Lianpeng Wu’s research while affiliated with Shantou Central Hospital and other places

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Publications (1)

Nanopore targeted sequencing process for drug resistance assay of Mycobacterium tuberculosis (MTB). (A) The drug resistance-associated genes marked by black triangles with their positions in the genome of the reference strain MTB H37Rv, including 18 highly recommended genes which were gyrB, gyrA, rpoB, mmpR5, rpsL, rplC, atpE, rrs, rrl, fabG1, inhA, rpsA, tlyA, katG, pncA, eis, embB, and ubiA, respectively. (B) Process of high-throughput nanopore sequencing combined with single-tube multiplex PCR-based target enrichment. After DNA extraction, the targeted regions of drug resistance-associated genes were amplified by multiplex PCR, followed by barcoding and adapter ligation, and the prepared library was then loaded onto the flow cell on the nanopore sequencer, which generated long-read sequence data finally analyzed with bioinformatics. The turnaround time of the NTS process was approximately 7.5 h.
Development of nanopore targeted sequencing with multiplex PCR for drug resistance assay of Mycobacterium tuberculosis (MTB). (A) Electrophoresis of multiplex PCR-based amplicons from five MTB samples and a negative control. The bands of electrophoretic lanes 1–5 from bottom to top were rpsL (560 bp), mmpR5 (600 bp), rplC (652 bp), atpE (744 bp), tlyA (827 bp), pncA (971 bp), ubiA (1,041 bp), gyrB (1,210 bp), rrl (1,275 bp), eis (1,322 bp), fabG1 and inhA (1,387 bp), rpsA (1,444 bp), gyrA (1,544 bp), katG (1,653 bp), rrs (1741 bp), rpoB (1845 bp), and embB (1925 bp), respectively. The ladder sizes of DNA markers (M) were from 600 bp to 4,000 bp. The negative control was nuclease-free water. (B) Bar chart of 18 drug resistance-associated genes arranged according to the sizes of gene fragments against the sequencing depth of each gene after nanopore sequencing. (C) Distribution of 18 drug resistance-associated genes in MTB genome with their corresponding sequencing depths. (D) NTS-based drug resistance data are available for isolates after using the TBProfiler. Each row is a drug, and the columns with filled cells represent the set of isolates possessing drug resistance information. The box below shows the number of isolates in the set. The bar plot in the left panel shows the number of isolates with resistance information for each drug.
Accuracy of multiple amplicons sequenced by nanopore targeted sequencing (NTS). (A) The density distribution comparison of raw sequence accuracy of multiple amplicons between ONT flow cell R9.4.1 and R10.4.1. (B) Per base sequence quality of the single-base Q-value corresponding to the base position in the multiple amplicons from ONT flow cell R10.4.1. The column area of the box-whisker plot represents the range of 25–75% of Q-value, and the area between the upper and lower black lines represents the range of 10–90%. The blue line represents the average Q-value in the position, and the red line represents the median of the Q-value in the position. The background color divides the picture into three parts: good base quality (green), medium base quality (yellow), and poor base quality (red).
Statistics of clinical samples and their processes of inclusion in nanopore targeted sequencing (NTS) assay. (A) Analysis workflow of the 99 clinical samples included in the study. (B) Contingency tables of MTB culture with NTS and Xpert MTB/RIF sets. Abbreviations: PPV, positive predictive value; NPV, negative predictive value; TCR, the total coincidence rate. (C) NTS-based drug resistance data are available for clinical samples after using the TBProfiler, with a total of 20 drug-resistant samples identified. Each row is a drug, and the columns with filled cells represent the set of samples possessing drug resistance information. The box below shows the number of samples in the set. The bar plot in the left panel shows the number of samples with resistance information for each drug. (D) The mutation at position 955 of the embB gene is detected using NTS and verified using Sanger sequencing.
High-throughput nanopore targeted sequencing for efficient drug resistance assay of Mycobacterium tuberculosis
  • Article
  • Full-text available

May 2024

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44 Reads

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5 Citations

Chen Tang

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Lianpeng Wu

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Machao Li

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Guangxin Xiang

Drug-resistant tuberculosis (TB), especially multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), is one of the urgent clinical problems and public health challenges. Culture-based phenotypic drug susceptibility testing (pDST) is time-consuming, and PCR-based assays are limited to hotspot mutations. In this study, we developed and validated a convenient and efficient approach based on high-throughput nanopore sequencing technology combined with multiplex PCR, namely nanopore targeted sequencing (NTS), to simultaneously sequence 18 genes associated with antibiotic resistance in Mycobacterium tuberculosis (MTB). The analytical performance of NTS was evaluated, and 99 clinical samples were collected to assess its clinical performance. The NTS results showed that MTB and its drug resistance were successfully identified in approximately 7.5 h. Furthermore, compared to the pDST and Xpert MTB/RIF assays, NTS provided much more drug resistance information, covering 14 anti-TB drugs, and it identified 20 clinical cases of drug-resistant MTB. The mutations underlying these drug-resistant cases were all verified using Sanger sequencing. Our approach for this TB drug resistance assay offers several advantages, including being culture-free, efficient, high-throughput, and highly accurate, which would be very helpful for clinical patient management and TB infection control.

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Citations (1)

... 6 WGS has emerged as a transformative approach in pathogen diagnostics, providing high-resolution genotyping and precise pathogen identification. 7,8 Unlike traditional DST methods, WGS provides a comprehensive analysis of MTB genomic DNA to prediction of drug resistance mechanisms across multiple antimicrobials. 9 While WGS has primarily been applied in economically developed regions with low TB burden, its potential in determining resistance to first-line drugs is increasingly being recognized. ...

Reference:

Genomic characterization of multidrug-resistant tuberculosis in Shanghai, China: antibiotic resistance, virulence and transmission
High-throughput nanopore targeted sequencing for efficient drug resistance assay of Mycobacterium tuberculosis
Chen Tang

·

Lianpeng Wu

·

Machao Li

·

[...]

·

Guangxin Xiang