop-out with mfA platform, which is impossible for conventional single-cell amplification methods.

Mechanism of inhibition of Ca2+-transport activity of sarcoplasmic reticulum Ca2+-ATPase by anisodamine.

The mechanism of inhibition of Ca2+-transport activity of rabbit sarcoplasmic reticulum Ca 2+-ATPase (SERCA) by anisodamine (a drug isolated from a medicinal herb Hyoscyamuns niger L) was investigated by using ANS (1-anilino-8-naphthalenesulfonate) fluorescence probe, intrinsic fluorescence quenching and Ca 2+-transport activity assays. The number of ANS binding sites for apo Ca2+-ATPase was determined as 8, using a multiple-identical binding site model. Both anisodamine and Ca2+ at millimolar level enhanced the ANS binding fluorescence intensities. Only anisodamine increased the number of ANS molecules bound by SERCA from 8 to 14. The dissociation constants of ANS to the enzyme without any ligand, with 30 mM anisodamine and with 15 mM Ca 2 were found to be 53.0 microM, 85.0 microM and 50.1 microM, respectively. Both anisodamine and Ca2+ enhanced the ANS binding fluorescenc with apparent dissociation constants of 7.6 mM and 2.3 mM, respectively, at a constant concentration of the enzyme. Binding of anisodamine significantly decreased the binding capacity of Ca2+ with the dissociation constant of 9.5 mM, but binding of Ca2+ had no obvious effect on binding of anisodamine. Intrinsic fluorescence quenching and Ca2+-transport activity assays gave the dissociation constants of anisodamine to SERCA as 9.7 and 5.4 mM, respectively, which were consistent with those obtained from ANS-binding fluorescence changes during titration of SERCA with anisodamine and anisodamine + 15 mM Ca2+, respectively. The results suggest that anisodamine regulates Ca2+-transport activity of the enzyme, by stabilizing the trans-membrane domain in an expanded, inactive conformation, at least at its annular ring region.