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J Med Virol ; 94(1): 327-334, 2022 01.
Article En | MEDLINE | ID: mdl-34524690

Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in COVID-19 pandemic control and elimination efforts, especially by elucidating its global transmission network and illustrating its viral evolution. The deployment of multiplex PCR assays that target SARS-CoV-2 followed by either massively parallel or nanopore sequencing is a widely-used strategy to obtain genome sequences from primary samples. However, multiplex PCR-based sequencing carries an inherent bias of sequencing depth among different amplicons, which may cause uneven coverage. Here we developed a two-pool, long-amplicon 36-plex PCR primer panel with ~1000-bp amplicon lengths for full-genome sequencing of SARS-CoV-2. We validated the panel by assessing nasopharyngeal swab samples with a <30 quantitative reverse transcription PCR cycle threshold value and found that ≥90% of viral genomes could be covered with high sequencing depths (≥20% mean depth). In comparison, the widely-used ARTIC panel yielded 79%-88% high-depth genome regions. We estimated that ~5 Mbp nanopore sequencing data may ensure a >95% viral genome coverage with a ≥10-fold depth and may generate reliable genomes at consensus sequence levels. Nanopore sequencing yielded false-positive variations with frequencies of supporting reads <0.8, and the sequencing errors mostly occurred on the 5' or 3' ends of reads. Thus, nanopore sequencing could not elucidate intra-host viral diversity.

Genome, Viral/genetics , Multiplex Polymerase Chain Reaction/methods , Nanopore Sequencing/methods , SARS-CoV-2/genetics , Whole Genome Sequencing/methods , COVID-19 , High-Throughput Nucleotide Sequencing/methods , Humans , Nasopharynx/virology , RNA, Viral/genetics , Sequence Analysis, RNA/methods
J Sci Food Agric ; 102(1): 147-155, 2022 Jan 15.
Article En | MEDLINE | ID: mdl-34057213

BACKGROUND: Bacterial community successions were surveyed during the processing stages of sugar production using high-throughput sequencing methods. Furthermore, the correlation between bacterial community and nitrate/nitrite content in beet sugar processing were investigated. RESULTS: In an analysis of the V3-V4 region of the 16S rDNA gene, 254 122 effective sequences were obtained from samples, which included sugar beet, cossettes, diffusion juice, second-phase diffusion juice, light juice and thick juice. The results showed that dominant genera included Pantoea, Pseudomonas, Leuconostoc and Burkholderia. Moreover, significant changes in bacterial communities were observed in samples. Regarding the relevant nitrogen metabolic potential, this study revealed communities with the ability for nitrate and nitrite metabolism. Furthermore, a shaking experiment involving diffusion juice and second-phase diffusion juice was performed, and results showed that the nitrate level declined 73% and 98% in 36 h, respectively. These results suggested that the bacterial communities contribute to nitrate and nitrite transformation. CONCLUSION: This study illustrated that the bacterial communities and their specific effects on the formation of nitrate and nitrite during beet sugar processing. The results presented the basic concept involving the nitrate- and nitrite-forming pathways directly related to the mechanism of bacterial community growth. This study could facilitate an understanding of the correlation between nitrite content and microorganisms to guide beet sugar manufacturers regarding the control of nitrite and nitrate content. © 2021 Society of Chemical Industry.

Bacteria/metabolism , Beta vulgaris/chemistry , Nitrates/analysis , Nitrites/analysis , Plant Tubers/microbiology , Sugars/isolation & purification , Bacteria/classification , Bacteria/genetics , Bacteria/isolation & purification , Beta vulgaris/microbiology , Biotransformation , Food Handling , High-Throughput Nucleotide Sequencing , Nitrates/metabolism , Nitrites/metabolism , Plant Tubers/chemistry , Sugars/chemistry
Food Chem ; 368: 130889, 2022 Jan 30.
Article En | MEDLINE | ID: mdl-34438175

Complex microbial community plays an important role for flavor formation in traditional dry-cured grass carp. To investigate the correlation between microorganisms and flavour development, the bacterial diversity and flavour quality of dry-cured fish at different stages of fermentation were analysed using high-throughput sequencing, volatile flavour analysis and metabolomics. Cobetia, Staphylococcus and Ralstonia were the dominant genera in dry-cured fish, with relative abundances of 37.78%, 34.46% and 3.2%, respectively. The flavour of dry-cured fish samples varied as the abundance of aldehydes, alcohols, small peptides, FAAs and carboxylic acids showed a great increase during fermentation. Moreover, there were significant correlations (P < 0.05) between specific microorganisms and volatile indicators, as well as flavour metabolites. Staphylococcus, as the dominant bacterial genus, is involved in the mechanism of flavour formation in dry-cured fish during fermentation. This information is useful for elucidating the mechanism of flavour formation in dry-cured fish.

Carps , Animals , Flavoring Agents , High-Throughput Nucleotide Sequencing , Metabolomics , Taste
J Sci Food Agric ; 102(1): 350-359, 2022 Jan 15.
Article En | MEDLINE | ID: mdl-34143449

BACKGROUND: The contribution of bacteria to fermented tea is not clear and the associated research is relatively limited. To reveal the role of microorganisms in fermented tea processing, the microbial community and metabolites of Fuzhuan brick tea (FBT), a Chinese traditional fermented tea, were revealed via high-throughput sequencing and liquid chromatography-mass spectrometry (LC-MS). RESULTS: In FBT, bacterial communities had a higher abundance and diversity, Lactococcus and Bacillus were the main bacteria, and Eurotium was the predominant fungus. The predictive metabolic function indicated the pathways of cellular growth, environmental information, genetics and material metabolism of bacterial communities were abundant, whereas the fungal community predictive metabolic function was almost saprotroph. Using LC-MS, 1143 and 536 metabolites were defined in positive and negative ion mode, respectively. There were essential correlations between bacterial populations and metabolites, such that Bacillus was correlated significantly with 44 metabolites (P < 0.05) and Enterococcus was significantly associated with 15 metabolites (P < 0.05). Some of the main active components were significantly correlated with the bacteria, such as Enterococcus, Lactococcus and Carnobacterium. CONCLUSION: Not only Eurotium, but also the bacteria were involved in the changes of metabolomics profile in fermented FBT. The present study assists in providing new insights into metabolomics profile generation in fermented tea. The present research lays a foundation for controlling the FBT fermentation by artificial inoculation to improve quality. © 2021 Society of Chemical Industry.

Bacteria/metabolism , Camellia sinensis/microbiology , Bacteria/chemistry , Bacteria/classification , Bacteria/genetics , Camellia sinensis/metabolism , Chromatography, Liquid , Fermentation , Fungi/chemistry , Fungi/classification , Fungi/genetics , Fungi/metabolism , High-Throughput Nucleotide Sequencing , Mass Spectrometry , Metabolomics , Tea/chemistry
Food Chem ; 371: 131066, 2022 Mar 01.
Article En | MEDLINE | ID: mdl-34543927

The adulteration of honey is common. Recently, High Throughput Sequencing (HTS)-based metabarcoding method has been applied successfully to pollen/honey identification to determine floral composition that, in turn, can be used to identify the geographical origins of honeys. However, the lack of local references materials posed a serious challenge for HTS-based pollen identification methods. Here, we sampled 28 honey samples from various geographic origins without prior knowledge of local floral information and applied a machine learning method to determine geographical origins. The machine learning method uses a resilient backpropagation algorithm to train a neural network. The results showed that biological components in honey provided characteristic traits that enabled accurate geographic tracing for nearly all honey samples, confidently discriminating honeys to their geographic origin with >99% success rates, including those separated by as little as 39 km.

Honey , High-Throughput Nucleotide Sequencing , Honey/analysis , Machine Learning , Metagenomics , Pollen
J Med Virol ; 94(1): 413-416, 2022 01.
Article En | MEDLINE | ID: mdl-34515998

In December 2020, Italy experienced the first case of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) B.1.1.7 lineage. In January 2021, we identified 21 cases of this variant in Corzano, defining the first outbreak of SARS-CoV-2 B.1.1.7 lineage in Italy. The high transmissibility of the B.1.1.7 variant represented an important benefit for the virus, which became rapidly dominant on the territory. Containment measures induced the epidemic curve onto a decreasing trajectory underlining the importance of appropriate control and surveillance for restraint of virus spread. Highlights The first Italian outbreak of SARS-CoV-2 B.1.1.7 lineage occurred in Lombardy in January 2021. The outbreak originated by a single introduction of the B.1.1.7 lineage. The genomic sequencing revealed, for the first time, the presence of the V551F mutation in the B.1.1.7 lineage in Italy. Surveillance, prompt sequencing and tracing efforts were fundamental to identify and to quickly contain the outbreak.

COVID-19 Nucleic Acid Testing , COVID-19/epidemiology , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Adolescent , Adult , COVID-19/transmission , Child , Child, Preschool , Disease Outbreaks/statistics & numerical data , Female , Genome, Viral/genetics , High-Throughput Nucleotide Sequencing , Humans , Infection Control/methods , Italy/epidemiology , Male , Middle Aged , Phylogeny , Sequence Analysis, RNA , Whole Genome Sequencing , Young Adult
Methods Mol Biol ; 2404: 189-205, 2022.
Article En | MEDLINE | ID: mdl-34694610

Individual-nucleotide crosslinking and immunoprecipitation (iCLIP) sequencing and its derivative enhanced CLIP (eCLIP) sequencing are methods for the transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). This chapter provides a stepwise tutorial for analyzing iCLIP and eCLIP data with replicates and size-matched input (SMI) controls after read alignment using our open-source tools htseq-clip and DEWSeq. This includes the preparation of gene annotation, extraction, and preprocessing of truncation sites and the detection of significantly enriched binding sites using a sliding window based approach suitable for different binding modes of RBPs.

High-Throughput Nucleotide Sequencing , Binding Sites , Immunoprecipitation , RNA , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Transcriptome
BMC Bioinformatics ; 22(1): 552, 2021 Nov 12.
Article En | MEDLINE | ID: mdl-34772337

BACKGROUND: With the rapid development of long-read sequencing technologies, it is possible to reveal the full spectrum of genetic structural variation (SV). However, the expensive cost, finite read length and high sequencing error for long-read data greatly limit the widespread adoption of SV calling. Therefore, it is urgent to establish guidance concerning sequencing coverage, read length, and error rate to maintain high SV yields and to achieve the lowest cost simultaneously. RESULTS: In this study, we generated a full range of simulated error-prone long-read datasets containing various sequencing settings and comprehensively evaluated the performance of SV calling with state-of-the-art long-read SV detection methods. The benchmark results demonstrate that almost all SV callers perform better when the long-read data reach 20× coverage, 20 kbp average read length, and approximately 10-7.5% or below 1% error rates. Furthermore, high sequencing coverage is the most influential factor in promoting SV calling, while it also directly determines the expensive costs. CONCLUSIONS: Based on the comprehensive evaluation results, we provide important guidelines for selecting long-read sequencing settings for efficient SV calling. We believe these recommended settings of long-read sequencing will have extraordinary guiding significance in cutting-edge genomic studies and clinical practices.

Benchmarking , Genomics , Diagnostic Tests, Routine , Genomic Structural Variation , High-Throughput Nucleotide Sequencing , Sequence Analysis, DNA
BMC Genomics ; 22(1): 822, 2021 Nov 14.
Article En | MEDLINE | ID: mdl-34773979

BACKGROUND: We benchmarked sequencing technology and assembly strategies for short-read, long-read, and hybrid assemblers in respect to correctness, contiguity, and completeness of assemblies in genomes of Francisella tularensis. Benchmarking allowed in-depth analyses of genomic structures of the Francisella pathogenicity islands and insertion sequences. Five major high-throughput sequencing technologies were applied, including next-generation "short-read" and third-generation "long-read" sequencing methods. RESULTS: We focused on short-read assemblers, hybrid assemblers, and analysis of the genomic structure with particular emphasis on insertion sequences and the Francisella pathogenicity island. The A5-miseq pipeline performed best for MiSeq data, Mira for Ion Torrent data, and ABySS for HiSeq data from eight short-read assembly methods. Two approaches were applied to benchmark long-read and hybrid assembly strategies: long-read-first assembly followed by correction with short reads (Canu/Pilon, Flye/Pilon) and short-read-first assembly along with scaffolding based on long reads (Unicyler, SPAdes). Hybrid assembly can resolve large repetitive regions best with a "long-read first" approach. CONCLUSIONS: Genomic structures of the Francisella pathogenicity islands frequently showed misassembly. Insertion sequences (IS) could be used to perform an evolutionary conservation analysis. A phylogenetic structure of insertion sequences and the evolution within the clades elucidated the clade structure of the highly conservative F. tularensis.

Francisella tularensis , Genome, Bacterial , DNA Transposable Elements , Francisella tularensis/genetics , Genomics , High-Throughput Nucleotide Sequencing , Phylogeny , Sequence Analysis, DNA
Yi Chuan ; 43(11): 1066-1077, 2021 Nov 20.
Article En | MEDLINE | ID: mdl-34815209

Castration can reduce odor and fights in boars, but the carcass yield is reduced, and the intramuscular fat content is increased. Understanding its molecular mechanism is of great significance for production. Recent studies have shown that circular RNAs (circRNAs) play an important role(s) in the regulation of muscle development. To explore the effects of circRNAs on the development of longissimus dorsi (LD) muscle after castration, six Huainan male pigs were selected and three of which were randomly castrated. Six pigs were slaughtered when their body weight reached around 130 kg, and the LD muscle samples were collected. The differentially expressed circRNAs (DECs) were screened by high-throughput sequencing and functionally analyzed using the KEGG databases. DECs-miRNAs network was constructed, and the expression profiles of candidate circRNAs and their interactions with miRNAs were verified in porcine skeletal muscle satellite cells. The results showed that a total of 5866 circRNAs were obtained, and 370 DECs were identified in LD muscle between the castrated and intact groups (| log2Foldchange | > 1, padj <0.8). KEGG enrichment indicated that the parental genes for the DECs were mainly enriched in the pathways associated with muscle development, muscle fiber type transformation, and energy metabolism. There were 8 miRNAs and 69 circRNAs enriched in the DECs-miRNA network. circRNA_2241 and circRNA_4237 were selected for verification, which showed that these two circRNAs really existed and their expression profiles were consistent with the sequencing results. Further, preliminary analysis showed that circRNA_2241 interacted with miR-1, and testosterone promoted circRNA_2241 but inhibited miR-1 expression. These results confirmed that circRNAs might participate in the regulation of LD muscle development after castration by interacting with miRNAs, thereby providing new materials and references for analyses on the molecular mechanisms of castration on the regulation of muscle development.

MicroRNAs , RNA, Circular , Animals , High-Throughput Nucleotide Sequencing , Male , MicroRNAs/genetics , Muscle Development , Muscles , Swine/genetics
Front Cell Infect Microbiol ; 11: 749905, 2021.
Article En | MEDLINE | ID: mdl-34790588

With the widespread use of antibacterial drugs and increasing number of immunocompromised patients, pulmonary fungal infections are becoming more common. However, the incidence of pulmonary fungal and bacterial co-infection is rarely reported. In this study, 119 patients definitively diagnosed with pulmonary fungal infections between July 2018 and March 2020 were assessed using metagenomic next-generation sequencing (mNGS) as well as traditional pathogen detection to gauge the incidence of fungal and bacterial co-infection and evaluate the associated risk factors. We found that of the 119 patients with fungal infections, 48 (40.3%) had pulmonary fungal and bacterial co-infection. We identified immunocompromised status and the presence of one or more pulmonary cavities as risk factors associated with fungal and bacterial co-infection. The most commonly isolated fungi species were Aspergillus, Pneumocystis, and Rhizopus. The most commonly isolated bacterial species were Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia. Seventy-nine (66.4%) patients had received empirical antibiotic treatment before their pathogenic test results became available, and 41.7% (fungal infection group) and 38.7% (fungal and bacterial co-infection group) of the patients had their antibacterial drug dosage changed accordingly. This mNGS-based study showed that the incidence of fungal and bacterial co-infection is significant. Our research outcomes can, thus, guide the use of antibacterial drugs in the treatment of clinical fungal infections.

Coinfection , Lung Diseases, Fungal , Coinfection/epidemiology , Fungi/genetics , High-Throughput Nucleotide Sequencing , Humans , Lung Diseases, Fungal/epidemiology , Metagenomics , Prevalence
BMC Bioinformatics ; 22(1): 559, 2021 Nov 22.
Article En | MEDLINE | ID: mdl-34809557

BACKGROUND: When analyzing DNA sequence data of an individual, knowing which nucleotide was inherited from each parent can be beneficial when trying to identify certain types of DNA variants. Mendelian inheritance logic can be used to accurately phase (haplotype) the majority (67-83%) of an individual's heterozygous nucleotide positions when genotypes are available for both parents (trio). However, when all members of a trio are heterozygous at a position, Mendelian inheritance logic cannot be used to phase. For such positions, a computational phasing algorithm can be used. Existing phasing algorithms use a haplotype reference panel, sequencing reads, and/or parental genotypes to phase an individual; however, they are limited in that they can only phase certain types of variants, require a specific genotype build, require large amounts of storage capacity, and/or require long run times. We created trioPhaser to address these challenges. RESULTS: trioPhaser uses gVCF files from an individual and their parents as initial input, and then outputs a phased VCF file. Input trio data are first phased using Mendelian inheritance logic. Then, the positions that cannot be phased using inheritance information alone are phased by the SHAPEIT4 phasing algorithm. Using whole-genome sequencing data of 52 trios, we show that trioPhaser, on average, increases the total number of phased positions by 21.0% and 10.5%, respectively, when compared to the number of positions that SHAPEIT4 or Mendelian inheritance logic can phase when either is used alone. In addition, we show that the accuracy of the phased calls output by trioPhaser are similar to linked-read and read-backed phasing. CONCLUSION: trioPhaser is a containerized software tool that uses both Mendelian inheritance logic and SHAPEIT4 to phase trios when gVCF files are available. By implementing both phasing methods, more variant positions are phased compared to what either method is able to phase alone.

Genome , Polymorphism, Single Nucleotide , Algorithms , Genomics , Haplotypes , High-Throughput Nucleotide Sequencing , Logic , Sequence Analysis, DNA
BMC Bioinformatics ; 22(1): 560, 2021 Nov 22.
Article En | MEDLINE | ID: mdl-34809571

BACKGROUND: Identifying haplotypes is central to sequence analysis in diploid or polyploid genomes. Despite this, there remains a lack of research and tools designed for physical phasing and its downstream analysis. RESULTS: HaplotypeTools is a new toolset to phase variant sites using VCF and BAM files and to analyse phased VCFs. Phasing is achieved via the identification of reads overlapping ≥ 2 heterozygous positions and then extended by additional reads, a process that can be parallelized across a computer cluster. HaplotypeTools includes various utility scripts for downstream analysis including crossover detection and phylogenetic placement of haplotypes to other lineages or species. HaplotypeTools was assessed for accuracy against WhatsHap using simulated short and long reads, demonstrating higher accuracy, albeit with reduced haplotype length. HaplotypeTools was also tested on real Illumina data to determine the ancestry of hybrid fungal isolate Batrachochytrium dendrobatidis (Bd) SA-EC3, finding 80% of haplotypes across the genome phylogenetically cluster with parental lineages BdGPL (39%) and BdCAPE (41%), indicating those are the parental lineages. Finally, ~ 99% of phasing was conserved between overlapping phase groups between SA-EC3 and either parental lineage, indicating mitotic gene conversion/parasexuality as the mechanism of recombination for this hybrid isolate. HaplotypeTools is open source and freely available from under the MIT License. CONCLUSIONS: HaplotypeTools is a powerful resource for analyzing hybrid or recombinant diploid or polyploid genomes and identifying parental ancestry for sub-genomic regions.

Genomics , High-Throughput Nucleotide Sequencing , Algorithms , Haplotypes , Phylogeny , Polymorphism, Single Nucleotide , Recombination, Genetic , Sequence Analysis, DNA
Medicine (Baltimore) ; 100(41): e27334, 2021 Oct 15.
Article En | MEDLINE | ID: mdl-34731104

RATIONALE: Pulmonary veno-occlusive disease (PVOD) is a kind of rare and fatal pulmonary arterial hypertension (PAH). Different from other subtypes of PAH, PVOD patients have a very poor prognosis because of the progressive nature of pulmonary vascular involvement and fatal pulmonary edema induced by PAH-targeted drugs. Lung transplantation is the only choice for these patients. PATIENT CONCERNS: We reported 2 cases of PVOD which was misdiagnosed as idiopathic pulmonary arterial hypertension initially due to the lack of typical findings of PVOD. Right heart catheterization was done. The results showed severe PAH with mean pulmonary artery pressure at 76 mmHg and 68 mmHg. DIAGNOSIS: The diagnosis of idiopathic pulmonary arterial hypertension was corrected by eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4) mutation screening. Biallelic mutations (c.1387delT (p. Arg463fs); c.989-990 delAA (p. Lys330fs)) were detected by next-generation sequencing for whole exome from blood sample. The presence of biallelic EIF2AK4 mutation was sufficient to confirm the diagnosis of PVOD. INTERVENTIONS: The 2 patients had good response to PAH-targeted therapy (Ambrisentan 10 mg once a day and tadalafil 20 mg once a day) in the following 1 year. OUTCOMES: Because the patients had a good response to targeted drugs, the treatment of the 2 cases was unchanged. Over 1-year period, they still have a good response to PAH-targeted drugs. There was no sign of pulmonary edema. LESSONS: All these results may indicate that PVOD is not so rare and typical findings of PVOD are lacking in some patients. EIF2AK4 mutation screening by next-generation sequencing maybe useful to differentiate PVOD from other PAH subtypes. PVOD is a heterogeneity population and different patients have different characteristics including response to PAH-targeted therapy. How to pick off this portion of patients timely is the core issue. Further study is necessary to answer this question.

Antihypertensive Agents/therapeutic use , Phenylpropionates/therapeutic use , Pulmonary Veno-Occlusive Disease/diagnosis , Pulmonary Veno-Occlusive Disease/drug therapy , Pyridazines/therapeutic use , Tadalafil/therapeutic use , Vasodilator Agents/therapeutic use , Adult , Diagnostic Errors , Drug Therapy, Combination , High-Throughput Nucleotide Sequencing , Humans , Male , Protein-Serine-Threonine Kinases , Pulmonary Arterial Hypertension/diagnosis , Pulmonary Veno-Occlusive Disease/genetics
Sci Rep ; 11(1): 21632, 2021 11 03.
Article En | MEDLINE | ID: mdl-34732835

At Wuhan, in December 2019, the SRAS-CoV-2 outbreak was detected and it has been the pandemic worldwide. This study aims to investigate the mutations in sequence of the SARS-CoV-2 genome and characterize the mutation patterns in Egyptian COVID-19 patients during different waves of infection. The samples were collected from 250 COVID-19 patients and the whole genome sequencing was conducted using Next Generation Sequencing. The viral sequence analysis showed 1115 different genome from all Egyptian samples in the second wave mutations including 613 missense mutations, 431 synonymous mutations, 25 upstream gene mutations, 24 downstream gene mutations, 10 frame-shift deletions, and 6 stop gained mutation. The Egyptian genomic strains sequenced in second wave of infection are different to that of the first wave. We observe a shift of lineage prevalence from the strain B.1 to B.1.1.1. Only one case was of the new English B.1.1.7. Few samples have one or two mutations of interest from the Brazil and South Africa isolates. New clade 20B appear by March 2020 and 20D appear by May 2020 till January 2021.

Genome, Viral , SARS-CoV-2 , Whole Genome Sequencing , COVID-19 , High-Throughput Nucleotide Sequencing , Humans , Pandemics , Phylogeny
Acta Chim Slov ; 68(2): 268-278, 2021 Jun.
Article En | MEDLINE | ID: mdl-34738119

Despite being around for more than 40 years, DNA sequencing is regarded as young technology in clinical medicine. As sequencing is becoming cheaper, faster and more accurate, it is rapidly being incorporated into clinical laboratories. In 2003, the completion of the first human genome opened the door to personalized medicine. Ever since it has been expected for genomics to widely impact clinical care and public health. However, many years can pass for genomic discoveries to reflect back and benefit the patients. DNA sequencing represents a less biased approach to diagnostics. It is not only a diagnostic tool, but can also influence clinical management and therapy. As new technologies rapidly emerge it is important for researchers and health professionals to have basic knowledge about the capabilities and drawbacks of the existing sequencing methods, and their use in clinical setting and research. This review provides an overview of nucleic acid sequencing technologies from historical perspective and later focuses on clinical utilization of sequencing. Some of the most promising areas are presented with selected examples from Slovenian researchers.

COVID-19 , Genome, Human , High-Throughput Nucleotide Sequencing , Humans , Precision Medicine , Sequence Analysis, DNA
Zhongguo Dang Dai Er Ke Za Zhi ; 23(11): 1154-1160, 2021 Nov 15.
Article En, Zh | MEDLINE | ID: mdl-34753548

OBJECTIVES: To investigate the diversity of peripheral blood T cell receptor (TCR) ß chain complementarity-determining region 3 (CDR3) based on immune repertoire sequencing in neonates with sepsis and the possible pathogenesis of neonatal sepsis. METHODS: A total of 12 neonates with sepsis were enrolled as the case group, and 9 healthy full-term infants, matched for gestational age, birth weight, and age, were enrolled as the control group. Omega nucleic acid purification kit (SQ blood DNA Kit II) was used to extract DNA from peripheral blood samples, TCR ß chain CDR3 was amplified by multiplex PCR, and then high-throughput sequencing was performed for the products to analyze the diversity of TCR ß chain CDR3 and the difference in expression. RESULTS: The length and type of TCR ß chain CDR3 were similar between the case and control groups, and Gaussian distribution was observed in both groups. With D50 and Shannon-Wiener index as the evaluation indices for diversity, the case group had a significantly lower diversity of TCR ß chain CDR3 than the control group (P<0.05). The frequency of 48 genes in TCR ß chain V segment was compared, and the results showed that compared with the control group, the case group had significantly higher frequencies of TRBV10-3, TRBV2, and TRBV20-1 (P<0.05). The frequency of 13 genes in TCR ß chain J segment were compared, and the results showed that compared with the control group, the case group had significantly higher frequencies of TRBJ2-3, TRBJ2-5, and TRBJ2-7 (P<0.05). CONCLUSIONS: There is a significant change in the diversity of TCR ß chain CDR3 in the peripheral blood of neonates with sepsis, suggesting that it might be associated with the immune pathogenesis of neonatal sepsis.

Complementarity Determining Regions , Neonatal Sepsis , Complementarity Determining Regions/genetics , High-Throughput Nucleotide Sequencing , Humans , Multiplex Polymerase Chain Reaction , Receptors, Antigen, T-Cell, alpha-beta/genetics