scholarly journals High-throughput sequencing of murine immunoglobulin heavy chain transcripts using single side unique molecular identifiers on an Ion Torrent PGM

2017 ◽  
Author(s):  
Jean-Philippe Bürckert ◽  
William J. Faison ◽  
Axel R. S. X. Dubois ◽  
Regina Sinner ◽  
Oliver Hunewald ◽  
...  
Keyword(s):  
High Throughput ◽  
Heavy Chain ◽  
High Throughput Sequencing ◽  
Gene Segment ◽  
Personal Genome ◽  
Sequencing Platform ◽  
Ion Torrent Pgm ◽  
Indel Detection ◽  
Processing Strategies ◽  
Nucleotide Insertions

AbstractWith the advent of high-throughput sequencing (HTS), profiling immunoglobulin (IG) repertoires has become an essential part of immunological research. Advances in sequencing technology enable the IonTorrent Personal Genome Machine (PGM) to cover the full-length of IG mRNA transcripts. Nucleotide insertions and deletions (indels) are the dominant errors of the PGM sequencing platform and can critically influence IG repertoire assessments. Here, we present a PGM-tailored IG repertoire sequencing approach combining error correction through unique molecular identifier (UID) barcoding and indel detection through ImMunoGeneTics (IMGT), the most commonly used sequence alignment database for IG sequences. Using artificially falsified sequences for benchmarking, we found that IMGT efficiently detects 98% of the introduced indels through gene-segment frameshifts. Undetected indels are either located at the ends of the sequences or produce masked frameshifts with an insertion and deletion in close proximity. IMGT’s indel correction algorithm resolves up to 87% of the tested insertions, but no deletions. The complementary determining regions 3 (CDR3s) are returned 100% correct for up to 3 insertions or 3 deletions through conservative culling. We further show, that our PGM-tailored unique molecular identifiers results in highly accurate HTS datasets if combined with the presented data processing. In this regard, considering sequences with at least two copies from datasets with UID families of minimum 3 reads result in correct sequences with over 99% confidence. The protocol and sample processing strategies described in this study will help to establish benchtop-scale sequencing of IG heavy chain transcripts in the field of IG repertoire research.

scholarly journals High-throughput sequencing of murine immunoglobulin heavy chain repertoires using single side unique molecular identifiers on an Ion Torrent PGM

Oncotarget ◽  
2018 ◽  
Vol 9 (54) ◽  
pp. 30225-30239 ◽  
Author(s):  
Jean-Philippe Bürckert ◽  
William J. Faison ◽  
Danielle E. Mustin ◽  
Axel R.S.X. Dubois ◽  
Regina Sinner ◽  
...  
Keyword(s):  
High Throughput ◽  
Heavy Chain ◽  
High Throughput Sequencing ◽  
Immunoglobulin Heavy Chain ◽  
Ion Torrent ◽  
Ion Torrent Pgm ◽  
Single Side

scholarly journals Comparisons of the antibody repertoires of a humanized rodent and humans by high throughput sequencing

2019 ◽  
Author(s):  
Collin Joyce ◽  
Dennis R. Burton ◽  
Bryan Briney
Keyword(s):  
High Throughput ◽  
Heavy Chain ◽  
High Throughput Sequencing ◽  
Somatic Hypermutation ◽  
Gene Segment ◽  
Great Promise ◽  
Antibody Diversity ◽  
Model Animal ◽  
Kappa Chain ◽  
Antibody Repertoires

ABSTRACTThe humanization of animal model immune systems by genetic engineering has shown great promise for antibody discovery, tolerance studies and for the evaluation of vaccines. Assessment of the baseline antibody repertoire of unimmunized model animals will be useful as a benchmark for future immunization experiments. We characterized the heavy chain and kappa chain antibody repertoires of a model animal, the OmniRat, by high throughput antibody sequencing and made use of two novel datasets for comparison to human repertoires. Intra-animal and inter-animal repertoire comparisons reveal a high level of conservation in antibody diversity between the lymph node and spleen and between members of the species. Multiple differences were found in both the heavy and kappa chain repertoires between OmniRats and humans including gene segment usage, CDR3 length distributions, class switch recombination, somatic hypermutation levels and in features of V(D)J recombination. The Inference and Generation of Repertoires (IGoR) software tool was used to model recombination in VH regions which allowed for the quantification of some of these differences. Diversity estimates of the OmniRat heavy chain repertoires almost reached that of humans, around two orders of magnitude less. Despite variation between the species repertoires, a high frequency of OmniRat clonotypes were also found in the human repertoire. These data give insights into the development and selection of humanized animal antibodies and provide actionable information for use in vaccine studies.

scholarly journals Application of High-Throughput Sequencing in the Diagnosis of Inherited Thrombocytopenia

2018 ◽  
Vol 24 (9_suppl) ◽  
pp. 94S-103S ◽  
Author(s):  
Qi Wang ◽  
Lijuan Cao ◽  
Guangying Sheng ◽  
Hongjie Shen ◽  
Jing Ling ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Gene Sequencing ◽  
Hereditary Diseases ◽  
Sequencing Platform ◽  
Pathogenic Variants ◽  
Diagnosis Method ◽  
Inherited Thrombocytopenia ◽  
Thrombotic Diseases ◽  
Next Generation Sequencing Ngs

Inherited thrombocytopenia is a group of hereditary diseases with a reduction in platelet count as the main clinical manifestation. Clinically, there is an urgent need for a convenient and rapid diagnosis method. We introduced a high-throughput, next-generation sequencing (NGS) platform into the routine diagnosis of patients with unexplained thrombocytopenia and analyzed the gene sequencing results to evaluate the value of NGS technology in the screening and diagnosis of inherited thrombocytopenia. From a cohort of 112 patients with thrombocytopenia, we screened 43 patients with hereditary features. For the blood samples of these 43 patients, a gene sequencing platform for hemorrhagic and thrombotic diseases comprising 89 genes was used to perform gene detection using NGS technology. When we combined the screening results with clinical features and other findings, 15 (34.9%) of 43patients were diagnosed with inherited thrombocytopenia. In addition, 19 pathogenic variants, including 8 previously unreported variants, were identified in these patients. Through the use of this detection platform, we expect to establish a more effective diagnostic approach to such disorders.

scholarly journals High-Throughput Sequencing to Investigate Phytopathogenic Fungal Propagules Caught in Baited Insect Traps

2019 ◽  
Vol 5 (1) ◽  
pp. 15 ◽  
Author(s):  
Émilie D. Tremblay ◽  
Troy Kimoto ◽  
Jean A. Bérubé ◽  
Guillaume J. Bilodeau
Keyword(s):  
High Throughput ◽  
Plant Pathogens ◽  
High Throughput Sequencing ◽  
Morphological Characteristics ◽  
Diagnostic Techniques ◽  
Phytopathogenic Fungi ◽  
Personal Genome ◽  
Annual Survey ◽  
Fungal Propagules ◽  
Wood Boring

Studying the means of dispersal of plant pathogens is crucial to better understand the dynamic interactions involved in plant infections. On one hand, entomologists rely mostly on both traditional molecular methods and morphological characteristics, to identify pests. On the other hand, high-throughput sequencing (HTS) is becoming the go-to avenue for scientists studying phytopathogens. These organisms sometimes infect plants, together with insects. Considering the growing number of exotic insect introductions in Canada, forest pest-management efforts would benefit from the development of a high-throughput strategy to investigate the phytopathogenic fungal and oomycete species interacting with wood-boring insects. We recycled formerly discarded preservative fluids from the Canadian Food Inspection Agency annual survey using insect traps and analysed more than one hundred samples originating from across Canada. Using the Ion Torrent Personal Genome Machine (PGM) HTS technology and fusion primers, we performed metabarcoding to screen unwanted fungi and oomycetes species, including Phytophthora spp. Community profiling was conducted on the four different wood-boring, insect-attracting semiochemicals; although the preservative (contained ethanol) also attracted other insects. Phytopathogenic fungi (e.g., Leptographium spp. and Meria laricis in the pine sawyer semiochemical) and oomycetes (mainly Peronospora spp. and Pythium aff. hypogynum in the General Longhorn semiochemical), solely associated with one of the four types of semiochemicals, were detected. This project demonstrated that the insect traps’ semiochemical microbiome represents a new and powerful matrix for screening phytopathogens. Compared to traditional diagnostic techniques, the fluids allowed for a faster and higher throughput assessment of the biodiversity contained within. Additionally, minimal modifications to this approach would allow it to be used in other phytopathology fields.

scholarly journals A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders

Blood ◽  
2016 ◽  
Vol 127 (23) ◽  
pp. 2791-2803 ◽  
Author(s):  
Ilenia Simeoni ◽  
Jonathan C. Stephens ◽  
Fengyuan Hu ◽  
Sri V. V. Deevi ◽  
Karyn Megy ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Genetic Test ◽  
Targeted Sequencing ◽  
Sequencing Platform ◽  
Key Points ◽  
Platelet Disorders

Key Points Developed a targeted sequencing platform covering 63 genes linked to heritable bleeding, thrombotic, and platelet disorders. The ThromboGenomics platform provides a sensitive genetic test to obtain molecular diagnoses in patients with a suspected etiology.

scholarly journals jackalope: a swift, versatile phylogenomic and high-throughput sequencing simulator

2019 ◽  
Author(s):  
Lucas A. Nell
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Population Genomics ◽  
R Package ◽  
Gene Trees ◽  
Sequencing Platform ◽  
Genomic Variants ◽  
Pacific Biosciences ◽  
Wide Range ◽  
Reference Genomes

AbstractHigh-throughput sequencing (HTS) is central to the study of population genomics and has an increasingly important role in constructing phylogenies. Choices in research design for sequencing projects can include a wide range of factors, such as sequencing platform, depth of coverage, and bioinformatic tools. Simulating HTS data better informs these decisions. However, current standalone HTS simulators cannot generate genomic variants under even somewhat complex evolutionary scenarios, which greatly reduces their usefulness for fields such as population genomics and phylogenomics. Here I present the R package jackalope that simply and efficiently simulates (i) variants from reference genomes and (ii) reads from both Illumina and Pacific Biosciences (PacBio) platforms. Genomic variants can be simulated using phylogenies, gene trees, coalescent-simulation output, population-genomic summary statistics, and Variant Call Format (VCF) files. jackalope can simulate single, paired-end, or mate-pair Illumina reads, as well as reads from Pacific Biosciences. These simulations include sequencing errors, mapping qualities, multiplexing, and optical/PCR duplicates. It can read reference genomes from FASTA files and can simulate new ones, and all outputs can be written to standard file formats. jackalope is available for Mac, Windows, and Linux systems.

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