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July 9, 2019

Single cell isoform sequencing (scIso-Seq) identifies novel full-length mRNAs and cell type-specific expression

Single cell RNA-seq (scRNA-seq) is an emerging field for characterizing cell heterogeneity in complex tissues. However, most scRNA-seq methodologies are limited to gene count information due to short read lengths. Here, we combine the microfluidics scRNA-seq technique, Drop-Seq, with PacBio Single Molecule, Real-Time (SMRT) Sequencing to generate full-length transcript isoforms that can be confidently assigned to individual cells. We generated single cell Iso-Seq (scIso-Seq) libraries for chimp and human cerebral organoid samples on the Dolomite Nadia platform and sequenced each library with two SMRT Cells 8M on the PacBio Sequel II System. We developed a bioinformatics pipeline to identify, classify,…

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October 1, 2018

Single-cell isoform RNA sequencing characterizes isoforms in thousands of cerebellar cells.

Full-length RNA sequencing (RNA-Seq) has been applied to bulk tissue, cell lines and sorted cells to characterize transcriptomes, but applying this technology to single cells has proven to be difficult, with less than ten single-cell transcriptomes having been analyzed thus far. Although single splicing events have been described for =200 single cells with statistical confidence, full-length mRNA analyses for hundreds of cells have not been reported. Single-cell short-read 3' sequencing enables the identification of cellular subtypes, but full-length mRNA isoforms for these cell types cannot be profiled. We developed a method that starts with bulk tissue and identifies single-cell types…

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February 3, 2017

Single-cell mRNA isoform diversity in the mouse brain.

Alternative mRNA isoform usage is an important source of protein diversity in mammalian cells. This phenomenon has been extensively studied in bulk tissues, however, it remains unclear how this diversity is reflected in single cells.Here we use long-read sequencing technology combined with unique molecular identifiers (UMIs) to reveal patterns of alternative full-length isoform expression in single cells from the mouse brain. We found a surprising amount of isoform diversity, even after applying a conservative definition of what constitutes an isoform. Genes tend to have one or a few isoforms highly expressed and a larger number of isoforms expressed at a…

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January 13, 2017

Single-cell multiomics: multiple measurements from single cells.

Single-cell sequencing provides information that is not confounded by genotypic or phenotypic heterogeneity of bulk samples. Sequencing of one molecular type (RNA, methylated DNA or open chromatin) in a single cell, furthermore, provides insights into the cell's phenotype and links to its genotype. Nevertheless, only by taking measurements of these phenotypes and genotypes from the same single cells can such inferences be made unambiguously. In this review, we survey the first experimental approaches that assay, in parallel, multiple molecular types from the same single cell, before considering the challenges and opportunities afforded by these and future technologies. Copyright © 2016.…

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September 29, 2016

Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq.

Parallel sequencing of a single cell's genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of…

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April 1, 2016

hybridSPAdes: an algorithm for hybrid assembly of short and long reads.

Recent advances in single molecule real-time (SMRT) and nanopore sequencing technologies have enabled high-quality assemblies from long and inaccurate reads. However, these approaches require high coverage by long reads and remain expensive. On the other hand, the inexpensive short reads technologies produce accurate but fragmented assemblies. Thus, a hybrid approach that assembles long reads (with low coverage) and short reads has a potential to generate high-quality assemblies at reduced cost.We describe hybridSPAdes algorithm for assembling short and long reads and benchmark it on a variety of bacterial assembly projects. Our results demonstrate that hybridSPAdes generates accurate assemblies (even in projects…

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June 1, 2015

G&T-seq: parallel sequencing of single-cell genomes and transcriptomes.

The simultaneous sequencing of a single cell's genome and transcriptome offers a powerful means to dissect genetic variation and its effect on gene expression. Here we describe G&T-seq, a method for separating and sequencing genomic DNA and full-length mRNA from single cells. By applying G&T-seq to over 220 single cells from mice and humans, we discovered cellular properties that could not be inferred from DNA or RNA sequencing alone.

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March 1, 2015

Draft genome sequence of Thauera sp. strain SWB20, isolated from a Singapore wastewater treatment facility using gel microdroplets.

We report here the genome sequence of Thauera sp. strain SWB20, isolated from a Singaporean wastewater treatment facility using gel microdroplets (GMDs) and single-cell genomics (SCG). This approach provided a single clonal microcolony that was sufficient to obtain a 4.9-Mbp genome assembly of an ecologically relevant Thauera species. Copyright © 2015 Dichosa et al.

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September 1, 2014

Single cell genomic study of Dehalococcoidetes species from deep-sea sediments of the Peruvian Margin.

The phylum Chloroflexi is one of the most frequently detected phyla in the subseafloor of the Pacific Ocean margins. Dehalogenating Chloroflexi (Dehalococcoidetes) was originally discovered as the key microorganisms mediating reductive dehalogenation via their key enzymes reductive dehalogenases (Rdh) as sole mode of energy conservation in terrestrial environments. The frequent detection of Dehalococcoidetes-related 16S rRNA and rdh genes in the marine subsurface implies a role for dissimilatory dehalorespiration in this environment; however, the two genes have never been linked to each other. To provide fundamental insights into the metabolism, genomic population structure and evolution of marine subsurface Dehalococcoidetes sp., we…

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April 1, 2014

Genomic and metabolic diversity of Marine Group I Thaumarchaeota in the mesopelagic of two subtropical gyres.

Marine Group I (MGI) Thaumarchaeota are one of the most abundant and cosmopolitan chemoautotrophs within the global dark ocean. To date, no representatives of this archaeal group retrieved from the dark ocean have been successfully cultured. We used single cell genomics to investigate the genomic and metabolic diversity of thaumarchaea within the mesopelagic of the subtropical North Pacific and South Atlantic Ocean. Phylogenetic and metagenomic recruitment analysis revealed that MGI single amplified genomes (SAGs) are genetically and biogeographically distinct from existing thaumarchaea cultures obtained from surface waters. Confirming prior studies, we found genes encoding proteins for aerobic ammonia oxidation and…

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January 1, 2014

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence.

Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over…

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January 1, 2014

An environmental bacterial taxon with a large and distinct metabolic repertoire.

Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such 'talented' producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus 'Entotheonella' with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei.…

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March 1, 2013

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome.

The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome amplification technique of multiple displacement amplification (MDA). However, MDA from a single chromosome copy suffers from amplification bias and a large loss of specificity from even very small amounts of DNA contamination, which makes assembling a genome difficult and completely finishing a genome impossible except in extraordinary circumstances. Gel microdrop cultivation…

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December 1, 2012

A single-cell genome for Thiovulum sp.

We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including…

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April 1, 2012

Capturing single cell genomes of active polysaccharide degraders: an unexpected contribution of Verrucomicrobia.

Microbial hydrolysis of polysaccharides is critical to ecosystem functioning and is of great interest in diverse biotechnological applications, such as biofuel production and bioremediation. Here we demonstrate the use of a new, efficient approach to recover genomes of active polysaccharide degraders from natural, complex microbial assemblages, using a combination of fluorescently labeled substrates, fluorescence-activated cell sorting, and single cell genomics. We employed this approach to analyze freshwater and coastal bacterioplankton for degraders of laminarin and xylan, two of the most abundant storage and structural polysaccharides in nature. Our results suggest that a few phylotypes of Verrucomicrobia make a considerable contribution…

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