Efficacy of next-generation sequencing in bacterial zoonoses diagnostics

Brucella , an extremely diverse but yet genetically highly homogenous genus of bacteria, has been a puzzle for scientists for many decades. These bacteria remain a prominent public health issue, particularly in the Balkan region. Correctly identifying and understanding the pathogen is a vital step in the epidemiology and epizootiology of any bacteria. Identification can be challenging, especially in the case of zoonotic species. This study aimed to implement fourth-generation sequencing in the typing of 11 Brucella suis strains kept in our archive and to compare this method to the classical and non-sequencing based molecular methods used to date. Classical biotyping is highly subjective and gave inconclusive results for 3 strains. Molecular methods used were multiplex PCR and RFLP methods since no one method can identify both species and biovar which is vital in the case of Brucella suis infections. Species and biovars of all the strains were successfully confirmed and in concordance with biotyping results. Oxford Nanopore long-read sequencing was used on a MinION device for next-generation sequencing (NGS). Various algorithms were implemented for genome assembly and BioNumerics 8.0 software was used for MLST identification and analysis. MLST 21 was used for biovar identification and epidemiological comparison of tested strains. The assembled genomes were 3,2 Mb in size and assembled into two chromosomes. MLST 21 analysis placed our strains into species and biovar clusters in concordance with other molecular tests used. To the extent of our knowledge, this is the first documented use of long-read sequencing in Brucella suis identification in this region. We conclude that bacteriological biotyping is outdated and host-specific identification in this genus is incorrect and that molecular characterisation is always the safer, faster and more suitable option. MinION sequencing proved to be a strong, accessible solution for species determination. Future study is required to determine how detailed genome information it can give, considering the error rate.

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Filling the gap of short-read next generation sequencing in PGD by long-read approach

10.26226/morressier.5af300b2738ab10027aa98ef ◽

2018 ◽

Author(s):

Dona Ngar Yin Ho

Keyword(s):

Next Generation Sequencing ◽

Next Generation ◽

Short Read ◽

Long Read ◽

Generation Sequencing

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Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009779 ◽

2021 ◽

Vol 15 (10) ◽

pp. e0009779

Author(s):

Fakhriddin Sarzhanov ◽

Funda Dogruman-Al ◽

Monica Santin ◽

Jenny G. Maloney ◽

Ayse Semra Gureser ◽

...

Keyword(s):

Next Generation Sequencing ◽

Gastrointestinal Symptoms ◽

Molecular Methods ◽

Clinical Samples ◽

Rrna Gene ◽

Next Generation ◽

Dientamoeba Fragilis ◽

Significant Difference ◽

Generation Sequencing ◽

Immunocompetent Patients

Introduction The clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing. Material and methods Individual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes. Results The prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients. Conclusion and recommendation Our findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.

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A Canadian guideline on the use of next-generation sequencing in oncology

Current Oncology ◽

10.3747/co.26.4731 ◽

2019 ◽

Vol 26 (2) ◽

Cited By ~ 6

Author(s):

S. Yip ◽

A. Christofides ◽

S. Banerji ◽

M. R. Downes ◽

I. Izevbaye ◽

...

Keyword(s):

Next Generation Sequencing ◽

Best Practices ◽

Sample Selection ◽

Molecular Profile ◽

Next Generation ◽

Canadian Guideline ◽

Molecular Tests ◽

Management Guidance ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Rapid advancements in next-generation sequencing (ngs) technology have created an unprecedented opportunity to decipher the molecular profile of tumours to more effectively prevent, diagnose, and treat cancer. Oncologists now have the option to order molecular tests that can guide treatment decisions. However, to date, most oncologists have received limited training in genomics, and they are now faced with the challenge of understanding how such tests and their interpretation align with patient management. Guidance on how to effectively use ngs technology is therefore needed to aid oncologists in applying the results of genomic tests. The Canadian guideline presented here describes best practices and unmet needs related to ngs-based testing for somatic variants in oncology, including clinical application, assay and sample selection, bioinformatics and interpretation of reports performed by laboratories, patient communication, and clinical trials.

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Methods for analyzing next-generation sequencing data VII. long-read assembly

Japanese Journal of Lactic Acid Bacteria ◽

10.4109/jslab.27.101 ◽

2016 ◽

Vol 27 (2) ◽

pp. 101-110

Author(s):

Yasuhiro Tanizawa ◽

Eli Kaminuma ◽

Yasukazu Nakamura ◽

Masanori Tohno ◽

Ken Osaki ◽

...

Keyword(s):

Next Generation Sequencing ◽

Next Generation Sequencing Data ◽

Next Generation ◽

Sequencing Data ◽

Long Read ◽

Generation Sequencing

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Exogene: A performant workflow for detecting viral integrations from paired-end next-generation sequencing data

10.1101/2021.04.19.440427 ◽

2021 ◽

Author(s):

Jean-Pierre Kocher ◽

Zachary Stephens ◽

Daniel O'Brien ◽

Mrunal Dehankar ◽

Lewis Roberts ◽

...

Keyword(s):

Next Generation Sequencing ◽

Sequence Data ◽

Next Generation Sequencing Data ◽

Next Generation ◽

Sequencing Data ◽

Long Read ◽

Breakpoint Detection ◽

Targeted Capture ◽

Genome Heterogeneity ◽

Generation Sequencing

The integration of viruses into the human genome is known to be associated with tumorigenesis in many cancers, but the accurate detection of integration breakpoints from short read sequencing data is made difficult by human-viral homologies, viral genome heterogeneity, coverage limitations, and other factors. To address this, we present Exogene, a sensitive and efficient workflow for detecting viral integrations from paired-end next generation sequencing data. Exogene's read filtering and breakpoint detection strategies yield integration coordinates that are highly concordant with those found in long read validation sets. We demonstrate this concordance across 6 TCGA Hepatocellular carcinoma (HCC) tumor samples, identifying integrations of hepatitis B virus that are validated by long reads. Additionally, we applied Exogene to targeted capture data from 426 previously studied HCC samples, achieving 98.9% concordance with existing methods and identifying 238 high-confidence integrations that were not previously reported. Exogene is applicable to multiple types of paired-end sequence data, including genome, exome, RNA-Seq or targeted capture.

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How Do Advanced Molecular Tests Compare to Routine Clinical Laboratory Evaluation of CSF in Meningoencephalitis? A Study in 10 Urban Emergency Departments Across the USA

Open Forum Infectious Diseases ◽

10.1093/ofid/ofx162.022 ◽

2017 ◽

Vol 4 (suppl_1) ◽

pp. S8-S9 ◽

Cited By ~ 1

Author(s):

Toby L Merlin ◽

Scott Chancey ◽

Yueli Zheng ◽

Brad Bowzard ◽

Leah Fischer ◽

...

Keyword(s):

Next Generation Sequencing ◽

Pilot Study ◽

Emergency Departments ◽

Clinical Laboratory ◽

Targeted Sequencing ◽

Laboratory Evaluation ◽

Next Generation ◽

Molecular Tests ◽

Causative Agents ◽

Generation Sequencing

Abstract Background The EMERGEncy ID Net Study Group is investigating whether advanced molecular tests (AMT) increase the detection of causative agents in the CSF of patients presenting with meningoencephalitis (ME). We report findings from a pilot study using AMT on 18 CSF samples from 10 US Urban Emergency Departments. The purpose of the pilot was to compare the performance of these four AMT to established clinical laboratory methods. Methods We investigated four AMT: (1) BioFire FilmArray ME Panel targeting 14 causative agents; (2) an in-house target-directed next generation sequencing assay targeting 25 agents; (3) a microarray capable of detecting >2,500 agents; and (4) deep metagenomic next generation sequencing. For targeted sequencing, loci from 12 DNA-based and 13 RNA-based pathogens were amplified from the extracts by multiplex PCR. All sequencing was performed on an Illumina MiSeq using 500 cycle v2 Reagent Kits. Reads from the targeted sequencing were aligned to the 25 specific reference target sequences using Bowtie2 while metagenomics reads were processed with the taxonomic sequence classifying software Kraken. For microarray analysis, Lawrence Livermore Microbial Detection Array v2 arrays were hybridized with Cy3-labeled DNA or cDNA. Scanned images of arrays were analyzed by CLiMax 3.1. Results Eight CSF samples had results positive for well-established causes of ME from prior testing (Table). The pilot study demonstrated none of the four AMT detected all causative agents in the eight CSF samples known to have well-established causes of ME. BioFire and targeted sequencing performed best, both detecting 6/8, metagenomics deep sequencing detected 3/8, and microarray detected 1/8. Conclusion Despite the sophistication of AMT, they cannot detect pathogens they do not target, that are present in small numbers, or that have been eliminated from the CSF by the immune response. Despite the theoretical potential for microarray and metagenomic sequencing to detect thousands of different agents, the agents probably must be present at high levels for detection. Disclosures All authors: No reported disclosures.

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B Chromosomes in Genus Sorghum (Poaceae)

Plants ◽

10.3390/plants10030505 ◽

2021 ◽

Vol 10 (3) ◽

pp. 505

Author(s):

Martina Bednářová ◽

Miroslava Karafiátová ◽

Eva Hřibová ◽

Jan Bartoš

Keyword(s):

Next Generation Sequencing ◽

B Chromosomes ◽

Molecular Methods ◽

Next Generation ◽

Scientific Interest ◽

Limited Knowledge ◽

Eukaryotic Species ◽

Available Information ◽

Generation Sequencing

B chromosomes (Bs) are supernumerary dispensable genomic elements that have been reported in several thousand eukaryotic species. Since their discovery, Bs have been subjected to countless studies aiming at the clarification of their origin, composition, and influence on the carriers. Despite these efforts, we still have very limited knowledge of the processes that led to the emergence of Bs, the mechanisms of their transmission, and the effects of Bs on the hosts. In the last decade, sophisticated molecular methods, including next-generation sequencing, have provided powerful tool to help answer some of these questions, but not many species have received much attention yet. In this review, we summarize the currently available information about Bs in the genus Sorghum, which has so far been on the periphery of scientific interest. We present an overview of the occurrence and characteristics of Bs in various Sorghum species, discuss the possible mechanisms involved in their maintenance and elimination, and outline hypotheses of the origin of Bs in this genus.

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Exogene: A performant workflow for detecting viral integrations from paired-end next-generation sequencing data

PLoS ONE ◽

10.1371/journal.pone.0250915 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0250915

Author(s):

Zachary Stephens ◽

Daniel O’Brien ◽

Mrunal Dehankar ◽

Lewis R. Roberts ◽

Ravishankar K. Iyer ◽

...

Keyword(s):

Next Generation Sequencing ◽

Sequence Data ◽

Next Generation Sequencing Data ◽

Next Generation ◽

Sequencing Data ◽

Long Read ◽

Breakpoint Detection ◽

Targeted Capture ◽

Genome Heterogeneity ◽

Generation Sequencing

The integration of viruses into the human genome is known to be associated with tumorigenesis in many cancers, but the accurate detection of integration breakpoints from short read sequencing data is made difficult by human-viral homologies, viral genome heterogeneity, coverage limitations, and other factors. To address this, we present Exogene, a sensitive and efficient workflow for detecting viral integrations from paired-end next generation sequencing data. Exogene’s read filtering and breakpoint detection strategies yield integration coordinates that are highly concordant with long read validation. We demonstrate this concordance across 6 TCGA Hepatocellular carcinoma (HCC) tumor samples, identifying integrations of hepatitis B virus that are also supported by long reads. Additionally, we applied Exogene to targeted capture data from 426 previously studied HCC samples, achieving 98.9% concordance with existing methods and identifying 238 high-confidence integrations that were not previously reported. Exogene is applicable to multiple types of paired-end sequence data, including genome, exome, RNA-Seq and targeted capture.

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Next-Generation Sequencing of Haematococcus lacustris Reveals an Extremely Large 1.35-Megabase Chloroplast Genome

Genome Announcements ◽

10.1128/genomea.00181-18 ◽

2018 ◽

Vol 6 (12) ◽

Cited By ~ 16

Author(s):

Nicholas Bauman ◽

Srividya Akella ◽

Elizabeth Hann ◽

Robert Morey ◽

Ariel S. Schwartz ◽

...

Keyword(s):

Next Generation Sequencing ◽

Chloroplast Genome ◽

Next Generation ◽

Content Type ◽

Haematococcus Lacustris ◽

Long Read ◽

Generation Sequencing

ABSTRACT Haematococcus lacustris is an industrially relevant microalga that is used for the production of the carotenoid astaxanthin. Here, we report the use of PacBio long-read sequencing to assemble the chloroplast genome of H. lacustris strain UTEX:2505. At 1.35 Mb, this is the largest assembled chloroplast of any plant or alga known to date.

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