scholarly journals Rapid and economical drug resistance profiling with Nanopore MinION for clinical specimens with low bacillary burden of Mycobacterium tuberculosis

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Wai Sing Chan ◽  
Chun Hang Au ◽  
Yvonne Chung ◽  
Henry Chi Ming Leung ◽  
Dona N. Ho ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Illumina Miseq ◽  
Allelic Frequency ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Clinical Specimens ◽  
Consensus Sequences ◽  
Reagent Cost ◽  
Sequencing Chemistry ◽  
Assay Time

Abstract Objective We designed and tested a Nanopore sequencing panel for direct tuberculosis drug resistance profiling. The panel targeted 10 resistance-associated loci. We assessed the feasibility of amplifying and sequencing these loci from 23 clinical specimens with low bacillary burden. Results At least 8 loci were successfully amplified from the majority for predicting first- and second-line drug resistance (14/23, 60.87%), and the 12 specimens yielding all 10 targets were sequenced with Nanopore MinION and Illumina MiSeq. MinION sequencing data was corrected by Nanopolish and recurrent variants were filtered. A total of 67,082 bases across all consensus sequences were analyzed, with 67,019 bases called by both MinION and MiSeq as wildtype. For the 41 single nucleotide variants (SNVs) called by MiSeq with 100% variant allelic frequency (VAF), 39 (95.1%) were called by MinION. For the 22 mixed bases called by MiSeq, a SNV with the highest VAF (70%) was called by MinION. With short assay time, reasonable reagent cost as well as continuously improving sequencing chemistry and signal correction pipelines, this Nanopore method can be a viable option for direct tuberculosis drug resistance profiling in the near future.

scholarly journals Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 694 ◽  
Author(s):  
Neil T. Parkin ◽  
Santiago Avila-Rios ◽  
David F. Bibby ◽  
Chanson J. Brumme ◽  
Susan H. Eshleman ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Illumina Miseq ◽  
Next Generation ◽  
Sequence Comparisons ◽  
Consensus Sequences ◽  
Drug Resistance Genotyping ◽  
Next Generation Sequencing Ngs ◽  
Sequence Quality ◽  
Average Sequence Identity ◽  
Hiv 1

Next-generation sequencing (NGS) is increasingly used for HIV-1 drug resistance genotyping. NGS methods have the potential for a more sensitive detection of low-abundance variants (LAV) compared to standard Sanger sequencing (SS) methods. A standardized threshold for reporting LAV that generates data comparable to those derived from SS is needed to allow for the comparability of data from laboratories using NGS and SS. Ten HIV-1 specimens were tested in ten laboratories using Illumina MiSeq-based methods. The consensus sequences for each specimen using LAV thresholds of 5%, 10%, 15%, and 20% were compared to each other and to the consensus of the SS sequences (protease 4–99; reverse transcriptase 38–247). The concordance among laboratories’ sequences at different thresholds was evaluated by pairwise sequence comparisons. NGS sequences generated using the 20% threshold were the most similar to the SS consensus (average 99.6% identity, range 96.1–100%), compared to 15% (99.4%, 88.5–100%), 10% (99.2%, 87.4–100%), or 5% (98.5%, 86.4–100%). The average sequence identity between laboratories using thresholds of 20%, 15%, 10%, and 5% was 99.1%, 98.7%, 98.3%, and 97.3%, respectively. Using the 20% threshold, we observed an excellent agreement between NGS and SS, but significant differences at lower thresholds. Understanding how variation in NGS methods influences sequence quality is essential for NGS-based HIV-1 drug resistance genotyping.

scholarly journals Whole Genome Sequencing of SARS-CoV-2: Assessment of the Ion Torrent AmpliSeq Panel and Comparison with the Illumina-MiSeq ARTIC Protocol

2021 ◽  
Author(s):  
Jonathan Plitnick ◽  
Sara Griesemer ◽  
Erica Lasek-Nesselquist ◽  
Navjot Singh ◽  
Daryl Lamson ◽  
...  
Keyword(s):  
Illumina Miseq ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Analysis Pipeline ◽  
Single Nucleotide ◽  
Clinical Specimens ◽  
Consensus Sequences ◽  
Viral Loads ◽  
Analysis Tools ◽  
Single Difference

Background: Fast and effective methods are needed for sequencing the SARS-CoV-2 genome to track genetic mutations and identify new and emerging variants during the ongoing pandemic. Objective: Assess the performance of the SARS-CoV-2 AmpliSeq Research Panel and S5 plug-in analysis tools for whole genome sequence analysis of SARS-CoV-2 and compare the results to those obtained with the MiSeq based ARTIC analysis pipeline, using metrics such as depth, coverage, and concordance of single nucleotide variant (SNV) calls. Methods: A total of 191 clinical specimens and a single cultured isolate were extracted and sequenced with AmpliSeq technology and analysis tools. Of the 191 clinical specimens, 83 (Ct 15.58 – 32.54) were also sequenced using an Illumina MiSeq based method with the ARTIC analysis pipeline for a direct comparison. Results: 176 of the 191 clinical specimens sequenced on the S5XL and prepared using the SARS-CoV-2 Research Panel, had near complete coverage (>98%) of the viral genome, with an average depth of 5031x. Similar coverage (>98%) levels were observed for 81/83 primary specimens sequenced with both methods tested. The sample with the lowest viral load (Ct of 32.54) achieved 89% coverage using the MiSeq method and failed to sequence with the AmpliSeq method. Consensus sequences produced by each method were identical in 81/82 samples, in areas of equal coverage, with a single difference present in one sample. Conclusions: The AmpliSeq approach is as effective as the Illumina based method using ARTIC V3 amplification for sequencing SARS-CoV-2 direct from patient specimens across a range of viral loads (Ct 15.56-32.54, median = 22.18). The AmpliSeq workflow is very easily automated with the Ion Chef and S5 instruments and requires less training and experience with NGS preparation than the Illumina workflow.

scholarly journals Genomic characterization and phylogenetic analysis of the first SARS-CoV-2 variants introduced in Lebanon

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11015
Author(s):  
Rita Feghali ◽  
Georgi Merhi ◽  
Aurelia Kwasiborski ◽  
Veronique Hourdel ◽  
Nada Ghosn ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Consensus Sequence ◽  
Unknown Origin ◽  
International Travel ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Genetic Lineages ◽  
Consensus Sequences

Background In December 2019, the COVID-19 pandemic initially erupted from a cluster of pneumonia cases of unknown origin in the city of Wuhan, China. Presently, it has almost reached 94 million cases worldwide. Lebanon on the brink of economic collapse and its healthcare system thrown into turmoil, has previously managed to cope with the initial SARS-CoV-2 wave. In this study, we sequenced 11 viral genomes from positive cases isolated between 2 February 2020 and 15 March 2020. Methods Sequencing data was quality controlled, consensus sequences generated, and a maximum-likelihood tree was generated with IQTREE v2. Genetic lineages were assigned with Pangolin v1.1.14 and single nucleotide variants (SNVs) were called from read files and manually curated from consensus sequence alignment through JalView v2.11 and the genomic mutational interference with molecular diagnostic tools was assessed with the CoV-GLUE pipeline. Phylogenetic analysis of whole genome sequences confirmed a multiple introduction scenario due to international travel. Results Three major lineages were identified to be circulating in Lebanon in the studied period. The B.1 (20A clade) was the most prominent, followed by the B.4 lineage (19A clade) and the B.1.1 lineage (20B clade). SNV analysis showed 15 novel mutations from which only one was observed in the spike region.

scholarly journals Limited SARS-CoV-2 diversity within hosts and following passage in cell culture

2020 ◽  
Author(s):  
Gage K. Moreno ◽  
Katarina M. Braun ◽  
Peter J. Halfmann ◽  
Trent M. Prall ◽  
Kasen K. Riemersma ◽  
...  
Keyword(s):  
Cell Culture ◽  
Evolutionary Dynamics ◽  
Purifying Selection ◽  
Illumina Miseq ◽  
Primary Isolate ◽  
Viral Diversity ◽  
Single Nucleotide Variants ◽  
Consensus Sequences

AbstractSince the first reports of pneumonia associated with a novel coronavirus (COVID-19) emerged in Wuhan, Hubei province, China, there have been considerable efforts to sequence the causative virus, SARS-CoV-2 (also referred to as hCoV-19) and to make viral genomic information available quickly on shared repositories. As of 30 March 2020, 7,680 consensus sequences have been shared on GISAID, the principal repository for SARS-CoV-2 genetic information. These sequences are primarily consensus sequences from clinical and passaged samples, but few reports have looked at diversity of virus populations within individual hosts or cultures. Understanding such diversity is essential to understanding viral evolutionary dynamics. Here, we characterize within-host viral diversity from a primary isolate and passaged samples, all originally deriving from an individual returning from Wuhan, China, who was diagnosed with COVID-19 and subsequently sampled in Wisconsin, United States. We use a metagenomic approach with Oxford Nanopore Technologies (ONT) GridION in combination with Illumina MiSeq to capture minor within-host frequency variants ≥1%. In a clinical swab obtained from the day of hospital presentation, we identify 15 single nucleotide variants (SNVs) ≥1% frequency, primarily located in the largest gene – ORF1a. While viral diversity is low overall, the dominant genetic signatures are likely secondary to population size changes, with some evidence for mild purifying selection throughout the genome. We see little to no evidence for positive selection or ongoing adaptation of SARS-CoV-2 within cell culture or in the primary isolate evaluated in this study.Author SummaryWithin-host variants are critical for addressing molecular evolution questions, identifying selective pressures imposed by vaccine-induced immunity and antiviral therapeutics, and characterizing interhost dynamics, including the stringency and character of transmission bottlenecks. Here, we sequenced SARS-CoV-2 viruses isolated from a human host and from cell culture on three distinct Vero cell lines using Illumina and ONT technologies. We show that SARS-CoV-2 consensus sequences can remain stable through at least two serial passages on Vero 76 cells, suggesting SARS-CoV-2 can be propagated in cell culture in preparation for in-vitro and in-vivo studies without dramatic alterations of its genotype. However, we emphasize the need to deep-sequence viral stocks prior to use in experiments to characterize sub-consensus diversity that may alter outcomes.

Common Genetic Variants in ABO and CLEC4M Modulate the Pharmacokinetics of Recombinant FVIII in Severe Hemophilia A Patients

2020 ◽  
Vol 120 (10) ◽  
pp. 1395-1406 ◽  
Author(s):  
Iris Garcia-Martínez ◽  
Nina Borràs ◽  
Marta Martorell ◽  
Rafael Parra ◽  
Carme Altisent ◽  
...  
Keyword(s):  
Blood Group ◽  
Half Life ◽  
Robust Regression ◽  
Hemophilia A ◽  
Illumina Miseq ◽  
Activity Levels ◽  
Severe Hemophilia ◽  
Single Nucleotide Variants ◽  
Common Genetic Variants ◽  
Recombinant Fviii

AbstractThe pharmacokinetic (PK) response of severe hemophilia A (HA) patients to infused factor VIII (FVIII) shows substantial variability. Several environmental and genetic factors are associated with changes in FVIII plasma levels and infused FVIII PK. Based on the hypothesis that factors influencing endogenous FVIII can affect FVIII PK, the contribution of single-nucleotide variants (SNVs) in candidate genes was investigated in 51 severe HA patients. The effects of blood group, F8 variant type, von Willebrand factor antigen and activity levels, age, and weight were also explored. The myPKFiT device was used to estimate individual PK parameters, and SNVs and clinically reportable F8 variants were simultaneously analyzed in an Illumina MiSeq instrument, using the microfluidics-based Fluidigm Access Array system. The contribution of SNVs to FVIII half-life and clearance was addressed by robust regression modeling, taking into account other modulators. In line with previous studies, we provide robust evidence that age, body weight, and blood group, as well as SNVs in ABO and CLEC4M, participate in the variability of FVIII PK in HA patients. Main results: each copy of the rs7853989 (ABO) allele increases FVIII half-life by 1.4 hours (p = 0.0131) and decreases clearance by 0.5 mL/h/kg (p = 5.57E-03), whereas each additional rs868875 (CLEC4M) allele reduces FVIII half-life by 1.1 hours (p = 2.90E-05) and increases clearance by 0.3 mL/h/kg (p = 1.01E-03). These results contribute to advancing efforts to improve FVIII replacement therapies by adjusting to each patient's PK profile based on pharmacogenomic data. This personalized medicine will decrease the burden of treatment and maximize the benefits obtained.

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 SARS-CoV-2 genomic analyses in cancer patients reveal elevated intrahost genetic diversity

2021 ◽  
Author(s):  
Juliana D Siqueira ◽  
Livia R Goes ◽  
Brunna M Alves ◽  
Pedro S de Carvalho ◽  
Claudia Cicala ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Phylogenetic Analysis ◽  
Cancer Patients ◽  
Disease Severity ◽  
Healthcare Workers ◽  
Viral Population ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Consensus Sequences ◽  
Vulnerable Patients

Abstract Numerous factors have been identified to influence susceptibility to SARS-CoV-2 infection and disease severity. Cancer patients are more prone to clinically evolve to more severe COVID-19 conditions, but the determinants of such a more severe outcome remain largely unknown. We have determined the full-length SARS-CoV-2 genomic sequences of cancer patients and healthcare workers (non-cancer controls) by deep sequencing and investigated the within-host viral population of each infection, quantifying intrahost genetic diversity. Naso- and oropharyngeal SARS-CoV-2+ swabs from 57 cancer patients and 14 healthcare workers from the Brazilian National Cancer Institute were collected in April–May 2020. Complete genome amplification using ARTIC network V3 multiplex primers was performed followed by next-generation sequencing. Assemblies were conducted in Geneious R11, where consensus sequences were extracted and intrahost single nucleotide variants were identified. Maximum likelihood phylogenetic analysis was performed using PhyMLv.3.0 and lineages were classified using Pangolin and CoV-GLUE. Phylogenetic analysis showed that all but one strain belonged to clade B1.1. Four genetically linked mutations known as the globally dominant SARS-CoV-2 haplotype (C241T, C3037T, C14408T and A23403G) were found in the majority of consensus sequences. SNV signatures of previously characterized Brazilian genomes were also observed in most samples. Another 85 SNVs were found at a lower frequency (1.4-19.7%) among the consensus sequences. Cancer patients displayed a significantly higher intrahost viral genetic diversity compared to healthcare workers. This difference was independent of SARS-CoV-2 Ct values obtained at the diagnostic tests, which did not differ between the two groups. The most common nucleotide changes of intrahost SNVs in both groups were consistent with APOBEC and ADAR activities. Intrahost genetic diversity in cancer patients was not associated with disease severity, use of corticosteroids, or use of antivirals, characteristics that could influence viral diversity. Moreover, the presence of metastasis, either in general or specifically in the lung, was not associated with intrahost diversity among cancer patients. Cancer patients carried significantly higher numbers of minor variants compared to non-cancer counterparts. Further studies on SARS-CoV-2 diversity in especially vulnerable patients will shed light onto the understanding of the basis of COVID-19 different outcomes in humans.

Nextera XT protocol for MiSeq HIVPR-RT sequencing v1

2021 ◽  
Author(s):  
Paula Aulicino
Keyword(s):  
Drug Resistance ◽  
Illumina Miseq ◽  
Hiv 1

The protocol allows amplification and NGS sequencing of a PR-RT HIV-1 sequence using tagmentation strategy and Illumina MiSeq equipment, followed by analysis of drug-resistance associated mutations by HyDRA

scholarly journals Global sequence characterization of rice centromeric satellite based on oligomer frequency analysis in large-scale sequencing data

2010 ◽  
Vol 26 (17) ◽  
pp. 2101-2108 ◽  
Author(s):  
Jiří Macas ◽  
Pavel Neumann ◽  
Petr Novák ◽  
Jiming Jiang
Keyword(s):  
Large Scale ◽  
Rice Genome ◽  
Supplementary Information ◽  
Sequencing Data ◽  
Satellite Repeat ◽  
Frequency Spectra ◽  
Consensus Sequences ◽  
Chip Sequencing ◽  
Conserved Sequence ◽  
Centromeric Satellite

Abstract Motivation: Satellite DNA makes up significant portion of many eukaryotic genomes, yet it is relatively poorly characterized even in extensively sequenced species. This is, in part, due to methodological limitations of traditional methods of satellite repeat analysis, which are based on multiple alignments of monomer sequences. Therefore, we employed an alternative, alignment-free, approach utilizing k-mer frequency statistics, which is in principle more suitable for analyzing large sets of satellite repeat data, including sequence reads from next generation sequencing technologies. Results: k-mer frequency spectra were determined for two sets of rice centromeric satellite CentO sequences, including 454 reads from ChIP-sequencing of CENH3-bound DNA (7.6 Mb) and the whole genome Sanger sequencing reads (5.8 Mb). k-mer frequencies were used to identify the most conserved sequence regions and to reconstruct consensus sequences of complete monomers. Reconstructed consensus sequences as well as the assessment of overall divergence of k-mer spectra revealed high similarity of the two datasets, suggesting that CentO sequences associated with functional centromeres (CENH3-bound) do not significantly differ from the total population of CentO, which includes both centromeric and pericentromeric repeat arrays. On the other hand, considerable differences were revealed when these methods were used for comparison of CentO populations between individual chromosomes of the rice genome assembly, demonstrating preferential sequence homogenization of the clusters within the same chromosome. k-mer frequencies were also successfully used to identify and characterize smRNAs derived from CentO repeats. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online.

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