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Nature Methods Jun 2021Cell atlas projects and high-throughput perturbation screens require single-cell sequencing at a scale that is challenging with current technology. To enable...
Cell atlas projects and high-throughput perturbation screens require single-cell sequencing at a scale that is challenging with current technology. To enable cost-effective single-cell sequencing for millions of individual cells, we developed 'single-cell combinatorial fluidic indexing' (scifi). The scifi-RNA-seq assay combines one-step combinatorial preindexing of entire transcriptomes inside permeabilized cells with subsequent single-cell RNA-seq using microfluidics. Preindexing allows us to load several cells per droplet and computationally demultiplex their individual expression profiles. Thereby, scifi-RNA-seq massively increases the throughput of droplet-based single-cell RNA-seq, and provides a straightforward way of multiplexing thousands of samples in a single experiment. Compared with multiround combinatorial indexing, scifi-RNA-seq provides an easy and efficient workflow. Compared to cell hashing methods, which flag and discard droplets containing more than one cell, scifi-RNA-seq resolves and retains individual transcriptomes from overloaded droplets. We benchmarked scifi-RNA-seq on various human and mouse cell lines, validated it for primary human T cells and applied it in a highly multiplexed CRISPR screen with single-cell transcriptome readout of T cell receptor activation.
Topics: Animals; Cell Line; Clustered Regularly Interspaced Short Palindromic Repeats; Cost-Benefit Analysis; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; Humans; Mice; Microfluidics; Receptors, Antigen, T-Cell; Single-Cell Analysis; Transcriptome
PubMed: 34059827
DOI: 10.1038/s41592-021-01153-z -
STAR Protocols Sep 2021The protocol allows for labeling nascent RNA without isolating nuclei. The cell-permeable uridine analog, 5-ethynyluridine (EU), is added to media to allow labeling of...
The protocol allows for labeling nascent RNA without isolating nuclei. The cell-permeable uridine analog, 5-ethynyluridine (EU), is added to media to allow labeling of nascent transcripts. Cells are lysed, total RNA is collected, and biotin is conjugated to EU-labeled RNAs. Custom biotin RNAs are added and biotinylated RNAs are isolated for generation of cDNA libraries. The sequencing data are normalized to controls for quantitative assessment of the nascent transcriptome. The protocol takes 4 days, not including sequencing and analysis. For complete details on the use and execution of this protocol, please refer to Palozola et al. (2017).
Topics: Biotin; Cell Line; Chemical Precipitation; High-Throughput Nucleotide Sequencing; Humans; Molecular Biology; RNA; RNA-Seq; Uridine
PubMed: 34485932
DOI: 10.1016/j.xpro.2021.100651 -
Acta Biochimica Et Biophysica Sinica May 2022High-throughput sequencing for B cell receptor (BCR) repertoire provides useful insights for the adaptive immune system. With the continuous development of the BCR-seq... (Review)
Review
High-throughput sequencing for B cell receptor (BCR) repertoire provides useful insights for the adaptive immune system. With the continuous development of the BCR-seq technology, many efforts have been made to develop methods for analyzing the ever-increasing BCR repertoire data. In this review, we comprehensively outline different BCR repertoire library preparation protocols and summarize three major steps of BCR-seq data analysis, . ., V(D)J sequence annotation, clonal phylogenetic inference, and BCR repertoire profiling and mining. Different from other reviews in this field, we emphasize background intuition and the statistical principle of each method to help biologists better understand it. Finally, we discuss data mining problems for BCR-seq data and with a highlight on recently emerging multiple-sample analysis.
Topics: Cells, Cultured; High-Throughput Nucleotide Sequencing; Phylogeny; Receptors, Antigen, B-Cell
PubMed: 35713313
DOI: 10.3724/abbs.2022062 -
Digestive Diseases and Sciences Mar 2020Over the past decade, it has become exceedingly clear that the microbiome is a critical factor in human health and disease and thus should be investigated to develop... (Review)
Review
Over the past decade, it has become exceedingly clear that the microbiome is a critical factor in human health and disease and thus should be investigated to develop innovative treatment strategies. The field of metagenomics has come a long way in leveraging the advances of next-generation sequencing technologies resulting in the capability to identify and quantify all microorganisms present in human specimens. However, the field of metagenomics is still in its infancy, specifically in regard to the limitations in computational analysis, statistical assessments, standardization, and validation due to vast variability in the cohorts themselves, experimental design, and bioinformatic workflows. This review summarizes the methods, technologies, computational tools, and model systems for characterizing and studying the microbiome. We also discuss important considerations investigators must make when interrogating the involvement of the microbiome in health and disease in order to establish robust results and mechanistic insights before moving into therapeutic design and intervention.
Topics: Animals; Computational Biology; High-Throughput Nucleotide Sequencing; Humans; Machine Learning; Metagenomics; Microbiota; Sequence Analysis, DNA
PubMed: 32002757
DOI: 10.1007/s10620-020-06091-y -
Human Mutation Aug 2022The success of many clinical, association, or population genetics studies critically relies on properly performed variant calling step. The variety of modern genomics... (Review)
Review
The success of many clinical, association, or population genetics studies critically relies on properly performed variant calling step. The variety of modern genomics protocols, techniques, and platforms makes our choices of methods and algorithms difficult and there is no "one size fits all" solution for study design and data analysis. In this review, we discuss considerations that need to be taken into account while designing the study and preparing for the experiments. We outline the variety of variant types that can be detected using sequencing approaches and highlight some specific requirements and basic principles of their detection. Finally, we cover interesting developments that enable variant calling for a broad range of applications in the genomics field. We conclude by discussing technological and algorithmic advances that have the potential to change the ways of calling DNA variants in the nearest future.
Topics: Algorithms; Genomics; High-Throughput Nucleotide Sequencing; Humans; Polymorphism, Single Nucleotide; Sequence Analysis, DNA; Software
PubMed: 34882898
DOI: 10.1002/humu.24311 -
Bioinformatics (Oxford, England) Apr 2022Over the past decade, short-read sequence alignment has become a mature technology. Optimized algorithms, careful software engineering and high-speed hardware have... (Review)
Review
SUMMARY
Over the past decade, short-read sequence alignment has become a mature technology. Optimized algorithms, careful software engineering and high-speed hardware have contributed to greatly increased throughput and accuracy. With these improvements, many opportunities for performance optimization have emerged. In this review, we examine three general-purpose short-read alignment tools-BWA-MEM, Bowtie 2 and Arioc-with a focus on performance optimization. We analyze the performance-related behavior of the algorithms and heuristics each tool implements, with the goal of arriving at practical methods of improving processing speed and accuracy. We indicate where an aligner's default behavior may result in suboptimal performance, explore the effects of computational constraints such as end-to-end mapping and alignment scoring threshold, and discuss sources of imprecision in the computation of alignment scores and mapping quality. With this perspective, we describe an approach to tuning short-read aligner performance to meet specific data-analysis and throughput requirements while avoiding potential inaccuracies in subsequent analysis of alignment results. Finally, we illustrate how this approach avoids easily overlooked pitfalls and leads to verifiable improvements in alignment speed and accuracy.
CONTACT
SUPPLEMENTARY INFORMATION
Appendices referenced in this article are available at Bioinformatics online.
Topics: Sequence Analysis, DNA; High-Throughput Nucleotide Sequencing; Software; Algorithms; Sequence Alignment
PubMed: 35139149
DOI: 10.1093/bioinformatics/btac066 -
Cellular and Molecular Life Sciences :... Aug 2022Single-cell sequencing is widely used in biological and medical studies. However, its application with multiple samples is hindered by inefficient sample processing,... (Review)
Review
Single-cell sequencing is widely used in biological and medical studies. However, its application with multiple samples is hindered by inefficient sample processing, high experimental costs, ambiguous identification of true single cells, and technical batch effects. Here, we introduce sample-multiplexing approaches for single-cell sequencing in transcriptomics, epigenomics, genomics, and multiomics. In single-cell transcriptomics, sample multiplexing uses variants of native or artificial features as sample markers, enabling sample pooling and decoding. Such features include: (1) natural genetic variation, (2) nucleotide-barcode anchoring on cellular or nuclear membranes, (3) nucleotide-barcode internalization to the cytoplasm or nucleus, (4) vector-based barcode expression in cells, and (5) nucleotide-barcode incorporation during library construction. Other single-cell omics methods are based on similar concepts, particularly single-cell combinatorial indexing. These methods overcome current challenges, while enabling super-loading of single cells. Finally, selection guidelines are presented that can accelerate technological application.
Topics: Epigenomics; Genomics; High-Throughput Nucleotide Sequencing; Nucleotides; Single-Cell Analysis
PubMed: 35927335
DOI: 10.1007/s00018-022-04482-0 -
Experimental & Molecular Medicine Oct 2022To date, more than 170 chemical modifications have been characterized in RNA, providing a new layer of gene expression regulation termed the 'epitranscriptome'. RNA... (Review)
Review
To date, more than 170 chemical modifications have been characterized in RNA, providing a new layer of gene expression regulation termed the 'epitranscriptome'. RNA modification detection methods and tools advance the functional studies of the epitranscriptome. According to the detection throughput and principles, existing RNA modification detection technologies can be categorized into four classes, including quantification methods, locus-specific detection methods, next-generation sequencing-based detection technologies and nanopore direct RNA sequencing-based technologies. In this review, we summarize the current knowledge about these RNA modification detection technologies and discuss the challenges for the existing detection tools, providing information for a comprehensive understanding of the epitranscriptome.
Topics: RNA; High-Throughput Nucleotide Sequencing; Sequence Analysis, RNA; Gene Expression Regulation
PubMed: 36266445
DOI: 10.1038/s12276-022-00821-0 -
Trends in Biotechnology Sep 2019A substantial amount of structural variation in the human genome remains uninvestigated due to the limitations of existing technologies, the presence of repetitive... (Review)
Review
A substantial amount of structural variation in the human genome remains uninvestigated due to the limitations of existing technologies, the presence of repetitive sequences, and the complexity of a diploid genome. New technologies have been developed, increasing resolution and appreciation of structural variation and how it affects human diversity and disease. The genetic etiology of most patients with complex disorders such as neurodegenerative brain diseases is not yet elucidated, complicating disease diagnosis, genetic counseling, and understanding of underlying pathological mechanisms needed to develop therapeutic interventions. Here, we focus on innovative progress and opportunities provided by the newest methods such as linked read sequencing, strand-specific sequencing, and long-read sequencing. Finally, we describe a strategy for generating a comprehensive catalog of structural variations across populations.
Topics: Base Sequence; Genetic Variation; Genome, Human; High-Throughput Nucleotide Sequencing; Humans; Sequence Analysis, DNA
PubMed: 30902345
DOI: 10.1016/j.tibtech.2019.02.003 -
Trends in Biotechnology Feb 2020The spatial distribution of molecules and cells is fundamental to understanding biological systems. Traditionally, microscopies based on electromagnetic waves such as... (Review)
Review
The spatial distribution of molecules and cells is fundamental to understanding biological systems. Traditionally, microscopies based on electromagnetic waves such as visible light have been used to localize cellular components by direct visualization. However, these techniques suffer from limitations of transmissibility and throughput. Complementary to optical approaches, biochemical techniques such as crosslinking can colocalize molecules without suffering the same limitations. However, biochemical approaches are often unable to combine individual colocalizations into a map across entire cells or tissues. Microscopy-by-sequencing techniques aim to biochemically colocalize DNA-barcoded molecules and, by tracking their thus unique identities, reconcile all colocalizations into a global spatial map. Here, we review this new field and discuss its enormous potential to answer a broad spectrum of questions.
Topics: DNA Barcoding, Taxonomic; High-Throughput Nucleotide Sequencing; Microscopy; Molecular Imaging
PubMed: 31416630
DOI: 10.1016/j.tibtech.2019.06.001