Dataset Information

GenSeizer: a Multiplex PCR-Based Targeted Gene Sequencing Platform for Rapid and Accurate Identification of Major Mycobacterium Species.

ABSTRACT: Mycobacterium tuberculosis and nontuberculous mycobacterium (NTM) infections often exhibit similar clinical symptoms. Timely and effective treatment relies on the rapid and accurate identification of species and resistance genotypes. In this study, a new platform (GenSeizer), which combines bioinformatics analysis of a large data set and multiplex PCR-based targeted gene sequencing, was developed to identify 10 major Mycobacterium species that cause pulmonary, as well as extrapulmonary, human diseases. The simultaneous detection of certain erm(41) and rrl resistance genotypes in M. abscessus was also feasible. This platform was specific and sensitive and exhibited no cross-reactivity among reference strains and a detection limit of 5 DNA copies or 50 CFU Mycobacterium/ml. In a blind comparison, GenSeizer and multigene sequencing showed 100% agreement in the ability to identify 88 clinical Mycobacterium isolates. The resistance genotypes, confirmed by whole-genome sequencing of 30 M. abscessus strains, were also correctly identified by GenSeizer 100% of the time. These results indicate that GenSeizer is an efficient, reliable platform for detecting major pathogenic Mycobacterium species.

SUBMITTER: Li B

PROVIDER: S-EPMC8111139 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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GenSeizer: a Multiplex PCR-Based Targeted Gene Sequencing Platform for Rapid and Accurate Identification of Major Mycobacterium Species.

Li Bing B Xu Liyun L Guo Qi Q Chen Jianhui J Zhang Yanan Y Huang Wenpan W Zhang Zhemin Z Han Lizhong L Xu Xiaogang X Chu Haiqing H

Journal of clinical microbiology 20210121 2

Mycobacterium tuberculosis and nontuberculous mycobacterium (NTM) infections often exhibit similar clinical symptoms. Timely and effective treatment relies on the rapid and accurate identification of species and resistance genotypes. In this study, a new platform (GenSeizer), which combines bioinformatics analysis of a large data set and multiplex PCR-based targeted gene sequencing, was developed to identify 10 major Mycobacterium species that cause pulmonary, as well as extrapulmo ...[more]

PMID: 33177124

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Project description:Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R² = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R² = 0.97-0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R² = 0.96) and whole transcriptome RNA-sequencing following "traditional" library preparation using Illumina NGS kits (R² = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 10⁷-fold range was reduced more than 10,000-fold, from 2.3×10⁹ to 1.4×10⁵ sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.

Dataset Information

GenSeizer: a Multiplex PCR-Based Targeted Gene Sequencing Platform for Rapid and Accurate Identification of Major Mycobacterium Species.

Publications

GenSeizer: a Multiplex PCR-Based Targeted Gene Sequencing Platform for Rapid and Accurate Identification of Major <i>Mycobacterium</i> Species.

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