Nanopore-Based Long-Read Sequencing Technology to Obtain Highly Contiguous Whole-Genome Sequence of Actinobacterial Genomes like Streptomyces Sp.: A Complete Guide for Actinobacterial Whole Genome Sequencing Project Using Nanopore

Abstract BackgroundWhole-genome sequencing has shown that the Mycobacterium tuberculosis infection process can be more heterogeneous than previously thought. Compartmentalized infections, exogenous reinfections, and microevolution are manifestations of this clonal complexity. The analysis of the mechanisms causing the microevolution —the genetic variability of M. tuberculosis at short time scales— of a parental strain into clonal variants with a patient is a relevant issue that has not been yet completely addressed. To our knowledge, a whole genome sequence microevolution analysis in a single patient with inadequate adherence to treatment has not been previously reported.Case Presentations In this work, we applied whole genome sequencing for a more in-depth analysis of the microevolution of a parental Mycobacterium tuberculosis strain into clonal variants within a patient with poor treatment compliance in Argentina. We analyzed the whole-genome sequence of 8 consecutive Mycobacterium. tuberculosis isolates obtained from a patient within 57-month of intermittent therapy. Nineteen mutations (9 short-term, 10 fixed variants) emerged, most of them associated with drug resistance. The first isolate was already resistant to isoniazid, rifampicin, and streptomycin, thereafter the strain developed resistance to fluoroquinolones and pyrazinamide. Surprisingly, isolates remained susceptible to the pro-drug ethionamide after acquiring a frameshift mutation in ethA, a gene required for its activation. We also found a novel variant, (T-54G), in the 5' untranslated region of whiB7 (T-54G), a region allegedly related to kanamycin resistance. Notably, discrepancies between canonical and phage-based susceptibility testing to kanamycin were previously found for the isolate harboring this mutation. In our patience, microevolution was mainly driven by drug selective pressure. Rare short-term mutations fixed together with resistance-conferring mutations during therapy.ConclusionsThis report highlights the relevance of whole-genome sequencing in the clinic for characterization of pre-XDR and MDR resistance profile, particularly in patients with incomplete and/or intermittent treatment.

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Exploring the Diversity of Bacillus whole genome sequencing projects using Peasant, the Prokaryotic Assembly and Annotation Tool

10.1101/132084 ◽

2017 ◽

Cited By ~ 6

Author(s):

Jonathon Brenner ◽

Laurynas Kalesinskas ◽

Catherine Putonti

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome Sequence ◽

Whole Genome ◽

Annotation Tool ◽

Illumina Platform ◽

Desktop Computer ◽

High Quality ◽

Sequencing Project ◽

Computational Resources

ABSTRACTBackgroundThe persistent decrease in cost and difficulty of whole genome sequencing of microbial organisms has led to a dramatic increase in the number of species and strains characterized from a wide variety of environments. Microbial genome sequencing can now be conducted by small laboratories and as part of undergraduate curriculum. While sequencing is routine in microbiology, assembly, annotation and downstream analyses still require computational resources and expertise, often necessitating familiarity with programming languages. To address this problem, we have created a light-weight, user-friendly tool for the assembly and annotation of microbial sequencing projects.ResultsThe Prokaryotic Assembly and Annotation Tool, Peasant, automates the processes of read quality control, genome assembly, and annotation for microbial sequencing projects. High-quality assemblies and annotations can be generated by Peasant without the need of programming expertise or high-performance computing resources. Furthermore, statistics are calculated so that users can evaluate their sequencing project. To illustrate the computational speed and accuracy of Peasant, the SRA records of 322 Illumina platform whole genome sequencing assays for Bacillus species were retrieved from NCBI, assembled and annotated on a single desktop computer. From the assemblies and annotations produced, a comprehensive analysis of the diversity of over 200 high-quality samples was conducted, looking at both the 16S rRNA phylogenetic marker as well as the Bacillus core genome.ConclusionsPeasant provides an intuitive solution for high-quality whole genome sequence assembly and annotation for users with limited programing experience and/or computational resources. The analysis of the Bacillus whole genome sequencing projects exemplifies the utility of this tool. Furthermore, the study conducted here provides insight into the diversity of the species, the largest such comparison conducted to date.

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Whole-genome sequencing reveals progressive versus stable myeloma precursor conditions as two distinct entities

Nature Communications ◽

10.1038/s41467-021-22140-0 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Bénedith Oben ◽

Guy Froyen ◽

Kylee H. Maclachlan ◽

Daniel Leongamornlert ◽

Federico Abascal ◽

...

Keyword(s):

Multiple Myeloma ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genome Sequence ◽

Monoclonal Gammopathy ◽

Whole Genome Sequence ◽

Whole Genome ◽

Driver Genes ◽

Smoldering Myeloma ◽

Undetermined Significance

AbstractMultiple myeloma (MM) is consistently preceded by precursor conditions recognized clinically as monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM). We interrogate the whole genome sequence (WGS) profile of 18 MGUS and compare them with those from 14 SMMs and 80 MMs. We show that cases with a non-progressing, clinically stable myeloma precursor condition (n = 15) are characterized by later initiation in the patient’s life and by the absence of myeloma defining genomic events including: chromothripsis, templated insertions, mutations in driver genes, aneuploidy, and canonical APOBEC mutational activity. This data provides evidence that WGS can be used to recognize two biologically and clinically distinct myeloma precursor entities that are either progressive or stable.

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Five-year microevolution of a multidrug-resistant Mycobacterium tuberculosis strain within a patient with inadequate compliance to treatment

BMC Infectious Diseases ◽

10.1186/s12879-021-06069-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Darío A. Fernandez Do Porto ◽

Johana Monteserin ◽

Josefina Campos ◽

Ezequiel J. Sosa ◽

Mario Matteo ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genome Sequence ◽

Whole Genome Sequence ◽

Intermittent Therapy ◽

Whole Genome ◽

Sequencing Analysis ◽

Short Term ◽

Depth Analysis

Abstract Background Whole-genome sequencing has shown that the Mycobacterium tuberculosis infection process can be more heterogeneous than previously thought. Compartmentalized infections, exogenous reinfections, and microevolution are manifestations of this clonal complexity. The analysis of the mechanisms causing the microevolution —the genetic variability of M. tuberculosis at short time scales— of a parental strain into clonal variants with a patient is a relevant issue that has not been yet completely addressed. To our knowledge, a whole genome sequence microevolution analysis in a single patient with inadequate adherence to treatment has not been previously reported. Case presentation In this work, we applied whole genome sequencing analysis for a more in-depth analysis of the microevolution of a parental Mycobacterium tuberculosis strain into clonal variants within a patient with poor treatment compliance in Argentina. We analyzed the whole-genome sequence of 8 consecutive Mycobacterium tuberculosis isolates obtained from a patient within 57-months of intermittent therapy. Nineteen mutations (9 short-term, 10 fixed variants) emerged, most of them associated with drug resistance. The first isolate was already resistant to isoniazid, rifampicin, and streptomycin, thereafter the strain developed resistance to fluoroquinolones and pyrazinamide. Surprisingly, isolates remained susceptible to the pro-drug ethionamide after acquiring a frameshift mutation in ethA, a gene required for its activation. We also found a novel variant, (T-54G), in the 5′ untranslated region of whiB7 (T-54G), a region allegedly related to kanamycin resistance. Notably, discrepancies between canonical and phage-based susceptibility testing to kanamycin were previously found for the isolate harboring this mutation. In our patient, microevolution was mainly driven by drug selective pressure. Rare short-term mutations fixed together with resistance-conferring mutations during therapy. Conclusions This report highlights the relevance of whole-genome sequencing analysis in the clinic for characterization of pre-XDR and MDR resistance profile, particularly in patients with incomplete and/or intermittent treatment.

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Whole-Genome Sequence of Streptococcus tigurinus Strain osk_001, Isolated from Postmortem Material

Genome Announcements ◽

10.1128/genomea.00878-17 ◽

2017 ◽

Vol 5 (35) ◽

Author(s):

Hidenori Yoshizawa ◽

Daisuke Motooka ◽

Ryuichi Katada ◽

Yuki Matsumoto ◽

Shota Nakamura ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genome Sequence ◽

Novel Species ◽

Infected Tissue ◽

Whole Genome Sequence ◽

Whole Genome ◽

Content Type ◽

Virulent Strains ◽

Highly Virulent

ABSTRACT Streptococcus tigurinus was recently described as a novel species, and some strains are highly virulent. We detected S. tigurinus in infected tissue sampled by necropsy. In order to characterize and confirm the virulence of this species, whole-genome sequencing of the pure cultured bacterium was performed. We found that the strain has specific and unique genetic elements contained in highly virulent strains of S. tigurinus.

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Whole-genome sequencing and de novo assembly of a 2019 novel coronavirus (SARS-CoV-2) strain isolated in Vietnam

Vietnam Journal of Biotechnology ◽

10.15625/1811-4989/18/2/15082 ◽

2020 ◽

Vol 18 (2) ◽

pp. 197-208

Author(s):

Le Tung Lam ◽

Nguyen Trung Hieu ◽

Nguyen Hong Trang ◽

Ho Thi Thuong ◽

Tran Huyen Linh ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Sanger Sequencing ◽

De Novo ◽

Whole Genome ◽

Smrt Sequencing ◽

Sequencing Technology ◽

High Quality ◽

Long Read ◽

Novel Coronavirus

The pandemic COVID-19 caused by the virus SARS-CoV-2 has devastated countries worldwide, infecting more than 4.5 million people and leading to more than 300,000 deaths as of May 16th, 2020. Whole-genome sequencing (WGS) is an effective tool to monitor emerging strains and provide information for intervention, thus help to inform outbreak control decisions. Here, we reported the first effort to sequence and de novo assemble the whole genome of SARS-CoV-2 using PacBio’s SMRT sequencing technology in Vietnam. We also presented the annotation results and a brief analysis of the variants found in our SARS-CoV-2 strain, which was isolated from a Vietnamese patient. The sequencing was successfully completed and de novo assembled in less than 30 hours, resulting in one contig with no gap and a length of 29,766 bp. All detected variants as compared to the NCBI reference were highly accurate, as confirmed by Sanger sequencing. The results have shown the potential of long read sequencing to provide high quality WGS data to support public health responses and advance understanding of this and future pandemics.

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Whole-Genome Sequence of the Mycoplasma mucosicanis Type Strain

Microbiology Resource Announcements ◽

10.1128/mra.00799-19 ◽

2019 ◽

Vol 8 (41) ◽

Author(s):

Rebecca L. Tallmadge ◽

Patrick K. Mitchell ◽

Renee Anderson ◽

Rebecca Franklin-Guild ◽

Laura B. Goodman

Keyword(s):

Whole Genome Sequencing ◽

Clinical Significance ◽

Genome Sequencing ◽

Genome Sequence ◽

Diagnostic Tests ◽

Type Strain ◽

Whole Genome Sequence ◽

Whole Genome ◽

Content Type

Whole-genome sequencing of Mycoplasma mucosicanis type strain 1642 was performed to support efforts to better understand the clinical significance of Mycoplasma infection in canine health. The availability of this sequence will also further the development of highly specific diagnostic tests.

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Advanced whole genome sequencing and analysis of fetal genomes from amniotic fluid

10.1101/173807 ◽

2017 ◽

Author(s):

Qing Mao ◽

Robert Chin ◽

Weiwei Xie ◽

Yuqing Deng ◽

Huixin Xu ◽

...

Keyword(s):

Autism Spectrum Disorder ◽

Amniotic Fluid ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genome Sequence ◽

Chromosomal Abnormalities ◽

Autism Spectrum ◽

Whole Genome Sequence ◽

Whole Genome ◽

First Time

Amniocentesis is typically performed to identify large chromosomal abnormalities within the fetus. Here we demonstrate that it is feasible to generate an accurate whole genome sequence (WGS) of a fetus from an amniotic sample. DNA from cells and the amniotic fluid were isolated and sequenced from 31 amniocenteses. Concordance of variant calls between the two DNA sources and with parental libraries was high. Two fetal genomes were found to harbor potentially detrimental variants in CHD8 and LRP1, variations in these genes have been associated with Autism Spectrum Disorder (ASD) and Keratosis pilaris atrophicans, respectively. We also discovered drug sensitivities and carrier information of fetuses for a variety of diseases. In this study, we demonstrate for the first time the sequencing of the whole genome of fetuses from amniotic fluid and show that much more information than large chromosomal abnormalities can be gained from an amniocentesis.

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WHOLE-GENOME SEQUENCING AND DE NOVO ASSEMBLY OF A 2019 NOVEL CORONAVIRUS (SARS-COV-2) STRAIN ISOLATED IN VIETNAM

10.1101/2020.06.12.149377 ◽

2020 ◽

Author(s):

Le Tung Lam ◽

Nguyen Trung Hieu ◽

Nguyen Hong Trang ◽

Ho Thi Thuong ◽

Tran Huyen Linh ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Sanger Sequencing ◽

De Novo ◽

Whole Genome ◽

Smrt Sequencing ◽

Sequencing Technology ◽

Zoonotic Virus ◽

Long Read ◽

Novel Coronavirus

ABSTRACTThe pandemic COVID-19 caused by the zoonotic virus SARS-CoV-2 has devastated countries worldwide, infecting more than 4.5 million people and leading to more than 300,000 deaths. Whole genome sequencing (WGS) is an effective tool to monitor emerging strains and provide information for intervention, thus help to inform outbreak control decisions. Here, we reported the first effort to sequence and de novo assemble the whole genome of SARS-CoV-2 using PacBio’s SMRT sequencing technology in Vietnam. We also presented the annotation results and a brief analysis of the variants found in our SARS-CoV-2 strain, which was isolated from a Vietnamese patient. The sequencing was successfully completed and de novo assembled in less than 30 hours, resulting in one contig with no gap and a length of 29,766 bp. All detected variants as compared to the NCBI reference were highly accurate as confirmed by Sanger sequencing. The results have shown the potential of long read sequencing to provide high quality WGS data to support public health responses, and advance understanding of this and future pandemics.

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Whole-Genome Sequencing of Pantoea sp. Strain RIT388, a Potential Oral Opportunistic Pathogen Isolated from a Chewing Stick (Distemonanthus benthamianus)

Microbiology Resource Announcements ◽

10.1128/mra.01468-19 ◽

2020 ◽

Vol 9 (9) ◽

Author(s):

Han Ming Gan ◽

Anutthaman Parthasarathy ◽

Kurtis R. Henry ◽

Michael A. Savka ◽

Bolaji N. Thomas ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genome Sequence ◽

Antibacterial Properties ◽

Opportunistic Pathogen ◽

Whole Genome Sequence ◽

Whole Genome ◽

Content Type ◽

Chewing Sticks

In this study, we report the isolation, identification, characterization, and whole-genome sequence of the endophyte Pantoea sp. strain RIT388, isolated from Distemonanthus benthamianus, a plant known for its antifungal and antibacterial properties that is commonly used for chewing sticks.

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