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

2021 ◽  
Author(s):  
Elizabeth Jaworski ◽  
Rose M. Langsjoen ◽  
Barbara Judy ◽  
Patrick Newman ◽  
Jessica A. Plante ◽  
...  
Keyword(s):  
Full Range ◽  
Virus Genome ◽  
Rna Recombination ◽  
Single Nucleotide ◽  
Reverse Transcription Reaction ◽  
Minority Variants ◽  
Next Generation Sequencing Ngs ◽  
Unique Molecular Identifier ◽  
Generation Sequencing ◽  
High Depth

AbstractHigh-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’. Tiled-ClickSeq 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, obviating the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended using click-chemistry and a PCR reaction using Illumina adaptors generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5’UTR, at high depth and specificity to virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and 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 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 Next-Generation Sequencing in Tumor Diagnosis and Treatment

Diagnostics ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 962
Author(s):  
Dario de Biase ◽  
Matteo Fassan ◽  
Umberto Malapelle
Keyword(s):  
Next Generation Sequencing ◽  
Tumor Diagnosis ◽  
Diagnosis And Treatment ◽  
Next Generation ◽  
Multiple Genes ◽  
Next Generation Sequencing Ngs ◽  
Very High ◽  
Generation Sequencing ◽  
High Depth

Next-Generation Sequencing (NGS) allows for the sequencing of multiple genes at a very high depth of coverage [...]

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 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.

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.

scholarly journals Technologies for Pharmacogenomics: A Review

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1456
Author(s):  
Maaike van der Lee ◽  
Marjolein Kriek ◽  
Henk-Jan Guchelaar ◽  
Jesse J. Swen
Keyword(s):  
Clinical Practice ◽  
Rare Variants ◽  
Turnaround Time ◽  
Sequencing Data ◽  
Single Nucleotide ◽  
Long Read ◽  
The Right ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Straightforward Interpretation

The continuous development of new genotyping technologies requires awareness of their potential advantages and limitations concerning utility for pharmacogenomics (PGx). In this review, we provide an overview of technologies that can be applied in PGx research and clinical practice. Most commonly used are single nucleotide variant (SNV) panels which contain a pre-selected panel of genetic variants. SNV panels offer a short turnaround time and straightforward interpretation, making them suitable for clinical practice. However, they are limited in their ability to assess rare and structural variants. Next-generation sequencing (NGS) and long-read sequencing are promising technologies for the field of PGx research. Both NGS and long-read sequencing often provide more data and more options with regard to deciphering structural and rare variants compared to SNV panels—in particular, in regard to the number of variants that can be identified, as well as the option for haplotype phasing. Nonetheless, while useful for research, not all sequencing data can be applied to clinical practice yet. Ultimately, selecting the right technology is not a matter of fact but a matter of choosing the right technique for the right problem.

scholarly journals A novel NGS library preparation method to characterize native termini of fragmented DNA

2020 ◽  
Vol 48 (8) ◽  
pp. e47-e47 ◽  
Author(s):  
Kelly M Harkins ◽  
Nathan K Schaefer ◽  
Christopher J Troll ◽  
Varsha Rao ◽  
Joshua Kapp ◽  
...  
Keyword(s):  
Library Preparation ◽  
Single Nucleotide ◽  
Preparation Methods ◽  
Fragmented Dna ◽  
Genomic Landscape ◽  
Dna Fragment ◽  
Nucleotide Resolution ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Biological and chemical DNA fragmentation generates DNA molecules with a variety of termini, including blunt ends and single-stranded overhangs. We have developed a Next Generation Sequencing (NGS) assay, XACTLY, to interrogate the termini of fragmented DNA, information traditionally lost in standard NGS library preparation methods. Here we describe the XACTLY method, showcase its sensitivity and specificity, and demonstrate its utility in in vitro experiments. The XACTLY assay is able to report relative abundances of all lengths and types (5′ and 3′) of single-stranded overhangs, if present, on each DNA fragment with an overall accuracy between 80–90%. In addition, XACTLY retains the sequence of each native DNA molecule after fragmentation and can capture the genomic landscape of cleavage events at single nucleotide resolution. The XACTLY assay can be applied as a novel research and discovery tool for fragmentation analyses and in cell-free DNA.

Ribosomal profiling adds new coding sequences to the proteome

2015 ◽  
Vol 43 (6) ◽  
pp. 1271-1276 ◽  
Author(s):  
Muhammad Ali S. Mumtaz ◽  
Juan Pablo Couso
Keyword(s):  
Single Nucleotide ◽  
Protein Coding ◽  
Large Numbers ◽  
New Findings ◽  
Genomic Scale ◽  
Bioinformatic Approaches ◽  
And Function ◽  
Next Generation Sequencing Ngs ◽  
Small Orfs ◽  
Generation Sequencing

Next generation sequencing (NGS) has enabled an in-depth look into genes, transcripts and their translation at the genomic scale. The application of NGS sequencing of ribosome footprints (Ribo-Seq) reveals translation with single nucleotide (nt) resolution, through the deep sequencing of ribosome-bound fragments (RBFs). Some results of Ribo-Seq challenge our understanding of the protein-coding potential of the genome. Earlier bioinformatic approaches had shown the presence of hundreds of thousands of putative small ORFs (smORFs) in eukaryotic genomes, but they had been largely ignored due to their large numbers and difficulty in determining their translation and function. Ribo-Seq has revealed that hundreds of putative smORFs within previously assumed long non-coding RNAs (lncRNAs) and UTRs of canonical mRNAs are associated with ribosomes, appearing to be translated. Here we review some of the approaches used to define translation within Ribo-Seq experiments and the challenges in defining translation of these novel smORFs in lncRNAs and UTRs. We also look at some of the bioinformatic and biochemical approaches used to independently corroborate these exciting new findings and elucidate real translation events.

scholarly journals Sanger sequencing is no longer always necessary based on a single-center validation of 1109 NGS variants in 825 clinical exomes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
A. Arteche-López ◽  
A. Ávila-Fernández ◽  
R. Romero ◽  
R. Riveiro-Álvarez ◽  
M. A. López-Martínez ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Turnaround Time ◽  
Copy Number Variations ◽  
Internal Quality ◽  
Single Nucleotide ◽  
Verification Method ◽  
Next Generation Sequencing Ngs ◽  
Improved Accuracy ◽  
Sample Set ◽  
Generation Sequencing

AbstractDespite the improved accuracy of next-generation sequencing (NGS), it is widely accepted that variants need to be validated using Sanger sequencing before reporting. Validation of all NGS variants considerably increases the turnaround time and costs of clinical diagnosis. We comprehensively assessed this need in 1109 variants from 825 clinical exomes, the largest sample set to date assessed using Illumina chemistry reported. With a concordance of 100%, we conclude that Sanger sequencing can be very useful as an internal quality control, but not so much as a verification method for high-quality single-nucleotide and small insertion/deletions variants. Laboratories might validate and establish their own thresholds before discontinuing Sanger confirmation studies. We also expand and validate 23 copy number variations detected by exome sequencing in 20 samples, observing a concordance of 95.65% (22/23).

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