scholarly journals Single‐cell based high‐throughput sequencing of full‐length immunoglobulin heavy and light chain genes

2013 ◽  
Vol 44 (2) ◽  
pp. 597-603 ◽  
Author(s):  
Christian E. Busse ◽  
Irina Czogiel ◽  
Peter Braun ◽  
Peter F. Arndt ◽  
Hedda Wardemann
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Light Chain ◽  
High Throughput Sequencing ◽  
Full Length

scholarly journals Optimization of single-cell plate sorting for high throughput sequencing applications

2019 ◽  
Vol 466 ◽  
pp. 17-23 ◽  
Author(s):  
Lauren E. Higdon ◽  
Corey J. Cain ◽  
Melissa A. Colden ◽  
Jonathan S. Maltzman
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cell Plate

scholarly journals Use of high throughput sequencing to observe genome dynamics at a single cell level

2009 ◽  
Vol 106 (49) ◽  
pp. 20830-20835 ◽  
Author(s):  
D. Parkhomchuk ◽  
V. Amstislavskiy ◽  
A. Soldatov ◽  
V. Ogryzko
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Single Cell Level ◽  
Cell Level ◽  
Genome Dynamics

scholarly journals Novel Method for High-Throughput Full-Length IGHV-D-J Sequencing of the Immune Repertoire from Bulk B-Cells with Single-Cell Resolution

2017 ◽  
Vol 8 ◽  
Author(s):  
Stefano Vergani ◽  
Ilya Korsunsky ◽  
Andrea Nicola Mazzarello ◽  
Gerardo Ferrer ◽  
Nicholas Chiorazzi ◽  
...  
Keyword(s):  
B Cells ◽  
Single Cell ◽  
High Throughput ◽  
Full Length ◽  
Immune Repertoire ◽  
Novel Method

scholarly journals Unraveling the Heterogeneity and Ontogeny of Dendritic Cells Using Single-Cell RNA Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Binyao Chen ◽  
Lei Zhu ◽  
Shizhao Yang ◽  
Wenru Su
Keyword(s):  
Dendritic Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Adaptive Immunity ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Future Trends ◽  
Innate And Adaptive Immunity ◽  
Sequencing Technologies ◽  
Single Cell Rna Sequencing

Dendritic cells (DCs) play essential roles in innate and adaptive immunity and show high heterogeneity and intricate ontogeny. Advances in high-throughput sequencing technologies, particularly single-cell RNA sequencing (scRNA-seq), have improved the understanding of DC subsets. In this review, we discuss in detail the remarkable perspectives in DC reclassification and ontogeny as revealed by scRNA-seq. Moreover, the heterogeneity and multifunction of DCs during diseases as determined by scRNA-seq are described. Finally, we provide insights into the challenges and future trends in scRNA-seq technologies and DC research.

scholarly journals Full-length immunoglobulin high-throughput sequencing reveals specific novel mutational patterns in POEMS syndrome

2019 ◽  
Author(s):  
Sébastien Bender ◽  
Vincent Javaugue ◽  
Alexis Saintamand ◽  
Maria Victoria Ayala ◽  
Mehdi Alizadeh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Plasma Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Clinical Manifestations ◽  
Diagnostic Value ◽  
Poems Syndrome ◽  
Full Length ◽  
Plasma Cell Dyscrasia ◽  
Multisystem Disease

AbstractPOEMS syndrome is a rare multisystem disease due to an underlying plasma cell (PC) dyscrasia. The pathophysiology of the disease remains unclear but the role of the monoclonal immunoglobulin (Ig) light chain (LC) is strongly suspected, due to the highly restrictive usage of two λ variable (V) domains (IGLV1-40 and IGLV1-44) and the general improvement of clinical manifestations following PC clone-targeted treatment. However, the diagnostic value of Ig LC sequencing, especially in case of incomplete forms of the disease, remains to be determined. Using a sensitive high-throughput Ig repertoire sequencing on RNA (RACE-RepSeq), we detected a λ LC monoclonal expansion in the bone marrow (BM) of 85% of patients with POEMS syndrome, including some in whom bone marrow tests routinely performed to diagnose plasma cell dyscrasia failed to detect λ+ monoclonal PCs. Twenty-four of the 30 LC clonal sequences found (80%) were derived from the IGLV1-40 and IGLV1-44 germline genes, two from the closely related IGLV1-36 gene, and all were associated with an IGLJ3*02 junction (J) gene, confirming the high restriction of VJ region usage in POEMS syndrome. RACE-RepSeq VJ full-length sequencing additionally revealed original mutational patterns, the strong specificity of which might crucially help establish or eliminate the diagnosis of POEMS syndrome in uncertain cases. Thus, RACE-RepSeq appears as a sensitive, rapid and specific tool to detect low-abundance PC clones in BM, and assign them to POEMS syndrome, with all the consequences for therapeutic options hereby.

scholarly journals High-Throughput BCR Sequencing and Single-Cell Transcriptomics Reveal Distinct Transcriptional Profiles Associated with Subclonal Evolution of Follicular Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 298-298
Author(s):  
Cornelis A.M. van Bergen ◽  
Marvyn T. Koning ◽  
Edwin Quinten ◽  
Agnieszka Mykowiecka ◽  
Julieta Sepulveda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lymph Node ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Single Cell ◽  
High Throughput ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Full Length

Objectives: Follicular lymphoma (FL) typically originates from premalignant mature B cells that carry the founder t(14;18) BCL2 translocation. Mutations in epigenetic modifiers and acquisition of N-glycosylation sites in CDR regions of the B-cell receptor (BCR) are recurrent secondary events in FL pathogenesis. Despite these oncogenic drivers, FL can remain indolent and clinically stable for years. The molecular events driving subclonal evolution into symptomatic progression and eventual transformation to aggressive lymphoma are insufficiently understood. FL cells are frozen in their B-cell development at the germinal center stage and undergo continuous somatic hypermutation mediated by expression of activation-induced deaminase (AID). We aim to identify crucial drivers of subclonal FL evolution by high-throughput mapping at single-cell resolution. Methods: Viable FL cells were isolated and cryopreserved from 23 histologically or immunocytologically confirmed FL samples from 13 patients with informed consent. Full-length VDJ/VJ transcripts were isolated by unbiased template-switching ARTISAN PCR and massive parallel NGS sequencing on the PacBio platform. The clonal primordial FL BCR (pBCR) was reconstructed from unmutated IGV/IGJ sequences with the CDR3 of the least mutated BCR. Since the IgTree program was unable to process the obtained numbers of BCR sequences, we developed the WILLOW algorithm for analysis of BCR evolution based on the principle of maximum parsimony and on distance from the pBCR. Intraclonal BCR variability was quantified by Shannon's diversity index. 5' single cell transcriptomics and VDJ/VJ sequencing was performed on 2 pools of highly purified FL cells from 5 lymph node biopsies on the 10x Genomics platform. Data were deconvoluted based on expressed variants by the Single Cell Sample Matcher (SCSM) algorithm. Clustering based on gene expression profiles was performed by shared nearest neighbour (SNN) modularity optimization within the R Seurat package. Genes whose expression differed significantly (adjusted p<0.05) between clusters were assigned to gene ontology terms. Results: ARTISAN PCR/PacBio NGS yielded a median of 743 full-length VDJ and VJ sequences (range 62-12782) per BCR chain with expected high intraclonal diversity (median 200 subclones, range 15-3301). WILLOW revealed dominant FL subclones with a subclonal hierarchy wherein multiple routes converged to offspring nodes with identical additional mutations rather than tree-like branching (Figure). In serial samples of 4 patients, lymph node biopsies had only marginally higher subclonal diversity than blood or bone marrow samples (p=0,055; Wilcoxon's matched-pairs signed rank test). Overall BCR mutational burden increased over time in sequential biopsies. Two cases of histological FL transformation were dominated by a single subclone (65% and 80% of all VDJ/VJ sequences, respectively) that was rare in the preceding FL BCR network (0.2% and 1.8%). Pooled transcriptomics data from 6050-6500 cells were assigned to individual samples by SCSM and revealed up to seven transcriptional clusters per FL. In 9 of 10 FL, genes assigned to immune function strongly contributed to separation into one or more clusters. Single cell VDJ/VJ sequencing yielded combined heavy and light chain BCR sequences for a median of 502 FL cells per biopsy (range 22 - 1919) that permitted mapping of subclonal evolution by WILLOW based on complete BCR information. Transcriptome clusters were not distributed evenly throughout the WILLOW FL BCR networks but rather statistically associated with distinct major FL subclones. Vice versa, major FL subclones within the same biopsy were distinguished by particular gene expression profiles. Conclusions: WILLOW facilitates mapping of subclonal FL evolution based on high-throughput BCR sequencing. FL evolution proceeds in networks rather than tree-like branching, whereby acquisition of certain combinations of several BCR mutations can occur in parallel in different trajectories. Transcriptomic profiling of single FL cells identifies distinct clusters within a single biopsy. Mapping of these clusters to the FL cell position in the subclonal FL evolutionary network identifies putative mechanisms that are associated with subclonal progression. These mechanisms involve physiological B-cell signalling pathways. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals A Scalable Strand-Specific Protocol Enabling Full-Length Total RNA Sequencing From Single Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Simon Haile ◽  
Richard D. Corbett ◽  
Veronique G. LeBlanc ◽  
Lisa Wei ◽  
Stephen Pleasance ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput Sequencing ◽  
Single Cells ◽  
Cell Types ◽  
Full Length ◽  
Sequencing Data ◽  
Total Rna ◽  
Specific Protocol

RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3′ or 5′ termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.

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