Atlas-scale single-cell chromatin accessibility using nanowell-based combinatorial indexing.

Here we present advancements in single-cell combinatorial indexed Assay for Transposase Accessible Chromatin (sciATAC) to measure chromatin accessibility that leverage nanowell chips to achieve atlas-scale cell throughput (>10 cells) at low cost. The platform leverages the core of the sciATAC workflow where multiple indexed tagmentation reactions are performed, followed by pooling and distribution to a second set of reaction wells for polymerase chain reaction (PCR)-based indexing. In this work, we instead leverage a chip containing 5184 nanowells at the PCR stage of indexing, enabling a 52-fold improvement in scale and reduction in per-cell preparation costs.

High-throughput robust single-cell DNA methylation profiling with sciMETv2.

DNA methylation is a key epigenetic property that drives gene regulatory programs in development and disease. Current single-cell methods that produce high quality methylomes are expensive and low throughput without the aid of extensive automation. We previously described a proof-of-principle technique that enabled high cell throughput; however, it produced only low-coverage profiles and was a difficult protocol that required custom sequencing primers and recipes and frequently produced libraries with excessive adapter contamination.

High-content single-cell combinatorial indexing.

Single-cell combinatorial indexing (sci) with transposase-based library construction increases the throughput of single-cell genomics assays but produces sparse coverage in terms of usable reads per cell. We develop symmetrical strand sci ('s3'), a uracil-based adapter switching approach that improves the rate of conversion of source DNA into viable sequencing library fragments following tagmentation. We apply this chemistry to assay chromatin accessibility (s3-assay for transposase-accessible chromatin, s3-ATAC) in human cortical and mouse whole-brain tissues, with mouse datasets demonstrating a six- to 13-fold improvement in usable reads per cell compared with other available methods.

Spatially mapped single-cell chromatin accessibility.

High-throughput single-cell epigenomic assays can resolve cell type heterogeneity in complex tissues, however, spatial orientation is lost. Here, we present single-cell combinatorial indexing on Microbiopsies Assigned to Positions for the Assay for Transposase Accessible Chromatin, or sciMAP-ATAC, as a method for highly scalable, spatially resolved, single-cell profiling of chromatin states. sciMAP-ATAC produces data of equivalent quality to non-spatial sci-ATAC and retains the positional information of each cell within a 214 micron cubic region, with up to hundreds of tracked positions in a single experiment.

A human cell atlas of fetal chromatin accessibility.

The chromatin landscape underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of chromatin accessibility and gene expression in fetal tissues. For chromatin accessibility, we devised a three-level combinatorial indexing assay and applied it to 53 samples representing 15 organs, profiling ~800,000 single cells.

Transcriptomic evidence that von Economo neurons are regionally specialized extratelencephalic-projecting excitatory neurons.

von Economo neurons (VENs) are bipolar, spindle-shaped neurons restricted to layer 5 of human frontoinsula and anterior cingulate cortex that appear to be selectively vulnerable to neuropsychiatric and neurodegenerative diseases, although little is known about other VEN cellular phenotypes. Single nucleus RNA-sequencing of frontoinsula layer 5 identifies a transcriptomically-defined cell cluster that contained VENs, but also fork cells and a subset of pyramidal neurons. Cross-species alignment of this cell cluster with a well-annotated mouse classification shows strong homology to extratelencephalic (ET) excitatory neurons that project to subcerebral targets.

High-Throughput Single-Cell Sequencing with Linear Amplification.

Conventional methods for single-cell genome sequencing are limited with respect to uniformity and throughput. Here, we describe sci-L3, a single-cell sequencing method that combines combinatorial indexing (sci-) and linear (L) amplification. The sci-L3 method adopts a 3-level (3) indexing scheme that minimizes amplification biases while enabling exponential gains in throughput.

Droplet-based combinatorial indexing for massive-scale single-cell chromatin accessibility.

Recent technical advancements have facilitated the mapping of epigenomes at single-cell resolution; however, the throughput and quality of these methods have limited their widespread adoption. Here we describe a high-quality (10 nuclear fragments per cell) droplet-microfluidics-based method for single-cell profiling of chromatin accessibility. We use this approach, named 'droplet single-cell assay for transposase-accessible chromatin using sequencing' (dscATAC-seq), to assay 46,653 cells for the unbiased discovery of cell types and regulatory elements in adult mouse brain.

The accessible chromatin landscape of the murine hippocampus at single-cell resolution.

Here we present a comprehensive map of the accessible chromatin landscape of the mouse hippocampus at single-cell resolution. Substantial advances of this work include the optimization of a single-cell combinatorial indexing assay for transposase accessible chromatin (sci-ATAC-seq); a software suite, , for the rapid processing and visualization of single-cell combinatorial indexing data sets; and a valuable resource of hippocampal regulatory networks at single-cell resolution. We used sci-ATAC-seq to produce 2346 high-quality single-cell chromatin accessibility maps with a mean unique read count per cell of 29,201 from both fresh and frozen hippocampi, observing little difference in accessibility patterns between the preparations.

The single-cell transcriptional landscape of mammalian organogenesis.

Mammalian organogenesis is a remarkable process. Within a short timeframe, the cells of the three germ layers transform into an embryo that includes most of the major internal and external organs. Here we investigate the transcriptional dynamics of mouse organogenesis at single-cell resolution.

Joint profiling of chromatin accessibility and gene expression in thousands of single cells.

Although we can increasingly measure transcription, chromatin, methylation, and other aspects of molecular biology at single-cell resolution, most assays survey only one aspect of cellular biology. Here we describe sci-CAR, a combinatorial indexing-based coassay that jointly profiles chromatin accessibility and mRNA (CAR) in each of thousands of single cells. As a proof of concept, we apply sci-CAR to 4825 cells, including a time series of dexamethasone treatment, as well as to 11,296 cells from the adult mouse kidney.

Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type.

We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large 'rosehip'-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1CCK, CNR1SSTCALB2PVALB) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex.

Cicero Predicts cis-Regulatory DNA Interactions from Single-Cell Chromatin Accessibility Data.

Linking regulatory DNA elements to their target genes, which may be located hundreds of kilobases away, remains challenging. Here, we introduce Cicero, an algorithm that identifies co-accessible pairs of DNA elements using single-cell chromatin accessibility data and so connects regulatory elements to their putative target genes. We apply Cicero to investigate how dynamically accessible elements orchestrate gene regulation in differentiating myoblasts.

A Single-Cell Atlas of In Vivo Mammalian Chromatin Accessibility.

We applied a combinatorial indexing assay, sci-ATAC-seq, to profile genome-wide chromatin accessibility in ∼100,000 single cells from 13 adult mouse tissues. We identify 85 distinct patterns of chromatin accessibility, most of which can be assigned to cell types, and ∼400,000 differentially accessible elements. We use these data to link regulatory elements to their target genes, to define the transcription factor grammar specifying each cell type, and to discover in vivo correlates of heterogeneity in accessibility within cell types.

Highly scalable generation of DNA methylation profiles in single cells.

We present a highly scalable assay for whole-genome methylation profiling of single cells. We use our approach, single-cell combinatorial indexing for methylation analysis (sci-MET), to produce 3,282 single-cell bisulfite sequencing libraries and achieve read alignment rates of 68 ± 8%. We apply sci-MET to discriminate the cellular identity of a mixture of three human cell lines and to identify excitatory and inhibitory neuronal populations from mouse cortical tissue.

The cis-regulatory dynamics of embryonic development at single-cell resolution.

Understanding how gene regulatory networks control the progressive restriction of cell fates is a long-standing challenge. Recent advances in measuring gene expression in single cells are providing new insights into lineage commitment. However, the regulatory events underlying these changes remain unclear.

Comprehensive single-cell transcriptional profiling of a multicellular organism.

To resolve cellular heterogeneity, we developed a combinatorial indexing strategy to profile the transcriptomes of single cells or nuclei, termed sci-RNA-seq (single-cell combinatorial indexing RNA sequencing). We applied sci-RNA-seq to profile nearly 50,000 cells from the nematode at the L2 larval stage, which provided >50-fold "shotgun" cellular coverage of its somatic cell composition. From these data, we defined consensus expression profiles for 27 cell types and recovered rare neuronal cell types corresponding to as few as one or two cells in the L2 worm.

Haplotype phasing of whole human genomes using bead-based barcode partitioning in a single tube.

Haplotype-resolved genome sequencing promises to unlock a wealth of information in population and medical genetics. However, for the vast majority of genomes sequenced to date, haplotypes have not been determined because of cumbersome haplotyping workflows that require fractions of the genome to be sequenced in a large number of compartments. Here we demonstrate barcode partitioning of long DNA molecules in a single compartment using "on-bead" barcoded tagmentation.

Contiguity-Preserving Transposition Sequencing (CPT-Seq) for Genome-Wide Haplotyping, Assembly, and Single-Cell ATAC-Seq.

Most genomes to date have been sequenced without taking into account the diploid nature of the genome. However, the distribution of variants on each individual chromosome can (1) significantly impact gene regulation and protein function, (2) have important implications for analyses of population history and medical genetics, and (3) be of great value for accurate interpretation of medically relevant genetic variation. Here, we describe a comprehensive and detailed protocol for an ultra fast (<3 h library preparation), cost-effective, and scalable haplotyping method, named Contiguity Preserving Transposition sequencing or CPT-seq (Amini et al.

Sequencing thousands of single-cell genomes with combinatorial indexing.

Single-cell genome sequencing has proven valuable for the detection of somatic variation, particularly in the context of tumor evolution. Current technologies suffer from high library construction costs, which restrict the number of cells that can be assessed and thus impose limitations on the ability to measure heterogeneity within a tissue. Here, we present single-cell combinatorial indexed sequencing (SCI-seq) as a means of simultaneously generating thousands of low-pass single-cell libraries for detection of somatic copy-number variants.

Massively multiplex single-cell Hi-C.

We present single-cell combinatorial indexed Hi-C (sciHi-C), a method that applies combinatorial cellular indexing to chromosome conformation capture. In this proof of concept, we generate and sequence six sciHi-C libraries comprising a total of 10,696 single cells. We use sciHi-C data to separate cells by karyotypic and cell-cycle state differences and identify cell-to-cell heterogeneity in mammalian chromosomal conformation.

Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing.

Technical advances have enabled the collection of genome and transcriptome data sets with single-cell resolution. However, single-cell characterization of the epigenome has remained challenging. Furthermore, because cells must be physically separated before biochemical processing, conventional single-cell preparatory methods scale linearly.

In vitro, long-range sequence information for de novo genome assembly via transposase contiguity.

We describe a method that exploits contiguity preserving transposase sequencing (CPT-seq) to facilitate the scaffolding of de novo genome assemblies. CPT-seq is an entirely in vitro means of generating libraries comprised of 9216 indexed pools, each of which contains thousands of sparsely sequenced long fragments ranging from 5 kilobases to > 1 megabase. These pools are "subhaploid," in that the lengths of fragments contained in each pool sums to ∼5% to 10% of the full genome.

Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing.

Haplotype-resolved genome sequencing enables the accurate interpretation of medically relevant genetic variation, deep inferences regarding population history and non-invasive prediction of fetal genomes. We describe an approach for genome-wide haplotyping based on contiguity-preserving transposition (CPT-seq) and combinatorial indexing. Tn5 transposition is used to modify DNA with adaptor and index sequences while preserving contiguity.