Nanopore sequencing for Mycobacterium tuberculosis : a critical review of the literature, new developments and future opportunities

The next-generation short-read sequencing technologies that generate comprehensive, whole-genome data with single-nucleotide resolution have already advanced tuberculosis diagnosis, treatment, surveillance and source investigation. Their high costs, tedious and lengthy processes, and large equipment remain major hurdles for research use in high tuberculosis burden countries and implementation into routine care. The portable next-generation sequencing devices developed by Oxford Nanopore Technologies (ONT) are attractive alternatives due to their long-read sequence capability, compact low-cost hardware, and continued improvements in accuracy and throughput. A systematic review of the published literature demonstrated limited uptake of ONT sequencing in tuberculosis research and clinical care. Of the 12 eligible articles presenting ONT sequencing data on at least one Mycobacterium tuberculosis sample, four addressed software development for long read ONT sequencing data with potential applications for M. tuberculosis . Only eight studies presented results of ONT sequencing of M. tuberculosis , of which five performed whole-genome and three did targeted sequencing. Based on these findings, we summarize the standard processes, reflect on the current limitations of ONT sequencing technology, and the research needed to overcome the main hurdles. Summary: The low capital cost, portable nature and continued improvement in the performance of ONT sequencing make it an attractive option for sequencing for research and clinical care, but limited data is available on its application in the tuberculosis field. Important research investment is needed to unleash the full potential of ONT sequencing for tuberculosis research and care.

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Phenotypic and genotypic features of the Mycobacterium tuberculosis lineage 1 subgroup in central Vietnam

Scientific Reports ◽

10.1038/s41598-021-92984-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nguyen Thi Le Hang ◽

Minako Hijikata ◽

Shinji Maeda ◽

Akiko Miyabayashi ◽

Keiko Wakabayashi ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Pulmonary Tuberculosis ◽

Genome Sequencing ◽

Whole Genome ◽

Sequencing Data ◽

Distinctive Features ◽

Sequencing Platform ◽

Central Vietnam ◽

Smear Positive Pulmonary Tuberculosis ◽

Long Read

AbstractMycobacterium tuberculosis (Mtb) has different features depending on different geographic areas. We collected Mtb strains from patients with smear-positive pulmonary tuberculosis in Da Nang, central Vietnam. Using a whole genome sequencing platform, including genome assembly complemented by long-read-sequencing data, genomic characteristics were studied. Of 181 Mtb isolates, predominant Vietnamese EAI4_VNM and EAI4-like spoligotypes (31.5%), ZERO strains (5.0%), and part of EAI5 (11.1%) were included in a lineage-1 (L1) sublineage, i.e., L1.1.1.1. These strains were found less often in younger people, and they genetically clustered less frequently than other modern strains. Patients infected with ZERO strains demonstrated less lung infiltration. A region in RD2bcg spanning six loci, i.e., PE_PGRS35, cfp21, Rv1985c, Rv1986, Rv1987, and erm(37), was deleted in EAI4_VNM, EAI4-like, and ZERO strains, whereas another 118 bp deletion in furA was specific only to ZERO strains. L1.1.1.1-sublineage-specific deletions in PE_PGRS4 and PE_PGRS22 were also identified. RD900, seen in ancestral lineages, was present in majority of the L1 members. All strains without IS6110 (5.0%) had the ZERO spoligo-pattern. Distinctive features of the ancestral L1 strains provide a basis for investigation of the modern versus ancestral Mtb lineages and allow consideration of countermeasures against this heterogeneous pathogen.

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Comprehensive identification of transposable element insertions using multiple sequencing technologies

Nature Communications ◽

10.1038/s41467-021-24041-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chong Chu ◽

Rebeca Borges-Monroy ◽

Vinayak V. Viswanadham ◽

Soohyun Lee ◽

Heng Li ◽

...

Keyword(s):

Transposable Element ◽

Structure And Function ◽

Endogenous Retroviruses ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data ◽

Short Read ◽

Sequencing Technologies ◽

Long Read ◽

And Function

AbstractTransposable elements (TEs) help shape the structure and function of the human genome. When inserted into some locations, TEs may disrupt gene regulation and cause diseases. Here, we present xTea (x-Transposable element analyzer), a tool for identifying TE insertions in whole-genome sequencing data. Whereas existing methods are mostly designed for short-read data, xTea can be applied to both short-read and long-read data. Our analysis shows that xTea outperforms other short read-based methods for both germline and somatic TE insertion discovery. With long-read data, we created a catalogue of polymorphic insertions with full assembly and annotation of insertional sequences for various types of retroelements, including pseudogenes and endogenous retroviruses. Notably, we find that individual genomes have an average of nine groups of full-length L1s in centromeres, suggesting that centromeres and other highly repetitive regions such as telomeres are a significant yet unexplored source of active L1s. xTea is available at https://github.com/parklab/xTea.

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Fast and low-cost decentralized surveillance of transmission of tuberculosis based on strain-specific PCRs tailored from whole genome sequencing data: a pilot study

Clinical Microbiology and Infection ◽

10.1016/j.cmi.2014.10.003 ◽

2015 ◽

Vol 21 (3) ◽

pp. 249.e1-249.e9 ◽

Cited By ~ 12

Author(s):

L. Pérez-Lago ◽

M. Martínez Lirola ◽

M. Herranz ◽

I. Comas ◽

E. Bouza ◽

...

Keyword(s):

Pilot Study ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Low Cost ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data

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Whole genome sequencing data of 1110 Mycobacterium tuberculosis isolates identifies insertions and deletions associated with drug resistance

BMC Genomics ◽

10.1186/s12864-018-4734-6 ◽

2018 ◽

Vol 19 (1) ◽

Cited By ~ 3

Author(s):

Xi Zeng ◽

Jamie Sui-Lam Kwok ◽

Kevin Yi Yang ◽

Kenneth Siu-Sing Leung ◽

Mai Shi ◽

...

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data ◽

Insertions And Deletions

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DNA Preparation In Vitis Vinifera L. for Third Generation Sequencing

10.21203/rs.3.rs-259540/v2 ◽

2021 ◽

Author(s):

Androniki C. Bibi ◽

Anastasios Kollias ◽

Maria Astrinaki ◽

Despoina Vassou ◽

Dimitris Kafetzopoulos ◽

...

Keyword(s):

Vitis Vinifera ◽

Low Cost ◽

Ease Of Use ◽

Vitis Vinifera L ◽

Full Potential ◽

Third Generation ◽

High Quality ◽

Fresh Tissue ◽

Long Read ◽

Young Leaves

Abstract Background: There have been several attempts to sequence the genome of Vitis vinifera L. (grapevine), utilizing low-resolution second-generation platforms. Nevertheless, the characterization of the grapevine genetic resources and its adaptation to vulnerable conditions could be better addressed through extensive and high-resolution genome sequencing.MinION is a third-generation sequencer preferred by many laboratories due to its relatively low cost, ease of use and small size. Even though this long-read technology has been rapidly improving, to reach its full potential requires high-quality DNA.Results: Here we establish a workflow for DNA extraction suitable for MinION sequencing long reads from grapevine. This protocol was tested with leaf samples from different positions on annual growing branches of grapevine, Purified nuclei from fresh young leaves that led to high quality, long DNA fragments, suitable for long-read sequencing were successfully generated. It is evident that longer reads in grapevine associate with both fresh tissue and adjusted conditions used for nuclei purification.Conclusions: We propose that this workflow presents a significant advancement for long-read quality DNA isolation for grapevine and likely other plant species.

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Rapid Mycobacterium tuberculosis spoligotyping from uncorrected long reads using Galru

10.1101/2020.05.31.126490 ◽

2020 ◽

Author(s):

Andrew J. Page ◽

Nabil-Fareed Alikhan ◽

Michael Strinden ◽

Thanh Le Viet ◽

Timofey Skvortsov

Keyword(s):

Mycobacterium Tuberculosis ◽

State Of The Art ◽

Sequence Data ◽

Human Pathogen ◽

Sequencing Data ◽

Short Read ◽

Short Read Sequencing ◽

Long Reads ◽

Long Read

AbstractSpoligotyping of Mycobacterium tuberculosis provides a subspecies classification of this major human pathogen. Spoligotypes can be predicted from short read genome sequencing data; however, no methods exist for long read sequence data such as from Nanopore or PacBio. We present a novel software package Galru, which can rapidly detect the spoligotype of a Mycobacterium tuberculosis sample from as little as a single uncorrected long read. It allows for near real-time spoligotyping from long read data as it is being sequenced, giving rapid sample typing. We compare it to the existing state of the art software and find it performs identically to the results obtained from short read sequencing data. Galru is freely available from https://github.com/quadram-institute-bioscience/galru under the GPLv3 open source licence.

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MTBseq: a comprehensive pipeline for whole genome sequence analysis of Mycobacterium tuberculosis complex isolates

PeerJ ◽

10.7717/peerj.5895 ◽

2018 ◽

Vol 6 ◽

pp. e5895 ◽

Cited By ~ 35

Author(s):

Thomas Andreas Kohl ◽

Christian Utpatel ◽

Viola Schleusener ◽

Maria Rosaria De Filippo ◽

Patrick Beckert ◽

...

Keyword(s):

Antibiotic Resistance ◽

Mycobacterium Tuberculosis ◽

Genome Sequence ◽

Sequence Data ◽

Whole Genome Sequence ◽

Whole Genome Sequencing Data ◽

Phylogenomic Analysis ◽

Whole Genome ◽

Sequencing Data ◽

Desktop Computer

Analyzing whole-genome sequencing data of Mycobacterium tuberculosis complex (MTBC) isolates in a standardized workflow enables both comprehensive antibiotic resistance profiling and outbreak surveillance with highest resolution up to the identification of recent transmission chains. Here, we present MTBseq, a bioinformatics pipeline for next-generation genome sequence data analysis of MTBC isolates. Employing a reference mapping based workflow, MTBseq reports detected variant positions annotated with known association to antibiotic resistance and performs a lineage classification based on phylogenetic single nucleotide polymorphisms (SNPs). When comparing multiple datasets, MTBseq provides a joint list of variants and a FASTA alignment of SNP positions for use in phylogenomic analysis, and identifies groups of related isolates. The pipeline is customizable, expandable and can be used on a desktop computer or laptop without any internet connection, ensuring mobile usage and data security. MTBseq and accompanying documentation is available from https://github.com/ngs-fzb/MTBseq_source.

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