scholarly journals Performance of Targeted Library Preparation Solutions for SARS-CoV-2 Whole Genome Analysis

Diagnostics ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 769
Author(s):  
Petr Klempt ◽  
Petr Brož ◽  
Martin Kašný ◽  
Adam Novotný ◽  
Kateřina Kvapilová ◽  
...  
Keyword(s):  
Genome Analysis ◽  
Library Preparation ◽  
Genome Sequences ◽  
Whole Genome Analysis ◽  
Input Sample ◽  
Variant Analysis ◽  
Global Initiative ◽  
Next Generation Sequencing Ngs ◽  
Targeted Library ◽  
Generation Sequencing

Single next-generation sequencing (NGS) proved to be an important tool for monitoring the SARS-CoV-2 outbreak at the global level Until today, thousands of SARS-CoV-2 genome sequences have been published at GISAID (Global Initiative on Sharing All Influenza Data) but only a portion are suitable for reliable variant analysis. Here we report on the comparison of three commercially available NGS library preparation kits. We discuss advantages and limitations from the perspective of required input sample quality and data quality for advanced SARS-CoV-2 genome analysis.

scholarly journals A20 Sample preparation for whole-genome next-generation sequencing (NGS) of hepatitis C virus (HCV) routine RNA samples

2019 ◽  
Vol 5 (Supplement_1) ◽  
Author(s):  
Julia Hillung ◽  
María Alma Bracho ◽  
Javier Pons Tamarit ◽  
Fernando González-Candelas
Keyword(s):  
Sample Preparation ◽  
Nucleic Acids ◽  
Blood Plasma ◽  
Reference Genome ◽  
Phylogenetic Analyses ◽  
Whole Genome ◽  
Library Preparation ◽  
Genome Sequences ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Next-generation sequencing (NGS) is a technique that can capture the variability of viral populations in transmission studies. The conventional sample preparation for NGS, based on amplicons, is a potential source of errors, derived from the variable affinity of specific primers for different viral variants and from irregular DNA polymerase efficiency. In this context, we propose a more reliable method for viral whole genome sample preparation, starting from nucleic acids obtained and stored with conventional procedures. Our goal was to obtain complete hepatitis C virus (HCV) genome sequences to subsequently perform extensive phylogenetic analyses. Additionally, we aimed to test the effectiveness of nuclease treatment used to remove contaminating host DNA. Nucleic acids were obtained from almost cell-free blood plasma of HCV-infected patients. As a source for Illumina library preparation, double-stranded cDNA was generated using random primers. The HCV genome was not amplified before library preparation, avoiding possible biases derived from unequal copying. To get rid of possible host contaminants in the samples, a DNase treatment step was added. Libraries were paired-end sequenced on the Illumina platform using MiSeq reagent kit v3. After conservative filtering of contaminant human reads by alignment with the human reference genome using Burrows-Wheeler Aligner (BWA), the remaining reads were mapped to the HCV reference genome using BWA. Primary maximum likelihood phylogenetic analyses were performed using ClustalW and IQTREE to infer the phylogenetic relationships of the sequenced samples in the context of complete genome sequences of the same genotype. NGS sample preparation method of HCV from blood plasma was established. Complete genome sequences of HCV could be obtained with variable coverage depending on the viral load of plasma samples. No significant reduction of host DNA proportion in DNase treated samples in comparison to the controls was observed. The new sequences clustered within the Los Alamos National Laboratory database-deposited HCV subtype 4d samples. The method can be used to obtain full-length sequences of HCV from nucleic acid samples not previously planned for NGS. No improvement was observed when DNase pre-treatment of nucleic acids extracted from blood plasma was performed.

scholarly journals Detection of SARS-CoV-2 from patient fecal samples by whole genome sequencing

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Andreas Papoutsis ◽  
Thomas Borody ◽  
Siba Dolai ◽  
Jordan Daniels ◽  
Skylar Steinberg ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Nasopharyngeal Swab ◽  
Whole Genome ◽  
Rt Pcr ◽  
Whole Genome Analysis ◽  
Fecal Samples ◽  
Oral Transmission ◽  
Study Participants ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Background SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. Here were sought to identify SARS-CoV-2 by enrichment next-generation sequencing (NGS) from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COVID-19 patients. Results Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n = 14), and RT-PCR nasopharyngeal swab analysis (n = 12). The concordance of SARS-CoV-2 detection by enrichment NGS from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS. Conclusion These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal–oral transmission. This study also elucidates the advantages of SARS-CoV-2 enrichment NGS, which may be a key methodology to document complete viral eradication. Trial registration ClinicalTrials.gov, NCT04359836, Registered 24 April 2020, https://clinicaltrials.gov/ct2/show/NCT04359836?term=NCT04359836&draw=2&rank=1).

scholarly journals benchNGS : An approach to benchmark short reads alignment tools

2015 ◽  
Author(s):  
Farzana Rahman ◽  
Mehedi Hassan ◽  
Alona Kryshchenko ◽  
Inna Dubchak ◽  
Tatiana V Tatarinova ◽  
...  
Keyword(s):  
Reference Genome ◽  
Global Alignment ◽  
Short Report ◽  
Related Genome ◽  
Genome Sequences ◽  
Short Reads ◽  
Relevant Reference ◽  
Next Generation Sequencing Ngs ◽  
Reference Genomes ◽  
Generation Sequencing

In the last decade a number of algorithms and associated software were developed to align next generation sequencing (NGS) reads to relevant reference genomes. The results of these programs may vary significantly, especially when the NGS reads are contain mutations not found in the reference genome. Yet there is no standard way to compare these programs and assess their biological relevance. We propose a benchmark to assess accuracy of the short reads mapping based on the pre-computed global alignment of closely related genome sequences. In this paper we outline the method and also present a short report of an experiment performed on five popular alignment tools .

scholarly journals Clinical results and importance of next-generation sequencing (NGS) in detecting targeted mutations in the treatment of metastatic Lung Cancer: Single center initial results

2019 ◽  
Vol 6 (12) ◽  
pp. 327-332
Author(s):  
Cem Mirili ◽  
Çiğdem Kahraman ◽  
Ali Yılmaz ◽  
Mehmet Bilici ◽  
Salim Başol Tekin ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Next Generation Sequencing ◽  
Genetic Variants ◽  
Genetic Variant ◽  
Clinical Results ◽  
Clinical Effects ◽  
Next Generation ◽  
Variant Analysis ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Objective:  In Lung cancer (LC), which is one of the most deadly cancers, longer survival has been achieved with targeted agents. For this reason, it is important to find the patients who are suitable for targeted therapies. Next-generation sequencing (NGS) is a method that allows multiple genetic variants to be detected simultaneously by performing massive parallel DNA sequencing at the same time. We wanted to reveal the clinical effects and benefits of genetic variant analysis with NGS for our patients. Material and Methods: Patients with stage 4 non-squamous and not otherwise specified (NOS) Non-small cell LC who underwent genetic variant analysis with NGS were included in the study, retrospectively. Results: Total of the 51 patients, 41 (80.4%) were male and the median age was 64 (35-85) years. According to TNM, 21 (41.2%) patients were stage 4A, 30 (58.8%) patients were stage 4B and 39 (76.5%) patients had adenocarcinoma and 12 (23.5%) had NOS histology. NGS analyzes were performed in median 14 days (8-43) and determined 24 pathogenic variants in 17 (%25) patients: 9EGFR (%17,6), 6PIKC3A (%11,7), 5KRAS (%9,8), 2PTEN (%3,9), 1BRAF (%1,9), 1MET (%1,6) (7 of them concomitantly). Cytotoxic chemotherapy was recommended in 41, anti-EGFR agents in 8 (afatinib in 4, erlotinib in 4 patients) patients and anti-BRAF+MEK inhibitor agent (dabrafenib+trametinib) in 1 patient. Conclusion: With the NGS, in just two weeks, both target and resistance genetic variants of our patients were detected at the same time and individualized treatments were applied. In this way, both time and cost were saved.

scholarly journals Whole-genome analysis reveals the complex evolutionary dynamics of Kenyan G2P[4] human rotavirus strains

2011 ◽  
Vol 92 (9) ◽  
pp. 2201-2208 ◽  
Author(s):  
Souvik Ghosh ◽  
Noriaki Adachi ◽  
Zipporah Gatheru ◽  
James Nyangao ◽  
Dai Yamamoto ◽  
...  
Keyword(s):  
Genome Analysis ◽  
Complete Genome ◽  
Evolutionary Dynamics ◽  
Genomic Analysis ◽  
Whole Genome ◽  
Genome Sequences ◽  
Whole Genome Analysis ◽  
Human Rotavirus ◽  
Genotype Constellation ◽  
Common Causes

Although G2P[4] rotaviruses are common causes of acute childhood diarrhoea in Africa, to date there are no reports on whole genomic analysis of African G2P[4] strains. In this study, the nearly complete genome sequences of two Kenyan G2P[4] strains, AK26 and D205, detected in 1982 and 1989, respectively, were analysed. Strain D205 exhibited a DS-1-like genotype constellation, whilst strain AK26 appeared to be an intergenogroup reassortant with a Wa-like NSP2 genotype on the DS-1-like genotype constellation. The VP2-4, VP6-7, NSP1, NSP3 and NSP5 genes of strain AK26 and the VP2, VP4, VP7 and NSP1–5 genes of strain D205 were closely related to those of the prototype or other human G2P[4] strains. In contrast, their remaining genes were distantly related, and, except for NSP2 of AK26, appeared to originate from or share a common origin with rotavirus genes of artiodactyl (ruminant and camelid) origin. These observations highlight the complex evolutionary dynamics of African G2P[4] rotaviruses.

scholarly journals Phylogenomic analysis of Pseudomonas nitroreducens strains FY43 and FY47

2022 ◽  
pp. 1-11
Author(s):  
Xue Li Tan ◽  
Wei Yee Wee ◽  
Boon Chin Tan ◽  
Chee How Teo
Keyword(s):  
Genome Analysis ◽  
Phylogenomic Analysis ◽  
Whole Genome ◽  
The Novel ◽  
Species Classification ◽  
Whole Genome Analysis ◽  
Pseudomonas Nitroreducens ◽  
High Level ◽  
Next Generation Sequencing Ngs ◽  
Diversity Studies

Proper identification of strain is essential in understanding the ecology of a bacteria species. The classification of Pseudomonas nitroreducens is still being questioned and revised until now. The novel P. nitroreducens strains FY43 and FY47 used in this study have been reported to show a high level of tolerance to glyphosate. In this study, next-generation sequencing (NGS) and whole genome analysis were used to clarify the delineation of the species. Whole genome analysis showed that P. nitroreducens strains FY43 and FY47 shared high homology to five reference genomes of P. nitroreducens: strain B, Aramco J, NBRC 12694, DF05, and TX01. Phylogenomic and phylogenetic analysis (average nucleotide identity based on BLAST (ANIb), genome-to-genome distance (GGDC) analysis) showed that both P. nitroreducens strains FY43 and FY47 are Pseudomonas nitroreducens members. However, strains DF05 and TX01 were not correctly assigned at the species level for all the analyses. The P. nitroreducens strain DF05 and TX01 should be further investigated for their classification as the correct species classification is the prerequisite for future diversity studies.

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