scholarly journals Intra-Host Diversity of SARS-Cov-2 Should Not Be Neglected: Case of the State of Victoria, Australia

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 133
Author(s):  
Alix Armero ◽  
Nicolas Berthet ◽  
Jean-Christophe Avarre
Keyword(s):  
Genetic Bottleneck ◽  
The State ◽  
Potential Importance ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Host Diversity ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Quasispecies Diversity ◽  
Over Time

Since the identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the etiological agent of the current COVID-19 pandemic, a rapid and massive effort has been made to obtain the genomic sequences of this virus to monitor (in near real time) the phylodynamic and diversity of this new pathogen. However, less attention has been given to the assessment of intra-host diversity. RNA viruses such as SARS-CoV-2 inhabit the host as a population of variants called quasispecies. We studied the quasispecies diversity in four of the main SARS-CoV-2 genes (ORF1a, ORF1b, S and N genes), using a dataset consisting of 210 next-generation sequencing (NGS) samples collected between January and early April of 2020 in the State of Victoria, Australia. We found evidence of quasispecies diversity in 68% of the samples, 76% of which was nonsynonymous variants with a higher density in the spike (S) glycoprotein and ORF1a genes. About one-third of the nonsynonymous intra-host variants were shared among the samples, suggesting host-to-host transmission. Quasispecies diversity changed over time. Phylogenetic analysis showed that some of the intra-host single-nucleotide variants (iSNVs) were restricted to specific lineages, highlighting their potential importance in the epidemiology of this virus. A greater effort must be made to determine the magnitude of the genetic bottleneck during transmission and the epidemiological and/or evolutionary factors that may play a role in the changes in the diversity of quasispecies over time.

scholarly journals Targeted Next-Generation Resequencing of Gene Identifies Novel Multiple Variants Pattern in Severe Hereditary Factor V Deficiency

2013 ◽  
Vol 2013 ◽  
pp. 1-7
Author(s):  
Piotr K. Janicki ◽  
Sonia Vaida ◽  
Hamid A. B. AL-Mondhiry
Keyword(s):  
Factor V ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Factor V Deficiency ◽  
Newborn Child ◽  
Next Generation Sequencing Ngs ◽  
Multiple Variants ◽  
Generation Sequencing ◽  
Entire Gene

The present study investigated the genetic defects underlying severe Factor V deficiency in a 26-year-old Columbian (South America) female and her immediate family (both parents and newborn child) by next generation sequencing (NGS) of the entire gene locus. Five mutations in the coding sequence of , including three missense single-nucleotide variants (R2102H, R513K, D107H) and two synonymous variants (A135A , S184S), were identified and confirmed by the Sanger sequencing in the investigated proband (homozygote for all detected mutations), her parents, and her newborn child (all heterozygotic carriers for identified mutations). Each of the three missense variants was previously associated with separate phenotypes, including Factor V deficiency (R2102H), thrombosis (R513K) and frequent miscarriages (D107H). In addition, at least 75 additional single-nucleotide variants (including six novels) were identified in untranslated region of .

scholarly journals Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Elizabeth Jaworski ◽  
Rose M Langsjoen ◽  
Brooke Mitchell ◽  
Barbara Judy ◽  
Patrick Newman ◽  
...  
Keyword(s):  
Full Range ◽  
Virus Genome ◽  
Clinical Samples ◽  
Rna Recombination ◽  
Single Nucleotide ◽  
Reverse Transcription Reaction ◽  
Minority Variants ◽  
Next Generation Sequencing Ngs ◽  
Unique Molecular Identifier ◽  
Generation Sequencing

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called ‘Tiled-ClickSeq’, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5’UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

scholarly journals Repli-seq: genome-wide analysis of replication timing by next-generation sequencing

2017 ◽  
Author(s):  
Claire Marchal ◽  
Takayo Sasaki ◽  
Daniel Vera ◽  
Korey Wilson ◽  
Jiao Sima ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Replication Timing ◽  
Nucleotide Polymorphisms ◽  
Robust Methods ◽  
Next Generation ◽  
Single Nucleotide ◽  
Cellular Processes ◽  
Genome Wide ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

ABSTRACTCycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early and late replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and sub-nuclear position. Moreover, RT is regulated during development and is altered in disease. Exploring mechanisms linking RT to other cellular processes in normal and diseased cells will be facilitated by rapid and robust methods with which to measure RT genome wide. Here, we describe a rapid, robust and relatively inexpensive protocol to analyze genome-wide RT by next-generation sequencing (NGS). This protocol yields highly reproducible results across laboratories and platforms. We also provide computational pipelines for analysis, parsing phased genomes using single nucleotide polymorphisms (SNP) for analyzing RT allelic asynchrony, and for direct comparison to Repli-chip data obtained by analyzing nascent DNA by microarrays.

scholarly journals A Model Study of In Silico Proficiency Testing for Clinical Next-Generation Sequencing

2016 ◽  
Vol 140 (10) ◽  
pp. 1085-1091 ◽  
Author(s):  
Eric J. Duncavage ◽  
Haley J. Abel ◽  
Jason D. Merker ◽  
John B. Bodner ◽  
Qin Zhao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Proficiency Testing ◽  
In Silico ◽  
Absolute Difference ◽  
Ion Torrent ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Clinical Laboratories ◽  
Generation Sequencing

Context.—Most current proficiency testing challenges for next-generation sequencing assays are methods-based proficiency testing surveys that use DNA from characterized reference samples to test both the wet-bench and bioinformatics/dry-bench aspects of the tests. Methods-based proficiency testing surveys are limited by the number and types of mutations that either are naturally present or can be introduced into a single DNA sample. Objective.—To address these limitations by exploring a model of in silico proficiency testing in which sequence data from a single well-characterized specimen are manipulated electronically. Design.—DNA from the College of American Pathologists reference genome was enriched using the Illumina TruSeq and Life Technologies AmpliSeq panels and sequenced on the MiSeq and Ion Torrent platforms, respectively. The resulting data were mutagenized in silico and 26 variants, including single-nucleotide variants, deletions, and dinucleotide substitutions, were added at variant allele fractions (VAFs) from 10% to 50%. Participating clinical laboratories downloaded these files and analyzed them using their clinical bioinformatics pipelines. Results.—Laboratories using the AmpliSeq/Ion Torrent and/or the TruSeq/MiSeq participated in the 2 surveys. On average, laboratories identified 24.6 of 26 variants (95%) overall and 21.4 of 22 variants (97%) with VAFs greater than 15%. No false-positive calls were reported. The most frequently missed variants were single-nucleotide variants with VAFs less than 15%. Across both challenges, reported VAF concordance was excellent, with less than 1% median absolute difference between the simulated VAF and mean reported VAF. Conclusions.—The results indicate that in silico proficiency testing is a feasible approach for methods-based proficiency testing, and demonstrate that the sensitivity and specificity of current next-generation sequencing bioinformatics across clinical laboratories are high.

scholarly journals Genome aging: somatic mutation in the brain links age-related decline with disease and nominates pathogenic mechanisms

2019 ◽  
Vol 28 (R2) ◽  
pp. R197-R206 ◽  
Author(s):  
Michael A Lodato ◽  
Christopher A Walsh
Keyword(s):  
Human Brain ◽  
Somatic Mutation ◽  
Somatic Mutations ◽  
Late Onset ◽  
Whole Genome ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Age Related ◽  
The Brain ◽  
Over Time

AbstractAging is a mysterious process, not only controlled genetically but also subject to random damage that can accumulate over time. While DNA damage and subsequent mutation in somatic cells were first proposed as drivers of aging more than 60 years ago, whether and to what degree these processes shape the neuronal genome in the human brain could not be tested until recent technological breakthroughs related to single-cell whole-genome sequencing. Indeed, somatic single-nucleotide variants (SNVs) increase with age in the human brain, in a somewhat stochastic process that may nonetheless be controlled by underlying genetic programs. Evidence from the literature suggests that in addition to demonstrated increases in somatic SNVs during aging in normal brains, somatic mutation may also play a role in late-onset, sporadic neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease. In this review, we will discuss somatic mutation in the human brain, mechanisms by which somatic mutations occur and can be controlled, and how this process can impact human health.

scholarly journals A customized scaffolds approach for the detection and phasing of complex variants by next-generation sequencing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Qiandong Zeng ◽  
Natalia T. Leach ◽  
Zhaoqing Zhou ◽  
Hui Zhu ◽  
Jean A. Smith ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Causative Mutation ◽  
Next Generation ◽  
Single Nucleotide ◽  
Highly Sensitive ◽  
Highly Sensitive Detection ◽  
Next Generation Sequencing Ngs ◽  
Reference Bias ◽  
Generation Sequencing

Abstract Next-generation sequencing (NGS) is widely used in genetic testing for the highly sensitive detection of single nucleotide changes and small insertions or deletions. However, detection and phasing of structural variants, especially in repetitive or homologous regions, can be problematic due to uneven read coverage or genome reference bias, resulting in false calls. To circumvent this challenge, a computational approach utilizing customized scaffolds as supplementary reference sequences for read alignment was developed, and its effectiveness demonstrated with two CBS gene variants: NM_000071.2:c.833T>C and NM_000071.2:c.[833T>C; 844_845ins68]. Variant c.833T>C is a known causative mutation for homocystinuria, but is not pathogenic when in cis with the insertion, c.844_845ins68, because of alternative splicing. Using simulated reads, the custom scaffolds method resolved all possible combinations with 100% accuracy and, based on > 60,000 clinical specimens, exceeded the performance of current approaches that only align reads to GRCh37/hg19 for the detection of c.833T>C alone or in cis with c.844_845ins68. Furthermore, analysis of two 1000 Genomes Project trios revealed that the c.[833T>C; 844_845ins68] complex variant had previously been undetected in these datasets, likely due to the alignment method used. This approach can be configured for existing workflows to detect other challenging and potentially underrepresented variants, thereby augmenting accurate variant calling in clinical NGS testing.

scholarly journals Prostate cancer heterogeneity assessment with multi-regional sampling and alignment-free methods

2020 ◽  
Vol 2 (3) ◽  
Author(s):  
Ross G Murphy ◽  
Aideen C Roddy ◽  
Shambhavi Srivastava ◽  
Esther Baena ◽  
David J Waugh ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Cancer Heterogeneity ◽  
Alignment Free ◽  
Treatment Indications ◽  
Patient Heterogeneity ◽  
Genomic Locations ◽  
Generation Sequencing

Abstract Combining alignment-free methods for phylogenetic analysis with multi-regional sampling using next-generation sequencing can provide an assessment of intra-patient tumour heterogeneity. From multi-regional sampling divergent branching, we validated two different lesions within a patient’s prostate. Where multi-regional sampling has not been used, a single sample from one of these areas could misguide as to which drugs or therapies would best benefit this patient, due to the fact these tumours appear to be genetically different. This application has the power to render, in a fraction of the time used by other approaches, intra-patient heterogeneity and decipher aberrant biomarkers. Another alignment-free method for calling single-nucleotide variants from raw next-generation sequencing samples has determined possible variants and genomic locations that may be able to characterize the differences between the two main branching patterns. Alignment-free approaches have been applied to relevant clinical multi-regional samples and may be considered as a valuable option for comparing and determining heterogeneity to help deliver personalized medicine through more robust efforts in identifying targetable pathways and therapeutic strategies. Our study highlights the application these tools could have on patient-aligned treatment indications.

Molekulare Blutgruppenbestimmung

2017 ◽  
Vol 7 (02) ◽  
pp. 87-94
Author(s):  
Christoph Gassner
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Next Generation Sequencing ◽  
Blood Transfusion ◽  
International Society ◽  
In Silico ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Molekulare Analyse ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

ZusammenfassungDie erste molekulare Analyse einer menschlichen Blutgruppe erfolgte 1983 mittels eines Restriktions-Fragment-Längen-Polymorphismus (RFLP) am System Xg. Seither wurden in unzähligen Studien die molekularen Ursachen für Blutgruppen und deren Antigene erforscht,und das resultierende Wissen für eine ständige Verbesserung der entsprechenden Analysemethoden verwendet. Die Untersuchung kausaler Punktmutationen (Single Nucleotide Polymorphism, SNP) aller 36 von der International Society for Blood Transfusion (ISBT) anerkannten Blutgruppensysteme erlaubt heute eine der Serologie ebenbürtige, exakte Vorhersage der Blutgruppenantigene. In Patienten wird die molekulare Blutgruppenbestimmung bevorzugt in Form von Einzelprobenanalytik für die Diagnose von RhD-Varianten eingesetzt, um damit transfusionsrelevante Entscheidungen bezüglich RhD zu treffen und um die Rh-Prophylaxe noch zielsicherer zu steuern. An Spenderproben und im Hochdurchsatz ermöglicht die Blutgruppen-Genotypisierung die Schaffung einer ausreichenden Anzahl von Spender-Datensätzen, um immunisierte Patienten bestverträglich zu transfundieren oder deren Immunisierung bereits im Ansatz zu vermeiden. Gleichzeitig werden heutzutage an den gleichen Proben zusätzlich eine Vielzahl weiterer SNPs zur Identifikation von Spendern mit seltener Negativität für hochfrequente Antigene getestet. Derartig umfassende Spender-Datensätze werden bereits ideal genutzt für „In-silico-Kreuzproben“ eingesetzt. Next Generation Sequencing (NGS) ist auch in der Transfusionsmedizin der „neue Stern am Horizont“ und wird vermutlich innert weniger Jahre eine wichtige Rolle in der Analyse kompletter Blutgruppengenome (chronischer) Empfänger spielen. Blutgruppenbestimmung als frühestes Beispiel echter personalisierter Medizin wird in ihrer molekularen Version mit dazu beitragen, den gebührenden Platz der Transfusionsmedizin in der modernen Medizin zu behaupten.

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