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Tuesday, April 21, 2020

RNA sequencing: the teenage years.

Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions…

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Tuesday, April 21, 2020

Combined Genome and Transcriptome (G&T) Sequencing of Single Cells.

The simultaneous examination of a single cell’s genome and transcriptome presents scientists with a powerful tool to study genetic variability and its effect on gene expression. In this chapter, we describe the library generation method for combined genome and transcriptome sequencing (G&T-seq) originally described by Macaulay et al. (Nat Protoc 11(11):2081-2103, 2016; Nat Methods 12(6):519-522, 2015). This includes some alterations we made to improve robustness of this process for both the novice user and laboratories that want to deploy this method at scale. Using this method, genomic DNA and full-length mRNA from single cells are separated, amplified, and converted into…

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Tuesday, April 21, 2020

Development of CRISPR-Cas systems for genome editing and beyond

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-Cas systems for genome editing has transformed the way life science research is conducted and holds enormous potential for the treatment of disease as well as for many aspects of biotech- nology. Here, I provide a personal perspective on the development of CRISPR-Cas9 for genome editing within the broader context of the field and discuss our work to discover novel Cas effectors and develop them into additional molecular tools. The initial demonstra- tion of Cas9-mediated genome editing launched the development of many other technologies, enabled new lines of biological inquiry, and motivated…

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Tuesday, April 21, 2020

Platanus-allee is a de novo haplotype assembler enabling a comprehensive access to divergent heterozygous regions.

The ultimate goal for diploid genome determination is to completely decode homologous chromosomes independently, and several phasing programs from consensus sequences have been developed. These methods work well for lowly heterozygous genomes, but the manifold species have high heterozygosity. Additionally, there are highly divergent regions (HDRs), where the haplotype sequences differ considerably. Because HDRs are likely to direct various interesting biological phenomena, many genomic analysis targets fall within these regions. However, they cannot be accessed by existing phasing methods, and we have to adopt costly traditional methods. Here, we develop a de novo haplotype assembler, Platanus-allee ( http://platanus.bio.titech.ac.jp/platanus2 ), which…

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Monday, March 30, 2020

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

In this AGBT presentation from AGBT 2019, Jason Underwood, shares information about single-cell isoform sequencing (scIso-Seq), focusing on a collaborative project with the labs of Evan Eichler and Alex Pollen. For this effort, scientists used Drop-seq sample prep and then loaded cDNA products onto the Sequel System. Results from a barnyard experiment using mouse and human cells as well as from cerebral organoids demonstrated that this approach could deliver cell type-specific gene expression data. Underwood also presents data from the Sequel II System comparing chimp and human organoids, resulting in information about 14,000 unique genes with important insights for post-transcriptional…

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Wednesday, February 26, 2020

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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Sunday, September 22, 2019

Searching for convergent pathways in autism spectrum disorders: insights from human brain transcriptome studies.

Autism spectrum disorder (ASD) is one of the most heritable neuropsychiatric conditions. The complex genetic landscape of the disorder includes both common and rare variants at hundreds of genetic loci. This marked heterogeneity has thus far hampered efforts to develop genetic diagnostic panels and targeted pharmacological therapies. Here, we give an overview of the current literature on the genetic basis of ASD, and review recent human brain transcriptome studies and their role in identifying convergent pathways downstream of the heterogeneous genetic variants. We also discuss emerging evidence on the involvement of non-coding genomic regions and non-coding RNAs in ASD.

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Sunday, September 22, 2019

The transcriptome of human pluripotent stem cells.

Human Embryonic Stem Cells (hESCs) are in vitro derivatives of the inner cell mass of the blastocyst and are characterized by an undifferentiated and pluripotent state that can be perpetuated in time, indefinitely. hESCs provide a unique opportunity to both dissect the molecular mechanisms that are predisposed to the maintenance of pluripotency and model the ability to initiate differentiation and cell commitment within the developing embryo. To fully understand these mechanisms, it is necessary to accurately identify the specific transcriptome of hESCs. Many distinct gene annotation methods, such as cDNA and EST sequencing and RNA-Seq, have been used to identify…

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Sunday, September 22, 2019

Alternative TSSs are co-regulated in single cells in the mouse brain.

Alternative transcription start sites (TSSs) have been extensively studied genome-wide for many cell types and have been shown to be important during development and to regulate transcript abundance between cell types. Likewise, single-cell gene expression has been extensively studied for many cell types. However, how single cells use TSSs has not yet been examined. In particular, it is unknown whether alternative TSSs are independently expressed, or whether they are co-activated or even mutually exclusive in single cells. Here, we use a previously published single-cell RNA-seq dataset, comprising thousands of cells, to study alternative TSS usage. We find that alternative TSS…

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Sunday, September 22, 2019

Alternative polyadenylation: methods, findings, and impacts.

Alternative polyadenylation (APA), a phenomenon that RNA molecules with different 3′ ends originate from distinct polyadenylation sites of a single gene, is emerging as a mechanism widely used to regulate gene expression. In the present review, we first summarized various methods prevalently adopted in APA study, mainly focused on the next-generation sequencing (NGS)-based techniques specially designed for APA identification, the related bioinformatics methods, and the strategies for APA study in single cells. Then we summarized the main findings and advances so far based on these methods, including the preferences of alternative polyA (pA) site, the biological processes involved, and the…

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Sunday, September 22, 2019

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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Sunday, September 22, 2019

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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Sunday, September 22, 2019

High-resolution comparative analysis of great ape genomes.

Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single- to mega-base pair-sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these…

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Sunday, September 22, 2019

Defining cell identity with single cell omics.

Cells are a fundamental unit of life, and the ability to study the phenotypes and behaviors of individual cells is crucial to understanding the workings of complex biological systems. Cell phenotypes (epigenomic, transcriptomic, proteomic, and metabolomic) exhibit dramatic heterogeneity between and within the different cell types and states underlying cellular functional diversity. Cell genotypes can also display heterogeneity throughout an organism, in the form of somatic genetic variation-most notably in the emergence and evolution of tumors. Recent technical advances in single-cell isolation and the development of omics approaches sensitive enough to reveal these aspects of cell identity have enabled a…

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Sunday, September 22, 2019

HapIso: An accurate method for the haplotype-specific isoforms reconstruction from long single-molecule reads

Sequencing of RNA provides the possibility to study an individual’s transcriptome landscape and determine allelic expression ratios. Single-molecule protocols generate multi-kilobase reads longer than most transcripts allowing sequencing of complete haplotype isoforms. This allows partitioning the reads into two parental haplotypes. While the read length of the single-molecule protocols is long, the relatively high error rate limits the ability to accurately detect the genetic variants and assemble them into the haplotype-specific isoforms. In this paper, we present HapIso (Haplotype-specific Isoform Reconstruction), a method able to tolerate the relatively high error-rate of the single-molecule platform and partition the isoform reads into…

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