scholarly journals The use of non-functional clonotypes as a natural spike-in for multiplex PCR bias correction in immune receptor repertoire profiling

2021 ◽  
Author(s):  
Alexander Komkov ◽  
Anastasia Smirnova ◽  
Anna Miroshnichenkova ◽  
Egor Volchkov ◽  
Yulia Olshanskaya ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
High Throughput Sequencing ◽  
Quantitative Measure ◽  
Immune Receptor ◽  
The Past ◽  
Receptor Repertoire ◽  
Amplification Rate ◽  
Rapid Amplification ◽  
Lymphocyte Clone ◽  
Bias Evaluation

AbstractHigh-throughput sequencing of immune receptor repertoires is a valuable tool for receiving insights in adaptive immunity studies. Several powerful methods for TCR/BCR repertoire reconstruction and analysis have been developed in the past decade. However, detection and correction of the discrepancy between real and experimentally observed lymphocyte clone frequencies are still challenging. Here we formulated a quantitative measure of V- and J-genes frequency bias driven by multiplex PCR during library preparation called Over Amplification Rate (OAR). Based on OAR concept, we developed an original software for multiplex PCR-specific bias evaluation and correction named iROAR: Immune Repertoire Over Amplification Removal (https://github.com/smiranast/iROAR). The iROAR algorithm was successfully tested on previously published TCR repertoires obtained using both 5’ RACE (Rapid Amplification of cDNA Ends)-based and multiplex PCR-based approaches. The developed tool can be used to increase the accuracy and consistency of repertoires reconstructed by different methods making them more applicable for comparative analysis.

Ultra-high-throughput sequencing of the immune receptor repertoire from millions of lymphocytes

2016 ◽  
Vol 11 (3) ◽  
pp. 429-442 ◽  
Author(s):  
Jonathan R McDaniel ◽  
Brandon J DeKosky ◽  
Hidetaka Tanno ◽  
Andrew D Ellington ◽  
George Georgiou
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Immune Receptor ◽  
Receptor Repertoire

scholarly journals OGRDB: a reference database of inferred immune receptor genes

2019 ◽  
Vol 48 (D1) ◽  
pp. D964-D970 ◽  
Author(s):  
William Lees ◽  
Christian E Busse ◽  
Martin Corcoran ◽  
Mats Ohlin ◽  
Cathrine Scheepers ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Reference Database ◽  
Supporting Evidence ◽  
Immune Receptor ◽  
Expert Review ◽  
Adaptive Immune ◽  
Receptor Repertoire ◽  
Public Resource ◽  
Animal Populations ◽  
Genomic Regions

Abstract High-throughput sequencing of the adaptive immune receptor repertoire (AIRR-seq) is providing unprecedented insights into the immune response to disease and into the development of immune disorders. The accurate interpretation of AIRR-seq data depends on the existence of comprehensive germline gene reference sets. Current sets are known to be incomplete and unrepresentative of the degree of polymorphism and diversity in human and animal populations. A key issue is the complexity of the genomic regions in which they lie, which, because of the presence of multiple repeats, insertions and deletions, have not proved tractable with short-read whole genome sequencing. Recently, tools and methods for inferring such gene sequences from AIRR-seq datasets have become available, and a community approach has been developed for the expert review and publication of such inferences. Here, we present OGRDB, the Open Germline Receptor Database (https://ogrdb.airr-community.org), a public resource for the submission, review and publication of previously unknown receptor germline sequences together with supporting evidence.

Efficient High-throughput Techniques for the Analysis of Disease-Resistant Plant Varieties and Detection of Food Adulteration

2021 ◽  
Vol 23 ◽  
Author(s):  
Romesh Kumar Salgotra ◽  
Rafiq Ahmad Bhat ◽  
Deyue Yu ◽  
Javaid Akhter Bhat
Keyword(s):  
Multiplex Pcr ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Crop Improvement ◽  
Small Sample ◽  
Food Crops ◽  
Genomic Resources ◽  
Breeding Programmes ◽  
Next Generation Sequencing Ngs

Abstract: Over the past two decades, the advances in the next generation sequencing (NGS) platforms have led to the identification of numerous genes/QTLs at high-resolution for their potential use in crop improvement. The genomic resources generated through these high-throughput sequencing techniques have been efficiently used in screening of particular gene of interest particularly for numerous types of plant stresses and quality traits. Subsequently, the identified-markers linked to a particular trait have been used in marker-assisted backcross breeding (MABB) activities. Besides, these markers are also being used to catalogue the food crops for detection of adulteration to improve the quality of food. With the advancement of technologies, the genomic resources are originating with new markers; however, to use these markers efficiently in crop breeding, high-throughput techniques (HTT) such as multiplex PCR and capillary electrophoresis (CE) can be exploited. Robustness, ease of operation, good reproducibility and low cost are the main advantages of multiplex PCR and CE. The CE is capable of separating and characterizing proteins with simplicity, speed and small sample requirements. Keeping in view the availability of vast data generated through NGS techniques and development of numerous markers, there is a need to use these resources efficiently in crop improvement programmes. In summary, this review describes the use of molecular markers in the screening of resistance genes in breeding programmes and detection of adulterations in food crops using high-throughput techniques.

scholarly journals Assessing pollution of aquatic environments with diatoms’ DNA metabarcoding: experience and developments from France water framework directive networks

2019 ◽  
Vol 3 ◽  
Author(s):  
Vasselon Valentin ◽  
Rimet Frédéric ◽  
Domaizon Isabelle ◽  
Monnier Olivier ◽  
Reyjol Yorick ◽  
...  
Keyword(s):  
Water Framework Directive ◽  
High Throughput Sequencing ◽  
Ecological Status ◽  
Aquatic Environments ◽  
Morphological Identification ◽  
The Past ◽  
Sequencing Technologies ◽  
Dna Metabarcoding ◽  
Morphological Approach ◽  
Status Assessment

Ecological status assessment of watercourses is based on the calculation of quality indices using pollution sensitivity of targeted biological groups, including diatoms. The determination and quantification of diatom species is generally based on microscopic morphological identification, which requires expertise and is time-consuming and costly. In Europe, this morphological approach is legally imposed by standards and regulatory decrees by the Water Framework Directive (WFD). Over the past decade, a DNA-based molecular biology approach has newly been developed to identify species based on genetic criteria rather than morphological ones (i.e. DNA metabarcoding). In combination with high throughput sequencing technologies, metabarcoding makes it possible both to identify all species present in an environmental sample and to process several hundred samples in parallel. This article presents the results of two recent studies carried out on the WFD networks of rivers of Mayotte (2013–2018) and metropolitan France (2016–2018). These studies aimed at testing the potential application of metabarcoding for biomonitoring in the context of the WFD. We discuss the various methodological developments and optimisations that have been made to make the taxonomic inventories of diatoms produced by metabarcoding more reliable, particularly in terms of species quantification. We present the results of the application of this DNA approach on more than 500 river sites, comparing them with those obtained using the standardised morphological method. Finally, we discuss the potential of metabarcoding for routine application, its limits of application and propose some recommendations for future implementation in WFD.

scholarly journals Complete genome sequence of GII.9 norovirus

2021 ◽  
Author(s):  
Zilong Zhang ◽  
Danlei Liu ◽  
Zilei Zhang ◽  
Peng Tian ◽  
Shenwei Li ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
High Throughput Sequencing ◽  
Open Reading Frames ◽  
Viral Sequence ◽  
Sequence Comparisons ◽  
Rapid Amplification ◽  
Genome Level ◽  
Reading Frames

AbstractNorovirus is recognized as one of the leading causes of acute gastroenteritis outbreaks. Genotype GII.9 was first detected in Norfolk, VA, USA, in 1997. However, the complete genome sequence of this genotype has not yet been determined. In this study, a complete genome sequence of GII.9[P7] norovirus, SCD1878_GII.9[P7], from a patient was determined using high-throughput sequencing and rapid amplification of cDNA ends (RACE) technology. The complete genome sequence of SCD1878_GII.9[P7] is 7544 nucleotides (nt) in length with a 3’ poly(A) tail and contains three open reading frames. Sequence comparisons indicated that SCD1878_GII.9[P7] shares 92.1%-92.3% nucleotide sequence identity with GII.P7 (AB258331 and AB039777) and 96.7%-97.4% identity with GII.9 (AY038599 and DQ379715). The results suggested that SCD1878_GII.9[P7] is a member of P genotype GII.P7 and G genotype GII.9. This viral sequence fills a gap at the whole-genome level for the GII.9 genotype.

scholarly journals The Bayesian optimist's guide to adaptive immune receptor repertoire analysis

2018 ◽  
Vol 284 (1) ◽  
pp. 148-166 ◽  
Author(s):  
Branden J. Olson ◽  
Frederick A. Matsen
Keyword(s):  
Immune Receptor ◽  
Adaptive Immune ◽  
Receptor Repertoire ◽  
Repertoire Analysis

scholarly journals Strategies for Building Computing Skills To Support Microbiome Analysis: a Five-Year Perspective from the EDAMAME Workshop

mSystems ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Ashley Shade ◽  
Taylor K. Dunivin ◽  
Jinlyung Choi ◽  
Tracy K. Teal ◽  
Adina C. Howe
Keyword(s):  
High Throughput Sequencing ◽  
Formal Training ◽  
Sufficient Time ◽  
Educational Approach ◽  
Research Staff ◽  
Microbiome Analysis ◽  
The Past ◽  
Computing Skills ◽  
Professional Learners ◽  
Training Opportunities

ABSTRACT Here, we report our educational approach and learner evaluations of the first 5 years of the Explorations in Data Analysis for Metagenomic Advances in Microbial Ecology (EDAMAME) workshop, held annually at Michigan State University’s Kellogg Biological Station from 2014 to 2018. We hope this information will be useful for others who want to organize computing-intensive workshops and will encourage quantitative skill development among microbiologists. IMPORTANCE High-throughput sequencing and related statistical and bioinformatic analyses have become routine in microbiology in the past decade, but there are few formal training opportunities to develop these skills. A weeklong workshop can offer sufficient time for novices to become introduced to best computing practices and common workflows in sequence analysis. We report our experiences in executing such a workshop targeted to professional learners (graduate students, postdoctoral scientists, faculty, and research staff).

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