scholarly journals Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

2017 ◽  
Author(s):  
Josh Quick ◽  
Nathan D Grubaugh ◽  
Steven T Pullan ◽  
Ingra M Claro ◽  
Andrew D Smith ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Genome Sequencing ◽  
Emerging Infectious Diseases ◽  
Design Tool ◽  
Clinical Samples ◽  
Metagenomic Sequencing ◽  
Bioinformatics Pipeline ◽  
Preparation Methods ◽  
Consensus Sequences ◽  
Viral Genomes

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples without isolation remains challenging for viruses such as Zika, where metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence complete genomes comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimised library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved starting with clinical samples in 1-2 days following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas.

scholarly journals Assessment of Metagenomic MinION and Illumina sequencing as an approach for the recovery of whole genome sequences of chikungunya and dengue viruses directly from clinical samples

2018 ◽  
Author(s):  
Liana E. Kafetzopoulou ◽  
Kyriakos Efthymiadis ◽  
Kuiama Lewandowski ◽  
Ant Crook ◽  
Dan Carter ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
Clinical Samples ◽  
Whole Genome ◽  
Metagenomic Sequencing ◽  
Front Line ◽  
Genome Sequences ◽  
Oxford Nanopore

AbstractThe recent global emergence and re-emergence of arboviruses has caused significant human disease. Common vectors, symptoms and geographical distribution make differential diagnosis both important and challenging. We performed metagenomic sequencing using both the Illumina MiSeq and the portable Oxford Nanopore MinION to study the feasibility of whole genome sequencing from clinical samples containing chikungunya or dengue virus, two of the most important arboviruses. Direct metagenomic sequencing of nucleic acid extracts from serum and plasma without viral enrichment allowed for virus and coinfection identification, subtype determination and in the majority of cases elucidated complete or near-complete genomes adequate for phylogenetic analysis. This work demonstrates that metagenomic whole genome sequencing is feasible for over 90% and 80% of chikungunya and dengue virus PCR-positive patient samples respectively. It confirms the feasibility of field metagenomic sequencing for these and likely other RNA viruses, highlighting the applicability of this approach to front-line public health.

scholarly journals Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 841 ◽  
Author(s):  
Carole Grädel ◽  
Miguel A. Terrazos Miani ◽  
Christian Baumann ◽  
Maria Teresa Barbani ◽  
Stefan Neuenschwander ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Rna Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
High Viral Load ◽  
Clinical Samples ◽  
Whole Genome ◽  
Consensus Sequences ◽  
Positive Stool ◽  
Stool Samples

Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains.

scholarly journals MINERVA: A facile strategy for SARS-CoV-2 whole genome deep sequencing of clinical samples

2020 ◽  
Author(s):  
Chen Chen ◽  
Jizhou Li ◽  
Lin Di ◽  
Qiuyu Jing ◽  
Pengcheng Du ◽  
...  
Keyword(s):  
High Performance ◽  
Vaccine Development ◽  
Viral Evolution ◽  
High Sensitivity ◽  
High Yield ◽  
Clinical Samples ◽  
High Coverage ◽  
Preparation Methods ◽  
Viral Genomes ◽  
Viral Spread

AbstractThe novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from clinical samples is crucial for the understanding of viral spread and viral evolution, as well as for vaccine development. Existing sample preparation methods for viral genome sequencing are demanding on user technique and time, and thus not ideal for time-sensitive clinical samples; these methods are also not optimized for high performance on viral genomes. We have developed MetagenomIc RNA EnRichment VirAl sequencing (MINERVA), a facile, practical, and robust approach for metagenomic and deep viral sequencing from clinical samples. This approach uses direct tagmentation of RNA/DNA hybrids using Tn5 transposase to greatly simplify the sequencing library construction process, while subsequent targeted enrichment can generate viral genomes with high sensitivity, coverage, and depth. We demonstrate the utility of MINERVA on pharyngeal, sputum and stool samples collected from COVID-19 patients, successfully obtaining both whole metatranscriptomes and complete high-depth high-coverage SARS-CoV-2 genomes from these clinical samples, with high yield and robustness. MINERVA is compatible with clinical nucleic extracts containing carrier RNA. With a shortened hands-on time from sample to virus-enriched sequencing-ready library, this rapid, versatile, and clinic-friendly approach will facilitate monitoring of viral genetic variations during outbreaks, both current and future.

scholarly journals Two-Pool Multiplex Long-Amplicon Amplification and Full-Genome Sequencing of SARS-CoV-2

2020 ◽  
Author(s):  
Hong-jie Liu ◽  
Jin-Hui Li ◽  
Yan-Feng Lin ◽  
Xiao-Chen Bo ◽  
Hong-Bin Song ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Genome Sequencing ◽  
Massively Parallel Sequencing ◽  
Clinical Samples ◽  
Nanopore Sequencing ◽  
Full Genome ◽  
Full Genome Sequencing ◽  
Genome Sequences ◽  
Genomic Variations ◽  
Pcr Targeting

Abstract BackgroundSince the first public genome of SARS-CoV-2, over 170,000 genome sequences of the virus have been shared by researchers world-wide (till November 1st 2020). Multiplex PCR targeting SARS-CoV-2 followed by massively parallel sequencing (MPS) and/or nanopore sequencing is a widely used strategy to recover the genome from primary samples. However, the bias of amplification among different amplicons should not be ignored, which might lead to uneven sequencing coverage on the viral genome.MethodsWe aim to develop a novel multiplex PCR panel to achieve an improved coverage evenness of SARS-CoV-2. We adapt long amplicons (~1000-bp) for the panel and thus reduced the number of primer pairs. The panel was validated with clinical samples and sequenced via MPS sequencing systems and a portable nanopore sequencing device MinION. We evaluated the full-genome coverage evenness and its dependence on viral loads of the long amplicon panel; we then compared it with a 98-plex panel provided by the ARTIC network. The accuracy to identify viral genomic variations based on the panel and sequencing with MinION was assessed.ResultsWe developed a two-pool 36-plex panel for full-genome sequencing of SARS-CoV-2, whose amplicon size ranged from 880 to 1027 bp. For samples with a <30 Ct value, >90% viral genome could be recovered with a high sequencing depth (>0.2 mean depth) by using the long-amplicon panel (n = 36), compared with 79-88% highly covered genome region for the ARTIC panel (n = 5). The coverage evenness of the long-amplicon panel was also less affected by low viral titers and not dependent on sequencing data amount. With MinION sequencing, the consensus viral genomes could be reliably recovered. However, a high false positive rate was observed to identify sub-clonal genomic variations with a <0.6 frequency.ConclusionA novel multiplex PCR panel for full-genome sequencing of SARS-CoV-2 with improved coverage evenness and low requirement of data throughput was validated with clinical samples. Amplification of SARS-CoV-2 with the panel followed by MinION sequencing could generate reliable consensus genome sequences, but the detection of non-dominating viral populations within host is error-prone.

scholarly journals Whole genome sequencing of human enteroviruses from clinical samples by nanopore direct RNA sequencing

2020 ◽  
Author(s):  
C. Grädel ◽  
M.A. Terrazos Miani ◽  
C. Baumann ◽  
MT Barbani ◽  
S. Neuenschwander ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Rna Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
High Viral Load ◽  
Clinical Samples ◽  
Whole Genome ◽  
Consensus Sequences ◽  
Positive Stool ◽  
Stool Samples

AbstractEnteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, those viruses are identified by PCR based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. Results of the approach were complemented with those obtained by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% to 99.6 % of the best reference genomes. The identification of the enterovirus sequences in the sample was confirmed by the short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94-97%. Here we show that nanopore DRS can be used to correctly identify the genotypes of enteroviruses from patient stool samples with high viral load.

scholarly journals Increased yields of duplex sequencing data by a series of quality control tools

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Gundula Povysil ◽  
Monika Heinzl ◽  
Renato Salazar ◽  
Nicholas Stoler ◽  
Anton Nekrutenko ◽  
...  
Keyword(s):  
Low Frequency ◽  
Variant Calling ◽  
Data Loss ◽  
Sequencing Data ◽  
Bioinformatics Pipeline ◽  
Consensus Sequences ◽  
Sequencing Errors ◽  
Data Output ◽  
Reverse Strand ◽  
Duplex Sequencing

Abstract Duplex sequencing is currently the most reliable method to identify ultra-low frequency DNA variants by grouping sequence reads derived from the same DNA molecule into families with information on the forward and reverse strand. However, only a small proportion of reads are assembled into duplex consensus sequences (DCS), and reads with potentially valuable information are discarded at different steps of the bioinformatics pipeline, especially reads without a family. We developed a bioinformatics toolset that analyses the tag and family composition with the purpose to understand data loss and implement modifications to maximize the data output for the variant calling. Specifically, our tools show that tags contain polymerase chain reaction and sequencing errors that contribute to data loss and lower DCS yields. Our tools also identified chimeras, which likely reflect barcode collisions. Finally, we also developed a tool that re-examines variant calls from raw reads and provides different summary data that categorizes the confidence level of a variant call by a tier-based system. With this tool, we can include reads without a family and check the reliability of the call, that increases substantially the sequencing depth for variant calling, a particular important advantage for low-input samples or low-coverage regions.

scholarly journals Cattle connection: molecular epidemiology of BVDV outbreaks via rapid nanopore whole-genome sequencing of clinical samples

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Jacqueline King ◽  
Anne Pohlmann ◽  
Kamila Dziadek ◽  
Martin Beer ◽  
Kerstin Wernike
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Economic Losses ◽  
Clinical Samples ◽  
Bovine Viral Diarrhea ◽  
Sequence Information ◽  
Whole Genome ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Loads

Abstract Background As a global ruminant pathogen, bovine viral diarrhea virus (BVDV) is responsible for the disease Bovine Viral Diarrhea with a variety of clinical presentations and severe economic losses worldwide. Classified within the Pestivirus genus, the species Pestivirus A and B (syn. BVDV-1, BVDV-2) are genetically differentiated into 21 BVDV-1 and four BVDV-2 subtypes. Commonly, the 5’ untranslated region and the Npro protein are utilized for subtyping. However, the genetic variability of BVDV leads to limitations in former studies analyzing genome fragments in comparison to a full-genome evaluation. Results To enable rapid and accessible whole-genome sequencing of both BVDV-1 and BVDV-2 strains, nanopore sequencing of twelve representative BVDV samples was performed on amplicons derived through a tiling PCR procedure. Covering a multitude of subtypes (1b, 1d, 1f, 2a, 2c), sample matrices (plasma, EDTA blood and ear notch), viral loads (Cq-values 19–32) and species (cattle and sheep), ten of the twelve samples produced whole genomes, with two low titre samples presenting 96 % genome coverage. Conclusions Further phylogenetic analysis of the novel sequences emphasizes the necessity of whole-genome sequencing to identify novel strains and supplement lacking sequence information in public repositories. The proposed amplicon-based sequencing protocol allows rapid, inexpensive and accessible obtainment of complete BVDV genomes.

scholarly journals CSMD: a computational subtraction-based microbiome discovery pipeline for species-level characterization of clinical metagenomic samples

2019 ◽  
Author(s):  
Yu Liu ◽  
Paul W Bible ◽  
Bin Zou ◽  
Qiaoxing Liang ◽  
Cong Dong ◽  
...  
Keyword(s):  
Species Level ◽  
Supplementary Information ◽  
Clinical Samples ◽  
Plasma Dna ◽  
Bioinformatics Pipeline ◽  
Microbial Identification ◽  
Reference Databases ◽  
Microbial Dna ◽  
Higher Sensitivity

Abstract Motivation Microbiome analyses of clinical samples with low microbial biomass are challenging because of the very small quantities of microbial DNA relative to the human host, ubiquitous contaminating DNA in sequencing experiments and the large and rapidly growing microbial reference databases. Results We present computational subtraction-based microbiome discovery (CSMD), a bioinformatics pipeline specifically developed to generate accurate species-level microbiome profiles for clinical samples with low microbial loads. CSMD applies strategies for the maximal elimination of host sequences with minimal loss of microbial signal and effectively detects microorganisms present in the sample with minimal false positives using a stepwise convergent solution. CSMD was benchmarked in a comparative evaluation with other classic tools on previously published well-characterized datasets. It showed higher sensitivity and specificity in host sequence removal and higher specificity in microbial identification, which led to more accurate abundance estimation. All these features are integrated into a free and easy-to-use tool. Additionally, CSMD applied to cell-free plasma DNA showed that microbial diversity within these samples is substantially broader than previously believed. Availability and implementation CSMD is freely available at https://github.com/liuyu8721/csmd. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Correction to: Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

Microbiome ◽  
2017 ◽  
Vol 5 (1) ◽  
Author(s):  
Dagmara W. Lewandowska ◽  
Osvaldo Zagordi ◽  
Fabienne-Desirée Geissberger ◽  
Verena Kufner ◽  
Stefan Schmutz ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Clinical Samples ◽  
Metagenomic Sequencing

scholarly journals Simplified Fluorescent Multiplex PCR Method for Evaluation of the T-Cell Receptor Vβ-Chain Repertoire

2005 ◽  
Vol 12 (4) ◽  
pp. 477-483 ◽  
Author(s):  
Sanjit Fernandes ◽  
Surendra Chavan ◽  
Vivek Chitnis ◽  
Nina Kohn ◽  
Savita Pahwa
Keyword(s):  
T Cell ◽  
Multiplex Pcr ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
Clinical Samples ◽  
Cdr3 Length ◽  
Pcr Products ◽  
Pcr Method ◽  
T Cell Receptor Vβ

ABSTRACTRationale: evaluation of the T-cell receptor (TCR) Vβ-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR Vβ repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream Vβ primers instead of the conventionally labeled downstream Cβ primer is described. Method: RNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a Cβ primer and an Omniscript RT kit. The 24 Vβ primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 Vβ primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled Cβ primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software. Results: Vβ spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled Vβ primers. The resolution was superior to that obtained with the labeled Cβ primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the Cβ labeling method, and the sample processing time was reduced by half. Conclusion: The method described for T-cell receptor Vβ-chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR Vβ repertoire analyses in clinical trials.

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