scholarly journals Ligation-anchored PCR unveils immune repertoire of TCR-beta from whole blood

2014 ◽  
Author(s):  
Fan Gao ◽  
Kai Wang
Keyword(s):  
High Throughput Sequencing ◽  
Read Length ◽  
Integrative Approach ◽  
Sequencing Data ◽  
Immune Repertoire ◽  
Universal Primer ◽  
Illumina Hiseq ◽  
Variable Regions ◽  
Anchored Pcr ◽  
Pcr Method

Background As one of the genetic mechanisms for adaptive immunity, V(D)J recombination generates an enormous repertoire of T-cell receptors (TCRs). With the development of high-throughput sequencing techniques, systematic exploration of V(D)J recombination becomes possible. Multiplex PCR method has been previously developed to assay immune repertoire, however the usage of primer pools has inherent bias in target amplification. In our study, we developed a ligation-anchored PCR method to unbiasedly amplify the repertoire. Results By utilizing a universal primer paired with a single primer targeting the conserved constant region, we amplified TCR-beta (TRB) variable regions from total RNA extracted from blood. Next-generation sequencing libraries were then prepared for Illumina HiSeq 2500 sequencer, which provided 151 bp read length to cover the entire V(D)J recombination region. We evaluated this approach on blood samples from patients with malignant and benign meningiomas. Mapping of sequencing data showed 64% to 91% of mapped TCRV-containing reads belong to TRB subtype. An increased usage of TRBV29-1 was observed in malignant meningiomas. Also distinct signatures were identified from CDR3 sequence logos, with predominant subset as 42 nt for benign and 45 nt for malignant samples, respectively. Conclusions In summary, we report an integrative approach to monitor immune repertoire in a systematic manner.

scholarly journals PIRD: Pan Immune Repertoire Database

2019 ◽  
Author(s):  
Wei Zhang ◽  
Longlong Wang ◽  
Ke Liu ◽  
Xiaofeng Wei ◽  
Kai Yang ◽  
...  
Keyword(s):  
Data Storage ◽  
High Throughput Sequencing ◽  
Homo Sapiens ◽  
Adaptive Immune System ◽  
B Cell Receptors ◽  
Sequencing Data ◽  
Immune Repertoire ◽  
Adaptive Immune ◽  
Potential Applications ◽  
Antibody Drug

Abstract Motivation T and B cell receptors (TCRs and BCRs) play a pivotal role in the adaptive immune system by recognizing an enormous variety of external and internal antigens. Understanding these receptors is critical for exploring the process of immunoreaction and exploiting potential applications in immunotherapy and antibody drug design. Although a large number of samples have had their TCR and BCR repertoires sequenced using high-throughput sequencing in recent years, very few databases have been constructed to store these kinds of data. To resolve this issue, we developed a database. Results We developed a database, the Pan Immune Repertoire Database (PIRD), located in China National GeneBank (CNGBdb), to collect and store annotated TCR and BCR sequencing data, including from Homo sapiens and other species. In addition to data storage, PIRD also provides functions of data visualization and interactive online analysis. Additionally, a manually curated database of TCRs and BCRs targeting known antigens (TBAdb) was also deposited in PIRD. Availability and implementation PIRD can be freely accessed at https://db.cngb.org/pird.

scholarly journals A Snapshot of the Genetic Diversity of Salmonella Enteritidis Population Involved in Human Infections in Romania Taken in the European Epidemiological Context

Pathogens ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1490
Author(s):  
Codruta-Romanita Usein ◽  
Mihaela Oprea ◽  
Adriana Simona Ciontea ◽  
Sorin Dinu ◽  
Daniela Cristea ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Salmonella Enteritidis ◽  
Tandem Repeats ◽  
High Throughput Sequencing ◽  
Minimum Spanning Tree ◽  
Public Health Practice ◽  
National Level ◽  
Variable Number ◽  
Integrative Approach ◽  
Sequencing Data

In the absence of consistent national molecular typing data to enhance the surveillance of Salmonella Enteritidis, it was considered useful to collect baseline information on the genetic diversity and antibiotic susceptibility of strains isolated in Romania between January 2016 and April 2020 and compare them to strains described in major international outbreaks of the same period. A collection of 245 clinical isolates were genotyped by a standardised multiple-locus variable-number of tandem repeats analysis (MLVA) 5-loci protocol and screened for antimicrobial resistance against 15 compounds. Twenty strains were further subjected to whole genome sequencing (WGS) and compared to epidemiologically relevant high-throughput sequencing data available in European databases. Twenty-seven MLVA genotypes were identified, of which three, commonly reported in Europe between 2016–2020, covered 72% of the collection. Antibiotic resistance was detected in 30% of the strains, with resistance to nalidixic acid and ciprofloxacin as the most common phenotype, and also associated with two prevalent MLVA clones. WGS-derived multilocus sequence typing (MLST) revealed a single sequence type (ST11) further resolved into 10 core-genome MLST complex types. The minimum spanning tree constructed from the cgMLST data clustered Romanian and international strains, which shared more than 95% of the core genes, revealing links with a contemporaneous multi-country outbreak. This study could be regarded as a forerunner to the advent of using this integrative approach in the public health practice at a national level and thus contribute to the concerted actions at a European level to stop outbreaks.

scholarly journals STRetch: detecting and discovering pathogenic short tandem repeat expansions

2017 ◽  
Author(s):  
Harriet Dashnow ◽  
Monkol Lek ◽  
Belinda Phipson ◽  
Andreas Halman ◽  
Simon Sadedin ◽  
...  
Keyword(s):  
Tandem Repeat ◽  
Short Tandem Repeat ◽  
High Throughput Sequencing ◽  
Read Length ◽  
Whole Genome Sequencing Data ◽  
Sequencing Data ◽  
Short Read ◽  
Dna Mutation ◽  
Str Loci ◽  
Short Tandem

AbstractShort tandem repeat (STR) expansions have been identified as the causal DNA mutation in dozens of Mendelian diseases. Historically, pathogenic STR expansions could only be detected by single locus techniques, such as PCR and electrophoresis. The ability to use short read sequencing data to screen for STR expansions has the potential to reduce both the time and cost to reaching diagnosis and enable the discovery of new causal STR loci. Most existing tools detect STR variation within the read length, and so are unable to detect the majority of pathogenic expansions. Those tools that can detect large expansions are limited to a set of known disease loci and as yet no new disease causing STR expansions have been identified with high-throughput sequencing technologies.Here we address this by presenting STRetch, a new genome-wide method to detect STR expansions at all loci across the human genome. We demonstrate the use of STRetch for detecting pathogenic STR expansions in short-read whole genome sequencing data with a very low false discovery rate. We further demonstrate the application of STRetch to solve cases of patients with undiagnosed disease and apply STRetch to the analysis of 97 whole genomes to reveal variation at STR loci. STRetch assesses expansions at all STR loci in the genome and allows screening for novel disease-causing STRs.STRetch is open source software, available from github.com/Oshlack/STRetch.

scholarly journals PIRD: Pan immune repertoire database

2018 ◽  
Author(s):  
Wei Zhang ◽  
Longlong Wang ◽  
Ke Liu ◽  
Xiaofeng Wei ◽  
Kai Yang ◽  
...  
Keyword(s):  
Data Storage ◽  
High Throughput Sequencing ◽  
Homo Sapiens ◽  
Adaptive Immune System ◽  
B Cell Receptors ◽  
Data Visualisation ◽  
Sequencing Data ◽  
Immune Repertoire ◽  
Adaptive Immune ◽  
Potential Applications

ABSTRACTMotivationT and B cell receptors (TCRs and BCRs) play a pivotal role in the adaptive immune system by recognizing an enormous variety of external and internal antigens. Understanding these receptors is critical for exploring the process of immunoreaction and exploiting potential applications in immunotherapy and antibody drug design. Although a large number of samples have had their TCR and BCR repertoires sequenced using high-throughput sequencing in recent years, very few databases have been constructed to store these kinds of data. To resolve this issue, we developed a database.ResultsWe developed a database, the Pan Immune Repertoire Database (PIRD), located in China National GeneBank (CNGBdb), to collect and store annotated TCR and BCR sequencing data, including from Homo sapiens and other species. In addition to data storage, PIRD also provides functions of data visualisation and interactive online analysis. Additionally, a manually curated database of TCRs and BCRs targeting known antigens (TBAdb) was also deposited in PIRD.Availability and ImplementationPIRD can be freely accessed at https://db.cngb.org/pird.

scholarly journals Improvement, identification, and target prediction for miRNAs in the porcine genome by using massive, public high-throughput sequencing data

2021 ◽  
Vol 99 (2) ◽  
Author(s):  
Yuhua Fu ◽  
Pengyu Fan ◽  
Lu Wang ◽  
Ziqiang Shu ◽  
Shilin Zhu ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Target Genes ◽  
Target Prediction ◽  
Large Data ◽  
Sequencing Data ◽  
Regulate Gene Expression ◽  
High Throughput Sequencing Data ◽  
Annotation Information ◽  
Public Data ◽  
Broad Variety

Abstract Despite the broad variety of available microRNA (miRNA) research tools and methods, their application to the identification, annotation, and target prediction of miRNAs in nonmodel organisms is still limited. In this study, we collected nearly all public sRNA-seq data to improve the annotation for known miRNAs and identify novel miRNAs that have not been annotated in pigs (Sus scrofa). We newly annotated 210 mature sequences in known miRNAs and found that 43 of the known miRNA precursors were problematic due to redundant/missing annotations or incorrect sequences. We also predicted 811 novel miRNAs with high confidence, which was twice the current number of known miRNAs for pigs in miRBase. In addition, we proposed a correlation-based strategy to predict target genes for miRNAs by using a large amount of sRNA-seq and RNA-seq data. We found that the correlation-based strategy provided additional evidence of expression compared with traditional target prediction methods. The correlation-based strategy also identified the regulatory pairs that were controlled by nonbinding sites with a particular pattern, which provided abundant complementarity for studying the mechanism of miRNAs that regulate gene expression. In summary, our study improved the annotation of known miRNAs, identified a large number of novel miRNAs, and predicted target genes for all pig miRNAs by using massive public data. This large data-based strategy is also applicable for other nonmodel organisms with incomplete annotation information.

scholarly journals High-precision and cost-efficient sequencing for real-time COVID-19 surveillance

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sung Yong Park ◽  
Gina Faraci ◽  
Pamela M. Ward ◽  
Jane F. Emerson ◽  
Ha Youn Lee
Keyword(s):  
Los Angeles ◽  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
High Precision ◽  
High Throughput Sequencing ◽  
Whole Genome ◽  
Sequencing Data ◽  
Public Health Response ◽  
Cost Efficient

AbstractCOVID-19 global cases have climbed to more than 33 million, with over a million total deaths, as of September, 2020. Real-time massive SARS-CoV-2 whole genome sequencing is key to tracking chains of transmission and estimating the origin of disease outbreaks. Yet no methods have simultaneously achieved high precision, simple workflow, and low cost. We developed a high-precision, cost-efficient SARS-CoV-2 whole genome sequencing platform for COVID-19 genomic surveillance, CorvGenSurv (Coronavirus Genomic Surveillance). CorvGenSurv directly amplified viral RNA from COVID-19 patients’ Nasopharyngeal/Oropharyngeal (NP/OP) swab specimens and sequenced the SARS-CoV-2 whole genome in three segments by long-read, high-throughput sequencing. Sequencing of the whole genome in three segments significantly reduced sequencing data waste, thereby preventing dropouts in genome coverage. We validated the precision of our pipeline by both control genomic RNA sequencing and Sanger sequencing. We produced near full-length whole genome sequences from individuals who were COVID-19 test positive during April to June 2020 in Los Angeles County, California, USA. These sequences were highly diverse in the G clade with nine novel amino acid mutations including NSP12-M755I and ORF8-V117F. With its readily adaptable design, CorvGenSurv grants wide access to genomic surveillance, permitting immediate public health response to sudden threats.

scholarly journals Transcriptomic Analysis of Rice Plants Overexpressing PsGAPDH in Response to Salinity Stress

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 641
Author(s):  
Hyemin Lim ◽  
Hyunju Hwang ◽  
Taelim Kim ◽  
Soyoung Kim ◽  
Hoyong Chung ◽  
...  
Keyword(s):  
Salt Stress ◽  
Abiotic Stress ◽  
Salinity Stress ◽  
Functional Enrichment ◽  
Differentially Expressed ◽  
Pathway Enrichment Analysis ◽  
Sequencing Data ◽  
Illumina Hiseq ◽  
Rice Plants ◽  
Significant Difference

In plants, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a main enzyme in the glycolytic pathway. It plays an essential role in glycerolipid metabolism and response to various stresses. To examine the function of PsGAPDH (Pleurotus sajor-caju GAPDH) in response to abiotic stress, we generated transgenic rice plants with single-copy/intergenic/homozygous overexpression PsGAPDH (PsGAPDH-OX) and investigated their responses to salinity stress. Seedling growth and germination rates of PsGAPDH-OX were significantly increased under salt stress conditions compared to those of the wild type. To elucidate the role of PsGAPDH-OX in salt stress tolerance of rice, an Illumina HiSeq 2000 platform was used to analyze transcriptome profiles of leaves under salt stress. Analysis results of sequencing data showed that 1124 transcripts were differentially expressed. Using the list of differentially expressed genes (DEGs), functional enrichment analyses of DEGs such as Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed. KEGG pathway enrichment analysis revealed that unigenes exhibiting differential expression were involved in starch and sucrose metabolism. Interestingly, trehalose-6-phosphate synthase (TPS) genes, of which expression was enhanced by abiotic stress, showed a significant difference in PsGAPDH-OX. Findings of this study suggest that PsGAPDH plays a role in the adaptation of rice plants to salt stress.

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