scholarly journals Comprehensive profiling of TCRβ repertoire in a non-model species (the bank vole) using high-throughput sequencing

2017 ◽  
Author(s):  
Magdalena Migalska ◽  
Alvaro Sebastian ◽  
Jacek Radwan
Keyword(s):  
High Throughput ◽  
Bank Vole ◽  
High Throughput Sequencing ◽  
Length Distribution ◽  
Amino Acid Sequences ◽  
Myodes Glareolus ◽  
Model Species ◽  
Immune Repertoire ◽  
Important Species ◽  
Repertoire Sequencing

AbstractIn recent years, immune repertoire profiling with high-throughput sequencing (HTS) has advanced our understanding of adaptive immunity. However, fast progress in the field applied mostly to human and mouse research, with only few studies devoted to other model vertebrates. We present the first in-depth characterization of the TCRβ repertoire in a non-model mammal with limited genomic resources available – the bank vole (Myodes glareolus). We used 5′RACE and Illumina HTS to describe V and J segments and to qualitatively characterize preferential V–J segment usage and CDR3 length distribution. Finally, a molecular protocol integrating unique molecular identifiers was used for quantitative analysis of CDR3 repertoire with stringent error correction. We found 37 V and 11 J genes that were orthologous to mice genes. A conservative, lower bound estimation of the TCRβ repertoire was 1.7–2.3×105 clonotypes, and the degree of sharing of the observed repertoire between any two individuals was 3.6% of nucleotide sequences and 14.3% of amino acid sequences. Our work adds a crucial element to the immunogenetic resources available for the bank vole, an important species in ecological and evolutionary research. The workflow that we developed can be applied for immune repertoire sequencing of non-model species, including endangered vertebrates.

scholarly journals Population genomics of bank vole populations reveals associations between immune related genes and the epidemiology of Puumala hantavirus in Sweden

2017 ◽  
Author(s):  
Audrey Rohfritsch ◽  
Maxime Galan ◽  
Mathieu Gautier ◽  
Karim Gharbi ◽  
Gert Olsson ◽  
...  
Keyword(s):  
High Throughput ◽  
Bank Vole ◽  
High Throughput Sequencing ◽  
Population Genomics ◽  
Reservoir Host ◽  
Model Organisms ◽  
Myodes Glareolus ◽  
A Genome ◽  
Outlier Loci ◽  
Immune Related Genes

AbstractInfectious pathogens are major selective forces acting on individuals. The recent advent of high-throughput sequencing technologies now enables to investigate the genetic bases of resistance/susceptibility to infections in non-model organisms. From an evolutionary perspective, the analysis of the genetic diversity observed at these genes in natural populations provides insight into the mechanisms maintaining polymorphism and their epidemiological consequences. We explored these questions in the context of the interactions between Puumala hantavirus (PUUV) and its reservoir host, the bank vole Myodes glareolus. Despite the continuous spatial distribution of M. glareolus in Europe, PUUV distribution is strongly heterogeneous. Different defence strategies might have evolved in bank voles as a result of co-adaptation with PUUV, which may in turn reinforce spatial heterogeneity in PUUV distribution. We performed a genome scan study of six bank vole populations sampled along a North/South transect in Sweden, including PUUV endemic and non-endemic areas. We combined candidate gene analyses (Tlr4, Tlr7, Mx2 genes) and high throughput sequencing of RAD (Restriction-site Associated DNA) markers. We found evidence for outlier loci showing high levels of genetic differentiation. Ten outliers among the 52 that matched to mouse protein-coding genes corresponded to immune related genes and were detected using ecological associations with variations in PUUV prevalence. One third of the enriched pathways concerned immune processes, including platelet activation and TLR pathway. In the future, functional experimentations should enable to confirm the role of these these immune related genes with regard to the interactions between M. glareolus and PUUV.

scholarly journals First isolation and genetic characterization of Puumala orthohantavirus strains from France

2020 ◽  
Author(s):  
Johann Vulin ◽  
Séverine Murri ◽  
Sarah Madrières ◽  
Maxime Galan ◽  
Caroline Tatard ◽  
...  
Keyword(s):  
Bank Vole ◽  
Endemic Area ◽  
High Throughput Sequencing ◽  
Hemorrhagic Fever ◽  
Amino Acid Sequences ◽  
Myodes Glareolus ◽  
Wild Rodents ◽  
Bank Voles ◽  
A Cell ◽  
The Impact

AbstractPuumala orthohantavirus (PUUV) causes a mild form of hemorrhagic fever with renal syndrome (HFRS) named nephropathia epidemica (NE), regularly diagnosed in Europe. France represents the Western frontier of NE expansion in Europe with two distinct areas: the endemic area (Northeast) where PUUV circulates in rodent populations and where many cases of NE are detected in humans and non-endemic area (Southwest) where the virus is not detected and only a few human cases have been reported. The country is a pertinent target to study factors that influence the evolution of PUUV distribution. In this study, we describe for the first time the isolation of two PUUV strains from two distinct French geographical areas: Ardennes (endemic area) and Loiret (non-endemic area). To isolate PUUV efficiently, we selected wild rodents (Myodes glareolus, the specific reservoir of PUUV) from these areas that were seronegative for anti-PUUV IgG (ELISA) but associated with viral RNA load in lung (qRT-PCR). With this design, we are able to cultivate and maintain these two strains in VeroE6 cells but also to propagate efficiently and rapidly both strains in a bank vole colony. Complete coding sequences of S and M segments were determined by Sanger sequencing of RNA extracted from positive bank voles (naturally and experimentally infected) and from supernatant of Vero E6. For the M segment, nucleotidic sequences were 100% identical for both strains. For the S segment, the amino acid sequences from each strain revealed one mismatch between sequences obtained from tissue and from supernatant, revealing a “bank vole” and a “cell” signature. High throughput sequencing confirmed Sanger results, and provided a better assessment of the impact of isolation methods on intra-host viral diversity.

High-throughput sequencing of the immune repertoire in oncology: Applications for clinical diagnosis, monitoring, and immunotherapies

2018 ◽  
Vol 416 ◽  
pp. 42-56 ◽  
Author(s):  
Baixin Ye ◽  
Daniel Smerin ◽  
Qingping Gao ◽  
Chunsheng Kang ◽  
Xiaoxing Xiong
Keyword(s):  
Clinical Diagnosis ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Immune Repertoire

scholarly journals High-Throughput Sequencing-Based Immune Repertoire Study during Infectious Disease

2016 ◽  
Vol 7 ◽  
Author(s):  
Dongni Hou ◽  
Cuicui Chen ◽  
Eric John Seely ◽  
Shujing Chen ◽  
Yuanlin Song
Keyword(s):  
Infectious Disease ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Immune Repertoire

scholarly journals Single cell based high-throughput Ig and TCR repertoire sequencing analysis in rhesus macaques

2021 ◽  
Author(s):  
Evan S Walsh ◽  
Tammy Tollison ◽  
Hayden Brochu ◽  
Brian Shaw ◽  
Kayliegh Diveley ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
High Throughput ◽  
Rhesus Macaques ◽  
Expression Profiles ◽  
Single Cells ◽  
Model Organisms ◽  
Sequencing Analysis ◽  
Immune Repertoire ◽  
Repertoire Sequencing

Recent advancements in microfluidics and high-throughput sequencing technologies have enabled recovery of paired heavy- and light- chains of immunoglobulins (Ig) and VDJ- and VJ- chains of T cell receptors (TCR) from thousands of single cells simultaneously in humans and mice. Despite rhesus macaques being one of the most well-studied model organisms for the human adaptive immune response, high-throughput single cell immune repertoire sequencing assays are not yet available due to the complexity of these polyclonal receptors. Here we employed custom primers that capture all known rhesus macaque Ig and TCR isotypes and chains that are fully compatible with a commercial solution for single cell immune repertoire profiling. Using these rhesus specific assays, we sequenced Ig and TCR repertoires in over 60,000 cells from cryopreserved rhesus PBMC, splenocytes, and FACS-sorted B and T cells. We were able to recover every Ig isotype and TCR chain, measure clonal expansion in proliferating T cells, and pair Ig and TCR repertoires with gene expression profiles of the same single cells. Our results establish the ability to perform high-throughput immune repertoire analysis in rhesus macaques at the single cell level.

Qualifying high-throughput immune repertoire sequencing

2014 ◽  
Vol 288 (1-2) ◽  
pp. 31-38 ◽  
Author(s):  
Norbert Niklas ◽  
Johannes Pröll ◽  
Johannes Weinberger ◽  
Agnes Zopf ◽  
Karin Wiesinger ◽  
...  
Keyword(s):  
High Throughput ◽  
Immune Repertoire ◽  
Repertoire Sequencing

scholarly journals IGoR: a tool for high-throughput immune repertoire analysis

2017 ◽  
Author(s):  
Quentin Marcou ◽  
Thierry Mora ◽  
Aleksandra M. Walczak
Keyword(s):  
T Cell ◽  
B Cells ◽  
High Throughput ◽  
T Cell Receptors ◽  
Diagnostic Tools ◽  
Biological Complexity ◽  
Immune Repertoire ◽  
Repertoire Analysis ◽  
Repertoire Sequencing ◽  
Immune Repertoire Analysis

High throughput immune repertoire sequencing is promising to lead to new statistical diagnostic tools for medicine and biology. Successful implementations of these methods require a correct characterization, analysis and interpretation of these datasets. We present IGoR - a new comprehensive tool that takes B or T-cell receptors sequence reads and quantitatively characterizes the statistics of receptor generation from both cDNA and gDNA. It probabilistically annotates sequences and its modular structure can investigate models of increasing biological complexity for different organisms. For B-cells IGoR returns the hypermutation statistics, which we use to reveal co-localization of hypermutations along the sequence. We demonstrate that IGoR outperforms existing tools in accuracy and estimate the sample sizes needed for reliable repertoire characterization.

scholarly journals Immunoglobulin variable domain high-throughput sequencing reveals specific novel mutational patterns in POEMS syndrome

Blood ◽  
2020 ◽  
Vol 135 (20) ◽  
pp. 1750-1758 ◽  
Author(s):  
Sébastien Bender ◽  
Vincent Javaugue ◽  
Alexis Saintamand ◽  
Maria Victoria Ayala ◽  
Mehdi Alizadeh ◽  
...  
Keyword(s):  
Plasma Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Clinical Manifestations ◽  
Diagnostic Value ◽  
Poems Syndrome ◽  
Plasma Cell Dyscrasia ◽  
Multisystem Disease ◽  
General Improvement ◽  
Repertoire Sequencing

Abstract Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare multisystem disease resulting from 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 because of the highly restrictive usage of 2 λ variable (V) domains (IGLV1-40 and IGLV1-44) and the general improvement of clinical manifestations after PC clone-targeted treatment. However, the diagnostic value of Ig LC sequencing, especially in the case of incomplete forms of the disease, remains to be determined. Using a sensitive high-throughput Ig repertoire sequencing on RNA (rapid amplification of cDNA ends-based repertoire sequencing [RACE-RepSeq]), we detected a λ LC monoclonal expansion in the bone marrow (BM) of 83% of patients with POEMS syndrome, including some in whom BM tests routinely performed to diagnose plasma cell dyscrasia failed to detect λ+ monoclonal PCs. Twenty-four (83%) of the 29 LC clonal sequences found were derived from the IGLV1-40 and IGLV1-44 germline genes, as well as 2 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.

High throughput sequencing of the Angiostrongylus cantonensis genome: a parasite spreading worldwide

Parasitology ◽  
2013 ◽  
Vol 140 (10) ◽  
pp. 1304-1309 ◽  
Author(s):  
ALESSANDRA L. MORASSUTTI ◽  
ANDREY PERELYGIN ◽  
MARCOS O. DE CARVALHO ◽  
LEANDRO NASCIMENTO LEMOS ◽  
PAULO MARCOS PINTO ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Parasitic Nematode ◽  
Definitive Diagnosis ◽  
Amino Acid Sequences ◽  
Angiostrongylus Cantonensis ◽  
Putative Orfs ◽  
Protein Targets ◽  
Antigenic Protein ◽  
Molecular Information

SUMMARYAngiostrongylus cantonensis is a parasitic nematode of rodents and a leading aetiological agent of eosinophilic meningitis in humans. Definitive diagnosis is difficult, often relying on immunodiagnostic methods which utilize crude antigens. New immunodiagnostic methods based on recombinant proteins are being developed, and ideally these methods would be made available worldwide. Identification of diagnostic targets, as well as studies on the biology of the parasite, are limited by a lack of molecular information on Angiostrongylus spp. available in databases. In this study we present data collected from DNA random high-throughput sequencing together with proteomic analyses and a cDNA walking methodology to identify and obtain the nucleotide or amino acid sequences of unknown immunoreactive proteins. 28 080 putative ORFs were obtained, of which 3371 had homology to other deposited protein sequences. Using the A. cantonensis genomic sequences, 156 putative ORFs, matching peptide sequences obtained from previous proteomic studies, were considered novel, with no homology to existing sequences. Full-length coding sequences of eight antigenic target proteins were obtained. In this study we generated not only the complete nucleotide sequences of the antigenic protein targets but also a large amount of genomic data which may help facilitate future genomic, proteomic, transcriptomic or metabolomic studies on Angiostrongylus.

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