amplicon-sequencing-using-the-long-read-sequencing-technologies

Amplicon Sequencing using the Long-Read Sequencing Technologies

The World Health Organization (WHO) continues to emphasize the urgent need for a rapid, cost-effective, and user-friendly diagnostic method for ...

Stellenbosch University

The aim of this study was to assess the effect of BC204 as a plant biostimulant on  plants under normal and NaCl-stressed conditions.

The Application of a Commercially Available Citrus-Based Extract Mitigates Moderate NaCl-Stress in  Plants.

Mycobacterium tuberculosis (Mtb) Central Asian Strain (CAS) Lineage 3 (L3) genotype is predominantly found in East-Africa, Central-Asia, Western-Asia, and South-Asia; however, a new spoligotyping CAS/SIT2545 was found in northern regions of Brazil. We aimed to characterize and describe the genetic diversity and perform a phylogenetic assessment of this novel genotype. We performed 24-MIRU-VNTR loci and Whole-genome sequencing (WGS) of six Brazilian isolates previously spoligotyped. The libraries were prepared using a Nextera-XT kit and sequenced in a NextSeq 550 Illumina instrument. We performed lineage assignment and genomic characterization. From publicly available genomes of Mtb L3 and other lineages, we created a robust dataset to run the MTBSeq pipeline and perform a phylogenetic analysis. MIRU-VNTR and WGS confirmed CAS/SIT2545 belongs to L3. Out of 1691 genomes, 1350 (79.83%) passed in quality control (genomic coverage > 95%). Strain 431 differed in 52 single nucleotide variants (SNV), confirming it does not belong to the same transmission chain. The eight genomes from a global dataset clustered closer to Brazilian strains differed in >52 SNVs. We hypothesized L3 and L1 were introduced in Brazilian Northern in the same historical event; however, there is a need for additional studies exploring the genetic diversity of Mtb Brazilian Northern.

Genomic Diversity of the Rarely Observed Genotype of the  Central Asian (CAS) Lineage 3 from North Brazil.

Molecular detection of bedaquiline resistant tuberculosis is challenging as only a small proportion of mutations in candidate bedaquiline resistance genes have been statistically associated with phenotypic resistance. We introduced two mutations, atpE Ile66Val and Rv0678 Thr33Ala, in the Mycobacterium tuberculosis H37Rv reference strain using homologous recombineering or recombination to investigate the phenotypic effect of these mutations. The genotype of the resulting strains was confirmed by Sanger- and whole genome sequencing, and bedaquiline susceptibility was assessed by minimal inhibitory concentration (MIC) assays. The impact of the mutations on protein stability and interactions was predicted using mutation Cutoff Scanning Matrix (mCSM) tools. The atpE Ile66Val mutation did not elevate the MIC above the critical concentration (MIC 0.25-0.5 µg/ml), while the MIC of the Rv0678 Thr33Ala mutant strains (> 1.0 µg/ml) classifies the strain as resistant, confirming clinical findings. In silico analyses confirmed that the atpE Ile66Val mutation minimally disrupts the bedaquiline-ATP synthase interaction, while the Rv0678 Thr33Ala mutation substantially affects the DNA binding affinity of the MmpR transcriptional repressor. Based on a combination of wet-lab and computational methods, our results suggest that the Rv0678 Thr33Ala mutation confers resistance to BDQ, while the atpE Ile66Val mutation does not, but definite proof can only be provided by complementation studies given the presence of secondary mutations.

Site-directed mutagenesis of Mycobacterium tuberculosis and functional validation to investigate potential bedaquiline resistance-causing mutations.

Animal tuberculosis is a significant infectious disease affecting both livestock and wildlife populations worldwide. Effective disease surveillance and characterization of  strains are essential for understanding transmission dynamics and implementing control measures. Currently, sequencing of genomic information has relied on culture-based methods, which are time-consuming, resource-demanding, and concerning in terms of biosafety. This study explores the use of culture-independent long-read whole-genome sequencing (WGS) for a better understanding of  epidemiology in African buffaloes (). By comparing two sequencing approaches, we evaluated the efficacy of Illumina WGS performed on culture extracts and culture-independent Oxford Nanopore adaptive sampling (NAS). Our objective was to assess the potential of NAS to detect genomic variants without sample culture. In addition, culture-independent amplicon sequencing, targeting mycobacterial-specific housekeeping and full-length 16S rRNA genes, was applied to investigate the presence of microorganisms, including nontuberculous mycobacteria. The sequencing quality obtained from DNA extracted directly from tissues using NAS is comparable to the sequencing quality of reads generated from culture-derived DNA using both NAS and Illumina technologies. We present a new approach that provides complete and accurate genome sequence reconstruction, culture independently, and using an economically affordable technique.

Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing.

 forms part of the  complex and has an extensive host range and zoonotic potential. Various genotyping methods (e.g., spoligotyping) have been used to describe the molecular epidemiology of  Advances in whole genome sequencing (WGS) have increased resolution to enable detection of genomic variants to the level of single nucleotide polymorphisms. This is especially relevant to One Health research on tuberculosis which benefits by being able to use WGS to identify epidemiologically linked cases, especially recent transmission. The use of WGS in molecular epidemiology has been extensively used in humans and cattle but is limited in wildlife. This approach appears to overcome the limitations of conventional genotyping methods due to lack of genetic diversity in .

Whole genome sequencing improves the discrimination between  strains on the southern border of Kruger National Park, South Africa.

Implementation of whole genome sequencing (WGS) for patient care is hindered by limited Mycobacterium tuberculosis (Mtb) in clinical specimens and slow Mtb growth. We evaluated droplet multiple displacement amplification (dMDA) for amplification of minute amounts of Mtb DNA to enable WGS as an alternative to other Mtb enrichment methods. Purified genomic Mtb-DNA (0.1, 0.5, 1, and 5 pg) was encapsulated and amplified using the Samplix Xdrop-instrument and sequenced alongside a control sample using standard Illumina protocols followed by MAGMA-analysis. The control and 5 pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400 ng DNA from the 0.1-5 pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7 × for 0.1 pg input DNA to ≥ 60 × for 5 pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5 pg input DNA. Nanopore sequencing of the 5 pg dMDA sample presented excellent coverage depth, breadth, and accurate strain characterization, albeit elevated false positive and false negative variants compared to Illumina-sequenced dMDA sample with identical Mtb DNA input. dMDA coupled with Illumina WGS for samples with ≥ 5 pg purified Mtb DNA, equating to approximately 1000 copies of the Mtb genome, offers precision for drug resistance, phylogeny, and transmission insights.

Droplet based whole genome amplification for sequencing minute amounts of purified Mycobacterium tuberculosis DNA.

Oxford Nanopore Technologies (ONT) sequencing is a promising technology. We assessed the performance of the new ONT R10 flowcells and V14 rapid sequencing chemistry for Mtb whole genome sequencing of Mycobacterium tuberculosis (Mtb) DNA extracted from clinical primary liquid cultures (CPLCs). Using the recommended protocols for MinION Mk1C, R10.4.1 MinION flowcells, and the ONT Rapid Sequencing Kit V14 on six CPLC samples, we obtained a pooled library yield of 10.9 ng/μl, generated 1.94 Gb of sequenced bases and 214k reads after 48h in a first sequencing run. Only half (49%) of all generated reads met the Phred Quality score threshold (>8). To assess if the low data output and sequence quality were due to impurities present in DNA extracted directly from CPLCs, we added a pre-library preparation bead-clean-up step and included purified DNA obtained from an Mtb subculture as a control sample in a second sequencing run. The library yield for DNA extracted from four CPLCs and one Mtb subculture (control) was similar (10.0 ng/μl), 2.38 Gb of bases and 822k reads were produced. The quality was slightly better with 66% of the produced reads having a Phred Quality >8. A third run of DNA from six CPLCs with bead clean-up pre-processing produced a low library yield (±1 Gb of bases, 166k reads) of low quality (51% of reads with a Phred Quality score >8). A median depth of coverage above 10× was only achieved for five of 17 (29%) sequenced libraries. Compared to Illumina WGS of the same samples, accurate lineage predictions and full drug resistance profiles from the generated ONT data could not be determined by TBProfiler. Further optimization of the V14 ONT rapid sequencing chemistry and library preparation protocol is needed for clinical Mtb WGS applications.

Exploring the potential of Oxford Nanopore Technologies sequencing for Mycobacterium tuberculosis sequencing: An assessment of R10 flowcells and V14 chemistry.

 Isoniazid-resistant tuberculosis (Hr-TB) is often overlooked in diagnostic algorithms because of reliance on first-line molecular assays testing only for rifampicin resistance.  To determine the prevalence, outcomes, and molecular mechanisms associated with rifampin-susceptible, isoniazid-resistant TB (Hr-TB) in the Eastern Cape, South Africa.  Between April 2016 and October 2017, sputum samples were collected from patients with rifampin-susceptible TB at baseline and at Weeks 7 and 23 of drug-susceptible TB treatment. We performed isoniazid phenotypic and genotypic drug susceptibility testing, including FluoroTypeMTBDR, Sanger sequencing, targeted next-generation sequencing, and whole-genome sequencing.  We analyzed baseline isolates from 766 patients with rifampin-susceptible TB. Of 89 patients (11.7%) who were found to have Hr-TB, 39 (44%) had canonical  or  promoter mutations; 35 (39%) had noncanonical  mutations (including 5 with underlying large deletions); 4 (5%) had mutations in other candidate genes associated with isoniazid resistance. For 11 (12.4%), no cause of resistance was found.  Among patients with rifampin-susceptible TB who were diagnosed using first-line molecular TB assays, there is a high prevalence of Hr-TB. Phenotypic drug susceptibility testing remains the gold standard. To improve the performance of genetic-based phenotyping tests, all isoniazid resistance-associated regions should be included, and such tests should have the ability to identify underlying mutations.

The Hidden Epidemic of Isoniazid-Resistant Tuberculosis in South Africa.

A previous study in Pará, Northern Brazil, described a strain of  with a unique genotype (SIT2517/T1) associated with multidrug-resistant tuberculosis (MDR-TB). To improve our understanding of MDR-TB transmission dynamics of these strains within this region, we performed phenotypic and genotypic drug susceptibility testing (pDST/gDST), 24-loci mycobacterial interspersed repetitive units (MIRU-VNTR) genotyping, whole-genome sequencing (WGS) and geo-epidemiology analysis. Of the 28 SIT2517/T1 isolates, 19 (67.9%) could be genotyped by 24-loci MIRU-VNTR and 15 by WGS. All belonged to sublineage 4.1.1.3, distinct from other representative Lineage 4 isolates identified in Brazil. The MDR phenotype determined by pDST was confirmed by gDST, the latter also demonstrating the presence of additional mutations conferring pre-extensively drug-resistance (pre-XDR). Discrepancies between gDST and pDST were observed for pyrazinamide and fluoroquinolones. Thirteen out of 15 isolates analyzed by WGS were clustered when applying a 12 single nucleotide polymorphisms (SNPs) cutoff. The SIT2517/T1 isolates were distributed across the metropolitan regions of Belém and Collares municipalities, showing no geographic clustering. WGS-transmission network analysis revealed a high likelihood of direct transmission and the formation of two closely linked transmission chains. This study highlights the need to implement TB genomic surveillance in the Brazilian Amazon region.

A Genome-Focused Investigation Reveals the Emergence of a  Strain Related to Multidrug-Resistant Tuberculosis in the Amazon Region of Brazil.

Current tuberculosis (TB) treatment is typically effective against drug-susceptible Mycobacterium tuberculosis, but can fail due to acquired drug resistance or phenotypic resistance. M. tuberculosis persisters, a subpopulation of viable but non-replicating (VBNR) antibiotic-tolerant bacteria, are thought to contribute to poor TB treatment outcomes. In this exploratory study, we investigated treatment-naïve drug-susceptible clinical isolates collected from people with TB, who subsequently had unsuccessful treatment outcomes. These were compared to isolates from cured individuals in terms of their ability to form VBNR subpopulations. Clinical isolates from individuals with unfavorable treatment outcomes form larger subpopulations of VBNR M. tuberculosis (2.67-13.71%) than clinical isolates from cured cases (0- 1.63%) following infection of THP-1 macrophages. All isolates were drug susceptible based on phenotypic and genotypic analysis. Whole genome sequencing identified 23 non-synonymous genomic variants shared by treatment failure clinical isolates, that were not present in isolates from cured cases. This exploratory study highlights the ability of treatment-naïve clinical isolates to form heterogeneous populations containing VBNR M. tuberculosis. We also demonstrate that clinical isolates from individuals with unsuccessful treatment outcomes form higher percentages of VBNR M. tuberculosis. The findings of this exploratory study suggest that an increased propensity to form VBNR subpopulations may impact TB treatment outcome.

Assessing the propensity of TB clinical isolates to form viable but non-replicating subpopulations.

The emergence of drug-resistant  () strains remains a threat to tuberculosis (TB) prevention and care. Understanding the drug resistance profiles of circulating strains is crucial for effective TB control. This study aimed to describe the genetic diversity of rifampicin-resistant  strains circulating in Botswana using whole genome sequencing (WGS).

Article	Year
The Application of a Commercially Available Citrus-Based Extract Mitigates Moderate NaCl-Stress in Plants. Plants (Basel, Switzerland)\| PubMed ID: 32785013	2020
Genomic Diversity of the Rarely Observed Genotype of the Central Asian (CAS) Lineage 3 from North Brazil. Microorganisms\| PubMed ID: 36677424	2023
Site-directed mutagenesis of Mycobacterium tuberculosis and functional validation to investigate potential bedaquiline resistance-causing mutations. Scientific reports\| PubMed ID: 37280265	2023
Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing. Frontiers in microbiology\| PubMed ID: 38075895	2023
Whole genome sequencing improves the discrimination between strains on the southern border of Kruger National Park, South Africa. One health (Amsterdam, Netherlands)\| PubMed ID: 38283183	2023
Droplet based whole genome amplification for sequencing minute amounts of purified Mycobacterium tuberculosis DNA. Scientific reports\| PubMed ID: 38689002	2024
Exploring the potential of Oxford Nanopore Technologies sequencing for Mycobacterium tuberculosis sequencing: An assessment of R10 flowcells and V14 chemistry. PloS one\| PubMed ID: 38843147	2024
The Hidden Epidemic of Isoniazid-Resistant Tuberculosis in South Africa. Annals of the American Thoracic Society\| PubMed ID: 38935769	2024
A Genome-Focused Investigation Reveals the Emergence of a Strain Related to Multidrug-Resistant Tuberculosis in the Amazon Region of Brazil. Microorganisms\| PubMed ID: 39338491	2024
Assessing the propensity of TB clinical isolates to form viable but non-replicating subpopulations. Scientific reports\| PubMed ID: 39532967	2024
Whole genomic analysis uncovers high genetic diversity of rifampicin-resistant strains in Botswana. Frontiers in microbiology\| PubMed ID: 40008038	2025

Johannes Loubser

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