Name: Amplicon Sequencing using the Long-Read Sequencing Technologies (Video) | JoVE Journal
Uploaded: 2025-08-29T13:40:00.000+00:00
Duration: 8 min 57 s
Description: Discover portable long-read sequencing for tuberculosis drug-resistance detection using targeted amplicon analysis. This method delivers rapid, accurate diagnostics without requiring extensive bioinformatics expertise.

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5 minutes

We would like to express our sincere gratitude to the SAMRC Centre for Tuberculosis at Stellenbosch University for providing the essential resources and facilities. We are also deeply thankful to the TS ELiOT Study for supplying the samples that were integral to this study.

This research was conducted as part of the TS ELiOT project, which received ethical approval from the Human Research Ethics Committee (HREC) at Stellenbosch University (N21/09/093) on October 20, 2021. Additionally, it obtained authorization from Western Cape Health Research for the use of government facilities. Samples were collected through a collaborative effort with the NHLS Green Point and Stellenbosch University, under the SU HREC approval number N09/11/296. The reagents and the equipment used are listed in the Table of Materials.
1. Sample preparation
<ol>
	<li>Heat inactivation
	<ol>
		<li>Incubate the MGIT cultures at 80&#8239;&#176;C for 1 h to achieve complete heat inactivation under BSL-3 conditions.</li>
		<li>Proceed with DNA extraction using the InstaGene method29.</li>
	</ol>
	</li>
	<li>DNA extraction
	<ol>
		<li>Place 5 mL of heat-inactivated MGIT culture into a 15 mL tube.</li>
		<li>Centrifuge at 4000 x g for 30 min at room temperature (RT).</li>
		<li>Carefully remove the supernatant by pipetting, leaving the sediment.</li>
		<li>Add 200 &#181;L of chelation-based DNA extraction reagent to the sediment and incubate at 56 &#176;C for 15 min.</li>
		<li>Transfer the solution to a 2 mL screw-cap tube and add three 2 mm diameter glass beads to each.</li>
		<li>Vortex for 10 s to spread cells and place the tubes in a dry bath incubator at 100 &#176;C for 8 min.</li>
		<li>Homogenize the samples using the bead-beating homogenizer with the following settings: 3 cycles of 20 s at 4.0 m/s.</li>
		<li>Centrifuge at 12,000 x g for 15 min at RT and carefully transfer 130 &#181;L of the supernatant (DNA-containing liquid) to a 1.5 mL tube, avoiding sediment and debris.</li>
		<li>Resuspend the beads by flicking or vortexing and add 156 &#181;L of the beads to the 1.5 mL tube containing the extracted DNA.</li>
		<li>Slowly pipette up and down 10 times to mix and incubate at RT for 5 min.</li>
		<li>Place the tubes on a magnetic rack for 5 min to separate the DNA-bound beads. 
		NOTE: A clear supernatant should appear above a compact pellet of dark magnetic beads at the side of the tube facing the magnet, indicating readiness for supernatant removal.</li>
		<li>Pipette off the supernatant without disturbing the beads.</li>
		<li>Add 100 &#181;L of freshly prepared 80% ethanol to the bead pellet while on the rack and leave for 30 s, then remove the ethanol. Repeat the wash step.</li>
		<li>Air-dry the bead pellet at RT for 5 min. Ensure the bead pellet appears matte and no visible liquid remains before proceeding to the next step.</li>
		<li>Remove the tube from the magnetic rack.</li>
		<li>Add 25 &#181;L of Nuclease Free Water (NFW) directly to the dried beads and resuspend by gently pipetting up and down 10 times.</li>
		<li>Incubate at RT for 5 min.</li>
		<li>Return the tube to the magnetic rack for another 5 min.</li>
		<li>Carefully transfer the eluted DNA (supernatant) to a new 1.5 mL tube.</li>
		<li>Measure the DNA concentration using the high-sensitivity fluorescence-based dsDNA quantification reagent. 
		NOTE: The DNA can be stored in a -20 &#176;C freezer or proceed with PCR.</li>
	</ol>
	</li>
	<li>Amplification (PCR)
	<ol>
		<li>Multiplex PCR and post-PCR cleanup, following manufacturer&#39;s instructions25.</li>
		<li>Measure the DNA concentration using the high-sensitivity fluorescence-based dsDNA quantification reagent.</li>
	</ol>
	</li>
	<li>Gel electrophoresis
	<ol>
		<li>Prepare a 2% agarose gel by dissolving agarose powder in 1x TAE buffer through microwave heating until fully melted.</li>
		<li>Add &#8239;5 &#181;L of nontoxic fluorescent DNA gel stain to the melted agarose and mix thoroughly.</li>
		<li>Pour the gel mixture into a gel casting tray fitted with a comb to form wells.</li>
		<li>Allow the gel to solidify at RT.</li>
		<li>Once solidified, remove the comb and place the gel tray in the electrophoresis tank filled with 1x TAE buffer.</li>
		<li>Load 5&#8239;&#181;L of a &#8239;kb DNA ladder mixed with 5&#8239;&#181;L of loading buffer into the first well.</li>
		<li>For each PCR product, mix 5 &#181;L of the product with 5&#8239;&#181;L of loading buffer and load into individual wells.</li>
		<li>Run the gel at 100 V for 1 h.</li>
		<li>Visualize the DNA bands using the gel documentation system with transillumination.</li>
	</ol>
	</li>
</ol>
2. ONT library preparation
<ol>
	<li>End-prep
	<ol>
		<li>Calculate 100 ng of each amplicon sample based on concentration and transfer the required volume into a clean PCR tube.</li>
		<li>To calculate the total mass (in ng) of amplicons in a given sample, multiply the concentration of the sample (ng/&#181;L) by its remaining volume (&#181;L) after quality control (QC). 
		NOTE: For example, if a sample has a concentration of 2&#8239;ng/&#181;L and a remaining volume of 1&#8239;&#181;L post-QC, the total amplicon mass is: 
		Total mass = 2&#8239;ng/&#181;L &#215; 1&#8239;&#181;L = 45&#8239;ng 
		To prepare 10&#8239;ng of amplicons for sequencing, use the dilution formula: 
		C1V1 = C2V2 
		Where: C1 = 45&#8239;ng, V1 = unknown, C2 = 10&#8239;ng, V2 = 1&#8239;&#181;L, V1 = (C2 &#215; V2) / C1 = (10&#8239;ng &#215; 1&#8239;&#181;L) / 45&#8239;ng = &#8239;&#181;L 
		&#8203;Thus, &#8239;&#181;L of the initial amplicon sample should be used to obtain 10&#8239;ng of DNA for sequencing.</li>
		<li>Adjust the total volume of each sample to 12.5 &#181;L using NFW.</li>
		<li>Mix the samples gently by pipetting up and down 10 times and briefly spin.</li>
		<li>Add 1.75 &#181;L of end-repair and A-tailing reaction buffer and 0.75 &#181;L of endrepair and A-tailing enzyme mix to each sample.</li>
		<li>Mix thoroughly by pipetting up and down 10 times and briefly spin down.</li>
		<li>Incubate the tubes in a thermocycler at 25 &#176;C for 30 min, followed by 65 &#176;C for 30min. 
		NOTE: At this stage, should the user decide to discontinue the process, it is advisable to utilize beads for the purification of the end-prepped DNA. The purified DNA can then be stored at 4 &#176;C for a maximum duration of seven days.</li>
	</ol>
	</li>
	<li>Native barcode ligation
	<ol>
		<li>Thaw and prepare the magnetic beads for DNA purification and size selection, paramagnetic DNA cleanup beads, EDTA, and native barcodes (NB01-96) according to the manufacturer&#39;s instructions, and then place them on ice.</li>
		<li>In new PCR tubes, add the following in sequence: 2.75 &#181;L of nuclease-free water,1 &#181;L of end-prepped DNA, 1.25 &#181;L native barcodes (NB01-96), and 5 &#181;L of DNA ligation enzyme mix.</li>
		<li>Mix each reaction by pipetting gently 10 times, spin briefly using a microcentrifuge, and incubate for 20 min at RT.</li>
		<li>Add 1 &#181;L of EDTA to each well, mix thoroughly by pipetting, and spin briefly.</li>
		<li>Pool-barcoded samples into a 1.5 mL microcentrifuge tube.</li>
		<li>Resuspend the DNA cleanup beads by vortexing and add 0.7x the total pooled sample volume (e.g., for 23 samples with a total volume of 253 &#181;L, add 177.1 &#181;L beads).</li>
		<li>Mix the solution by pipetting and incubate on a HulaMixer for 10 min at RT.</li>
		<li>Prepare 2 mL of 80% ethanol by mixing 1600 &#181;L pure ethanol with 400 &#181;L NFW.</li>
		<li>Place the tube on a magnetic rack for 5 min until the eluate is clear and colourless.</li>
		<li>Remove and discard the supernatant without disturbing the pellet.</li>
		<li>Wash the beads with 700 &#181;L freshly prepared 80% ethanol without disturbing the pellet, then remove and discard the ethanol.</li>
		<li>Repeat the ethanol wash step.</li>
		<li>Centrifuge the tube briefly, return to the magnetic rack, and remove residual ethanol.</li>
		<li>Allow the beads to air dry for 30 s.</li>
		<li>Remove the tube from the magnetic rack, resuspend the beads in 35 &#181;L nuclease-free water by gentle flicking, and incubate at 37 &#176;C for 10 min.</li>
		<li>Return the tube to the magnetic rack until the eluate is clear and colourless.</li>
		<li>Transfer 35 &#181;L of the eluate into a clean 1.5 mL microcentrifuge tube for subsequent steps. 
		NOTE: The library can be stored at 4 &#176;C overnight at this stage.</li>
	</ol>
	</li>
	<li>Adapter ligation and cleanup
	<ol>
		<li>Prepare the rapid DNA ligation reagent kit according to the manufacturer&#39;s instructions and place it on ice.</li>
		<li>Mix the Native Adapter (NA) and Quick Ligase by pipetting, then place on ice.</li>
		<li>Thaw the elution buffer (EB) at RT, vortex, centrifuge, and place it on ice.</li>
		<li>Thaw Short Fragment Buffer (SFB) at RT, vortex, centrifuge, and place on ice.</li>
		<li>In a 1.5 mL low-binding microcentrifuge tube, mix the following: 30 &#181;L of pooled barcoded sample, 5 &#181;L of Native Adapter (NA), 10 &#181;L of rapid ligation reaction buffer, and 5 &#181;L of rapid-acting T4 DNA ligase.</li>
		<li>Gently pipette to mix, briefly centrifuge, and incubate for 20 min at RT.</li>
		<li>Resuspend DNA cleanup beads by vortexing and add 20 &#181;L (0.4x) of beads to the reaction.</li>
		<li>Mix gently by pipetting and incubate on a HulaMixer for 10 min at RT.</li>
		<li>Pellet the beads on a magnetic rack and aspirate the supernatant without disturbing the pellet.</li>
		<li>Wash the beads by adding 125 &#181;L of Short Fragment Buffer (SFB), resuspend by flicking, centrifuge briefly, and return to the magnetic rack.</li>
		<li>Pellet the beads and aspirate the supernatant; repeat the washing step once.</li>
		<li>Briefly centrifuge, return the tube to the magnetic rack, and aspirate any residual supernatant.</li>
		<li>Remove the tube from the magnetic rack and resuspend the beads in 15 &#181;L of Elution Buffer (EB).</li>
		<li>Briefly centrifuge and incubate at 37 &#176;C for 10 min to elute the DNA.</li>
		<li>Place the tube on the magnetic rack, pellet the beads until the eluate is clear and colorless, and aspirate 15 &#181;L of the eluate into a clean 1.5 mL microcentrifuge tube.</li>
		<li>Quantify 1 &#181;L of the eluate.</li>
		<li>Dilute the library to 20 fmol (calculate using the NEB calculator) and use 12 &#181;L of the 20 fmol prepared library for sequencing on the R10.4.1 flow cell. 
		NOTE: The library can now be stored at -20 &#176;C, allowing for repeated use.</li>
	</ol>
	</li>
	<li>Priming and loading the nanopore sequencing flow cell
	<ol>
		<li>Thaw the Sequencing Buffer (SB), Library Beads (LIB), Flow Cell Tether (FCT), and Flow Cell Flush (FCF) at RT, mix them by vortexing, and spin them down.</li>
		<li>Prepare the flow cell priming mix by combining 5 &#181;L of Bovine Serum Albumin (BSA, 50 mg/mL), 30 &#181;L of Flow Cell Tether (FCT), and 1170 &#181;L of Flow Cell Flush (FCF), mixing the solution by inversion and pipetting.</li>
		<li>Open the priming port of the flow cell, aspirate ~20 &#181;L of buffer to remove air bubbles, load 800 &#181;L of the priming mix into the priming port without introducing air bubbles, and wait for 5 min.</li>
		<li>Prepare the library by mixing 37.5 &#181;L of Sequencing Buffer (SB), 25.5 &#181;L of Library Beads (LIB) or Library Solution (LIS), and 12 &#181;L of the 20 fmol DNA library in a 1.5 mL microcentrifuge tube, mixing thoroughly by pipetting.</li>
		<li>Prime the flow cell again by gently lifting the flow cell sample port cap, loading 200 &#181;L of the priming mix into the priming port without introducing air bubbles, and preparing the library for loading.</li>
		<li>Mix the prepared library gently by pipetting and load 75 &#181;L of the library into the sequencing flow cell sample port dropwise, ensuring each drop flows into the port before adding the next.</li>
		<li>Close the flow cell sample port cap, ensuring the bung enters the flow cell sample port, then close the priming port.</li>
		<li>Connect the portable sequencing device to a computer (with an uninterrupted power supply) and initiate sequencing using a sequencing device control and analysis software.</li>
	</ol>
	</li>
	<li>Software parameters 
	NOTE: This software application, developed by ONT, is utilized for the comprehensive management of sequencing runs and the generation of detailed reports.
	<ol>
		<li>To initiate the sequencing process, please select the Start option followed by Start sequencing. 
		NOTE: One will then be prompted to designate a folder for file storage and provide a name for your sequencing run, for example, &#34;sequencing_run_1.&#34; Afterward, click on Continue to settings to select the appropriate kit name, such as library-sequencing-amplicons-native-barcoding-v14-sqk-nbd114-96, and customize the desired run time, for instance, 20 h (the maximum run time is 72 h).</li>
		<li>Next, choose the output file type, selecting from options such as POD5 for raw output reads and FASTQ for basecalled outputs. Subsequently, select the FAST-Basecalling model.</li>
		<li>Please note that some of the default settings include frequent pore scanning every 1.5 h, a minimum read length of 200 bp, and a minimum quality score of 8. Lastly, review the settings and commence the reading process.</li>
	</ol>
	</li>
</ol>
3. Post-sequencing analysis
<ol>
	<li>System requirements 
	NOTE: Guide for setting up the environment and analyzing Mycobacterium tuberculosis amplicon sequencing data using the ONT-TB-NF pipeline. The instructions cater to both Windows (via WSL) and macOS users.
	<ol>
		<li>Use the following - Operating System: Windows 10/11 (with WSL2) or macOS; Memory: Minimum 8 GB RAM (16 GB recommended); Storage: At least 50 GB of free disk space; Internet: Required for downloading packages and data; Permissions: Ability to install software and manage environments.</li>
	</ol>
	</li>
	<li>Ensure the following environment setup: Windows Users (Using WSL2). Enable WSL2.
	<ol>
		<li>Open PowerShell as Administrator and run: wsl - install.</li>
		<li>Restart the computer when prompted.</li>
	</ol>
	</li>
	<li>Install Ubuntu
	<ol>
		<li>After restarting, launch the Microsoft Store, search for Ubuntu, and install the preferred version (e.g., Ubuntu 22.04 LTS).</li>
		<li>Launch Ubuntu from the Start Menu.</li>
		<li>Create a new UNIX username and password when prompted.</li>
		<li>Update package Lists: sudo apt update &#38;&#38; sudo apt upgrade -y.</li>
	</ol>
	</li>
	<li>macOS Users
	<ol>
		<li>Install Homebrew (if not already installed): /bin/bash -c &#34;$(curl -fsSL https://raw.githubusercontent.com/Homebrew/install/HEAD/install.sh)&#34;.</li>
		<li>Install required packages: brew install git wget</li>
	</ol>
	</li>
	<li>Installing Conda or Mamba
	<ol>
		<li>Visit the Miniconda download page to download the correct installer for the operating system.</li>
		<li>To install Miniconda, run the installer and follow the on-screen instructions. After the installation is complete, please restart the terminal.</li>
		<li>Configure Conda Channels: 
		conda config --add channels defaults 
		conda config --add channels bioconda 
		&#8203;conda config --add channels conda-forge</li>
		<li>Installing Mambaforge (Mamba) 
		For Linux: 
		wget 
		https://github.com/conda-forge/miniforge/releases/latest/download/Mambaforge-Linux-x86_64.sh 
		For macOS: 
		wget https://github.com/conda-forge/miniforge/releases/latest/download/Mambaforge-MacOSX-arm64.sh</li>
		<li>Install Mambaforge: bash Mambaforge-*.sh</li>
		<li>Follow the prompts to complete the installation, and restart your terminal to activate Mamba.</li>
	</ol>
	</li>
	<li>Setting up the ONT-TB-NF pipeline
	<ol>
		<li>Create a new Conda environment: conda create -n ont_tb_env samtools=1.15.1 minimap2=2.24 nanoplot=1.40.2 mosdepth=0.3.3 flye=2.9.1 nanofilt fastqc bedtools -c bioconda</li>
		<li>Alternatively, use Mamba: mamba create -n ont_tb_env samtools=1.15.1 minimap2=2.24 nanoplot=1.40.2 mosdepth=0.3.3 flye=2.9.1 nanofilt fastqc bedtools -c bioconda</li>
		<li>Activate the environment: conda activate ont_tb_env</li>
		<li>Install Nextflow: curl -s&#160;https://get.nextflow.io | bash, sudo mv nextflow /usr/local/bin/</li>
		<li>Clone the ONT-TB-NF repository: git clone&#160;https://github.com/HKU-BAL/ONT-TB-NF.git 
		cd ONT-TB-NF</li>
		<li>Pull Required Docker Images (if using Docker) 
		docker pull hkubal/clair3:v0.1-r12 
		&#8203;docker pull quay.io/biocontainers/tb-profiler:4.3.0--pypyh5e36f6f_0</li>
	</ol>
	</li>
	<li>Running the pipeline
	<ol>
		<li>Concatenate each samples&#39;s Basecalled FASTQ files independently using the following command. 
		cat *.fastq.gz &#62; concatenated.fastq.gz 
		nextflow run run_tb_amplicon.nf \ 
		read_fq /path/to/concatenated.fastq.gz \ 
		sample_name SAMPLE_ID \ 
		amplicon_bed /path/to/amplicon_regions.bed \ 
		threads 16 \ 
		&#8203;output_dir /absolute/path/to/output_directory</li>
	</ol>
	</li>
	<li>Output files and interpretation
	<ol>
		<li>Upon successful execution, ensure that the pipeline generates the following outputs in the specified output directory:
		<ol>
			<li>Aligned Reads: *.bam files.</li>
			<li>Variant Calls: *.vcf files containing SNPs and indels.</li>
			<li>Summary Reports: *.csv or *.tsv files summarizing drug resistance mutations and other relevant metrics.</li>
			<li>Quality Control Reports: *.htmL files generated by tools like MultiQC.</li>
			<li>Logs: Detailed logs of the pipeline execution.</li>
			<li>Intermediate Files: Stored in the work/ directory (can be deleted after successful run). 
			NOTE: In the event of encountering permission errors, it is essential to verify that one possess the necessary permissions to read and write files, as well as to execute scripts. Regarding environmental issues, please ensure that the appropriate Conda environment is activated prior to executing the pipeline. For those utilizing Docker profiles, it is imperative to confirm that Docker is both installed and operational. Additionally, one must monitor system resources meticulously to avert any memory or storage limitations during execution. An overview of these methodologies is presented in Figure 1.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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<li>Shea, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+wholegenome+sequencing+and+reporting+of+drug+resistance+profiles+on+clinical+cases+of+Mycobacterium+tuberculosis+in+New+York+State%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Comprehensive wholegenome sequencing and reporting of drug resistance profiles on clinical cases of Mycobacterium tuberculosis in New York State.</a> J Clin Microbiol. 55 (6), 1871-1882 (2017).</li>
<li>Meehan, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+genome+sequencing+of+Mycobacterium+tuberculosis%3A+Current+standards+and+open+issues%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">Whole genome sequencing of Mycobacterium tuberculosis: Current standards and open issues.</a> Nat Rev Microbiol. 17 (10), 533-545 (2019).</li>
<li>Heupink, T. H., Verboven, L., Warren, R. M., Van Rie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+and+accurate+genetic+variant+identification+from+contaminated+and+lowcoverage+Mycobacterium+tuberculosis+whole+genome+sequencing+data%5BTitle%5D)+AND+%22Microb+Genom%22%5BJournal%5D)">Comprehensive and accurate genetic variant identification from contaminated and lowcoverage Mycobacterium tuberculosis whole genome sequencing data.</a> Microb Genom. 7 (1), e000555(2021).</li>
<li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interpretation+of+the+third+edition+of+WHO+consolidated+guidelines+on+tuberculosis%3A+Module+3%3A+Diagnosis%3A+Rapid+diagnostics+for+tuberculosis+detection%5BTitle%5D)+AND+%22Chin+J+Antituberc%22%5BJournal%5D)">Interpretation of the third edition of WHO consolidated guidelines on tuberculosis: Module 3: Diagnosis: Rapid diagnostics for tuberculosis detection.</a> Chin J Antituberc. 46 (2), 120-130 (2024).</li>
<li>Kambli, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+nextgeneration+sequencing+directly+from+sputum+for+comprehensive+genetic+information+on+drugresistant+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22Tuberculosis%22%5BJournal%5D)">Targeted nextgeneration sequencing directly from sputum for comprehensive genetic information on drugresistant Mycobacterium tuberculosis.</a> Tuberculosis. 127 (1), 102051(2021).</li>
<li>Use of targeted nextgeneration sequencing to detect drugresistant tuberculosis. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Rapid+Communication%22">Rapid Communication</a>. , WHO. 1-8 (2023).</li>
<li>Nandlal, L., Perumal, R., Naidoo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+molecular+assays+for+the+diagnosis+of+drugresistant+tuberculosis%5BTitle%5D)+AND+%22Infect+Drug+Resist%22%5BJournal%5D)">Rapid molecular assays for the diagnosis of drugresistant tuberculosis.</a> Infect Drug Resist. 15 (1), 4971-4984 (2022).</li>
<li>Dippenaar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanopore+sequencing+for+Mycobacterium+tuberculosis%3A+A+critical+review+of+the+literature%2C+new+developments%2C+and+future+opportunities%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Nanopore sequencing for Mycobacterium tuberculosis: A critical review of the literature, new developments, and future opportunities.</a> J Clin Microbiol. 60 (5), e0123422(2022).</li>
<li>Chan, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+economical+drug+resistance+profiling+with+nanopore+MinION+for+clinical+specimens+with+low+bacillary+burden+of+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22BMC+Res+Notes%22%5BJournal%5D)">Rapid and economical drug resistance profiling with nanopore MinION for clinical specimens with low bacillary burden of Mycobacterium tuberculosis.</a> BMC Res Notes. 13 (1), 123(2020).</li>
<li>Feuerriegel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+genomic+first+and+secondline+drug+resistance+prediction+from+clinical+Mycobacterium+tuberculosis+specimens+using+DeeplexMycTB%5BTitle%5D)+AND+%22Eur+Respir+J%22%5BJournal%5D)">Rapid genomic first and secondline drug resistance prediction from clinical Mycobacterium tuberculosis specimens using DeeplexMycTB.</a> Eur Respir J. 57 (4), 2004784(2021).</li>
<li>GenoScreen product brochure. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Deeplex+MycTB%22">Deeplex MycTB</a>. , GenoScreen. (2023).</li>
<li>Mtafya, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+practical+approach+to+render+tuberculosis+samples+safe+for+application+of+tuberculosis+molecular+bacterial+load+assay+in+clinical+settings+without+a+biosafety+level+3+laboratory%5BTitle%5D)+AND+%22Tuberculosis%22%5BJournal%5D)">A practical approach to render tuberculosis samples safe for application of tuberculosis molecular bacterial load assay in clinical settings without a biosafety level 3 laboratory.</a> Tuberculosis. 138 (1), 102194(2023).</li>
<li>Suganthi, P., Usharani, B., Venkateswari, R., Muthuraj, M. Biosafety practices in mycobacteriology laboratory. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSuganthi%2C+P+%22Research+Advances+in+Microbiology+and+Biotechnology%22">Research Advances in Microbiology and Biotechnology</a>. 3, B P International (a part of SCIENCEDOMAIN International). Chennai. 1-39 (2023).</li>
<li>consolidated guidelines on tuberculosis. Module 4: Treatment and care. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22WHO+guideline%22">WHO guideline</a>. , WHO. (2022).</li>
<li>Williams, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrixbased+DNA+extraction+for+targeted+nextgeneration+sequencing+on+decontaminated+sputum+samples%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Matrixbased DNA extraction for targeted nextgeneration sequencing on decontaminated sputum samples.</a> J Vis Exp. (181), e68147(2025).</li>
<li>Delahaye, C., Nicolas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequencing+DNA+with+nanopores%3A+Troubles+and+biases%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Sequencing DNA with nanopores: Troubles and biases.</a> PLoS One. 16 (2), e0245534(2021).</li>
<li>Zhao, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+identification+of+drugresistant+tuberculosis+genes+using+direct+PCR+amplification+and+Oxford+Nanopore+Technology+sequencing%5BTitle%5D)+AND+%22Can+J+Infect+Dis+Med+Microbiol%22%5BJournal%5D)">Rapid identification of drugresistant tuberculosis genes using direct PCR amplification and Oxford Nanopore Technology sequencing.</a> Can J Infect Dis Med Microbiol. 2022, 8845672(2022).</li>
<li>Tagliani, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culture+and+nextgeneration+sequencingbased+drug+susceptibility+testing+unveil+high+levels+of+drugresistantTB+in+Djibouti%3A+Results+from+the+first+national+survey%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Culture and nextgeneration sequencingbased drug susceptibility testing unveil high levels of drugresistantTB in Djibouti: Results from the first national survey.</a> Sci Rep. 7 (1), 15076(2017).</li>
<li>Smith, C., Halse, T. A., Shea, J., Modestil, H., Fowler, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+nanopore+sequencing+for+clinical+diagnostics%3A+For+Mycobacterium+tuberculosis%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Assessing nanopore sequencing for clinical diagnostics: For Mycobacterium tuberculosis.</a> J Clin Microbiol. 59 (7), e0123421(2021).</li>
</ol>

The long-read sequencing platform demonstrated notable advantages in this study, particularly its ability to achieve rapid sequencing with consistently high coverage breadth. Across all successfully sequenced isolates, a coverage breadth of 99.9% was achieved, indicating near-complete representation of resistance-associated genomic regions. The average coverage depth across all targeted genes was 4462X, with the eis gene showing the highest depth at 22,242.08X and the rrs gene the lowest at 99.46X. Thes...

Tuberculosis (TB) continues to be a major cause of mortality globally and presents an ongoing public health challenge, especially in low- and middle-income countries1,2. Despite initiatives aimed at enhancing TB control measures, advancements have been slow, partly due to difficulties in achieving rapid, accurate, cost-effective, and comprehensive diagnostic techniques3,4,5. While molecular diagnostic methods like line probe assays (LPAs) and automated nucleic acid amplification tests (NAATs) have sped up the identification of TB and drug-resistant TB (DR-TB), their limited mutation detection restricts thorough drug susceptibility testing (DST). This limitation is particularly significant in TB high-burden countries6,7,8,9.
To address these limitations, the World Health Organization (WHO) has recommended the use of next-generation sequencing (NGS) technologies, which have emerged as powerful tools for TB surveillance and resistance detection10,11. Both whole genome sequencing (WGS) and targeted NGS (tNGS) approaches offer broader mutation coverage, enabling the identification of resistance-associated variants across multiple drugs8,12,13,14,15. While whole genome sequencing (WGS) provides a comprehensive DST report, its reliance on culture makes it unsuitable for rapid diagnostics, as culture can take up to six weeks15,16,17. Targeted NGS offers a better alternative since it does not depend on culture; sequencing can be performed directly from clinical specimens18. As a result, the WHO has recommended the use of three tNGS assays for DST: Deeplex Myc-TB assay: This assay detects resistance to rifampicin, isoniazid, fluoroquinolones, aminoglycosides (amikacin, kanamycin, capreomycin), ethambutol, pyrazinamide, and ethionamide19,20,21. Sentinel TB assay: This targets resistance to rifampicin, isoniazid, fluoroquinolones, and second-line injectable drugs18,20. AQ-TB assay: This identifies resistance to rifampicin, isoniazid, fluoroquinolones, and second-line injectable drugs. These assays have been optimized for use on the short-read platform due to its high accuracy20. ONT MinION long-read platforms, on the other hand, provide real-time data, portability, and the potential for cost-effective implementation22,23. However, the widespread use of NGS is limited by the demands of infrastructure and cost, especially with short-read systems10.
The Deeplex assay is the most comprehensive of the three recommended assays. The assay was originally designed to perform multiplex PCR on 18 regions associated with resistance to 13 anti-TB drugs. However, the WHO recommended it for 10 anti-TB drugs, indicating sufficient sensitivity and specificity. This study optimized it for use with the ONT MK1B MinION, a long-read sequencing platform. The portable and reusable flow-cell system of this platform reduces both infrastructure and potentially sequencing costs per sample4,22,23. While other long-read-compatible assays have been developed, they tend to have a limited DST scope, typically targeting only a narrow range of resistance genes. In contrast, the assay offers broad target coverage, making it a more comprehensive diagnostic tool for decentralized DST24,25. A detailed list of the genomic targets amplified by the assay and their corresponding drug associations is provided in Table 1, highlighting the assay&#39;s diagnostic capabilities25.
Prior to DNA extraction, samples must be decontaminated and inactivated under Biosafety Level 3 (BSL-3) conditions, following the necessary safety precautions25,26,27. Internal amplification and external controls should be incorporated in each run, with contamination tracked through negative controls and routine laboratory evaluations25. Important quality metrics, including read depth, coverage breadth, and amplification success, should be assessed after sequencing. For targeted sequencing, the WHO advises a minimum read depth of 100x for each amplicon and adequate coverage of all targeted drug-resistance regions to allow for the detection of minor variants that are present at &#8805;5%28. The World Health Organization endorses these targeted sequencing methods for identifying resistance to first- and second-line drugs, assuming that quality assurance systems are established and that all target regions are sufficiently covered28.

<table><tbody><tr><td>15 mL &amp;amp; 1.5 mL or 2 mL Lo-binding Tubes</td><td>Eppendorf</td><td>EP0030108051-250EA</td><td></td></tr><tr><td>1kb ladder</td><td>Thermo Fisher Scientific</td><td>SM0311</td><td></td></tr><tr><td>2 mL screw cap Tubes</td><td>Scientific Specialties Inc</td><td>P2TUB056C-0001.5ST</td><td></td></tr><tr><td>2 mm sterile glass beads</td><td>Sigma-Aldrich</td><td>Z273627-1EA</td><td></td></tr><tr><td>AMPure XP Bead</td><td>Beckman Colter</td><td>A63881</td><td></td></tr><tr><td>Deeplex Myc-TB&amp;nbsp;</td><td>GenoScreen</td><td>20090205</td><td></td></tr><tr><td>Electrophoresis chamber</td><td>Thermo Fisher Scientific</td><td>A25977</td><td></td></tr><tr><td>Ethyl alcohol, Pure</td><td>Sigma-Aldrich</td><td>E7023-500ML</td><td></td></tr><tr><td>FastPrep Homogenizer</td><td>MP Biomedicals</td><td>116005500</td><td></td></tr><tr><td>Filter pipette tips (10 &amp;micro;L)&amp;nbsp;</td><td>Bio-Smart Scientific</td><td>FT-10-R</td><td></td></tr><tr><td>Filter pipette tips (1000 &amp;micro;L)&amp;nbsp;</td><td>Bio-Smart Scientific</td><td>FT-1000-R</td><td></td></tr><tr><td>Filter pipette tips (20 &amp;micro;L)&amp;nbsp;</td><td>Bio-Smart Scientific</td><td>FT-20-R</td><td></td></tr><tr><td>Filter pipette tips (200 &amp;micro;L)&amp;nbsp;</td><td>Bio-Smart Scientific</td><td>FT-200-R</td><td></td></tr><tr><td>Flow-cell&amp;nbsp;</td><td>Oxford Nanopore Technologies</td><td>FLO-MIN114</td><td></td></tr><tr><td>Heating-block</td><td>Eppendorf</td><td>5382000031</td><td></td></tr><tr><td>InstaGene Matrix</td><td>Bio-Rad</td><td>BBRD7326030</td><td></td></tr><tr><td>Magnetic Rack&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>12321D</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf&amp;nbsp;</td><td>5406000046</td><td></td></tr><tr><td>MinION</td><td>Oxford Nanopore Technologies</td><td>MK1B</td><td></td></tr><tr><td>Native-barcoding kit V14&amp;nbsp;</td><td>Oxford Nanopore Technologies</td><td>SQK-NBD114.24</td><td></td></tr><tr><td>NEB Blunt/TA Ligase Master Mix</td><td>NEB</td><td>M0367</td><td></td></tr><tr><td>NEBNext Quick Ligation Module</td><td>NEB</td><td>E6056</td><td></td></tr><tr><td>NEBNext Ultra II End repair/dA-tailing Module</td><td>NEB</td><td>E7546</td><td></td></tr><tr><td>Nuclease-free water (NFW)</td><td>Thermo Fisher Scientific</td><td>AM9937</td><td></td></tr><tr><td>Pipettes (P10, P20, P200 and P1000)</td><td>Eppendorf</td><td>EP3123000918-1EA</td><td></td></tr><tr><td>Qubit Assay Tubes</td><td>Thermo Fisher Scientific</td><td>Q32856</td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher Scientific</td><td>Q33231</td><td></td></tr><tr><td>Qubit fluorometer</td><td>Thermo Fisher Scientific</td><td>Q33226</td><td></td></tr><tr><td>SYBR Safe DNA gel Strain</td><td>Thermo Fisher Scientific</td><td>S33102</td><td></td></tr><tr><td>UltraPure Agarose</td><td>Thermo Fisher Scientific</td><td>17852</td><td></td></tr><tr><td>Universal Hood III transilluminator (Molecular Imager Gel Doc XR System)</td><td>Bio-Rad</td><td>170-8170</td><td></td></tr><tr><td>Vortex mixer</td><td>Lasec</td><td>WMBB0L0E0216</td><td></td></tr></tbody></table>

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 tuberculosis (TB) and drug-resistant TB (DR-TB). Next-generation sequencing (NGS) technologies, endorsed by the WHO, have significantly improved the detection of DR-TB. Among these, targeted NGS (tNGS) enables focused detection of genetic mutations associated with drug resistance, eliminating the need for traditional culture-based diagnostics. One widely used tNGS assay provides rapid and comprehensive drug susceptibility testing but has been primarily optimized for sequencing platforms with high accuracy. However, the high cost of these sequencing systems has limited accessibility in low- and middle-income countries, particularly across Africa. Portable sequencing technologies present a promising alternative, offering flexibility and reduced infrastructure requirements. In this study, DNA was extracted from rifampicin-resistant TB (RR-TB) isolates, amplified using a tNGS assay, and sequenced on a portable sequencing platform. The same amplification products were also sequenced on a high-accuracy short-read sequencing platform to serve as a reference. Data from the portable sequencer were processed using a bioinformatics pipeline designed for long-read sequencing, while short-read sequencing data were analyzed using an established web-based application. The analysis showed that the long-read sequencing approach successfully identified high-frequency resistance-associated variants detected by short-read sequencing but exhibited limitations in detecting low-frequency variants.

The selected DNA extraction method was chosen for its efficiency and superior yield compared to other rapid techniques. It consistently produced high-quality genomic DNA (gDNA), and integrating a bead-based cleanup step helped minimize processing time while reducing per-sample costs. This was achieved by eliminating the need for additional quality control assessments such as spectrophotometric measurements, with fluorometry as the sole DNA quantification tool. Post-PCR concentrations were found to be optimal for sequenci...

This protocol was optimized for targeted deep sequencing of 18 drug-resistance regions in Mycobacterium tuberculosis using a long-read sequencing platform, followed by analysis with a tuberculosis-specific bioinformatics pipeline designed for long-read data.

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

I investigate whether portable long-read sequencing can provide TB drug-resistant susceptibility results in resource-limited settings with accuracy comparable to gold standards method, advancing accessible, high-quality tuberculosis diagnostics. Utilizing targeted next-generation sequencing as a diagnostic tool for drug-resistant tuberculosis by offering a comprehensive resistance profile directly from clinical sample without bioinformatics expertise. Our protocol utilizing long-read sequencing has the potential to provide quicker turnaround times, simpler workflow than in real-time resistance detection, facilitating comprehensive TB diagnostics and enhancing outbreak tracking in resource-limited environment with minimal infrastructure.To begin, calculate 100 nanograms of each amplicon sample based on the concentration and transfer the required volume into a clean PCR tube. To determine the total mass of amplicons in the sample, use the given formula. Adjust the total volume of each sample to 12.5 microliters using nuclease-free water.Mix the samples gently by pipetting up and down 10 times and briefly spin them in a microcentrifuge. Next, add 1.75 microliters of end-repair and adenylation reaction buffer to each sample, followed by 0.75 microliters of end-repair and adenylation enzyme mix. After mixing and spinning the samples, place the tubes in a thermocycler at 25 degrees Celsius for 30 minutes, followed by 65 degrees Celsius for 30 minutes.For native barcode ligation, add the components shown on the screen in new PCR tubes. Mix the reactions gently by pipetting and spin briefly using a microcentrifuge. Next, add one microliter of EDTA to each tube.Then, mix thoroughly and spin briefly as shown earlier. Pull the barcoded samples into a 1.5 milliliter microcentrifuge tube. Vortex the DNA cleanup beads to resuspend them completely and add a volume equal to 0.7 times the pooled sample volume.Mix the bead sample solution and incubate it on a HulaMixer at room temperature for 10 minutes. Next, to prepare two milliliters of 80%ethanol, mix 1, 600 microliters of pure ethanol with 400 microliters of nuclease-free water. Place the tube on a magnetic rack for five minutes until the eluate appears clear and colorless.Then, remove and discard the supernatant without disturbing the pellet. Wash the beads with 700 microliters of freshly prepared 80%ethanol and centrifuge the tube briefly. After placing the tube on the magnetic rack, remove the residual ethanol.Now, remove the tube from the magnetic rack. Resuspend the beads in 35 microliters of nuclease-free water by gentle flicking and incubate at 37 degrees Celsius for 10 minutes. Return the tube to the magnetic rack until the eluate becomes clear and colorless.Then, transfer 35 microliters of the eluate into a clean 1.5 milliliter microcentrifuge tube for further use. For adapter ligation, mix the given components in a 1.5 milliliter low-binding microcentrifuge tube. Centrifuge the tube briefly and incubate at room temperature for 20 minutes.Vortex the DNA cleanup beads to resuspend them completely. Add 20 microliters of beads to the tube and mix gently. Incubate on a HulaMixer for 10 minutes at room temperature.Then, place the tube on a magnetic rack to pellet the beads and aspirate the supernatant without disturbing the pellet. Add 125 microliters of short fragment buffer and gently flick the tube to resuspend the beads. Centrifuge briefly and return the tube to the magnetic rack.Aspirate the supernatant again after the beads have pelleted. Now, remove the tube from the magnetic rack and resuspend the beads in 15 microliters of elution buffer by gentle pipetting or flicking. Briefly centrifuge the tube to collect the contents, then incubate it at 37 degrees Celsius for 10 minutes to elute the DNA.Place the tube on a magnetic rack until the eluate is clear and colorless. Then, aspirate 15 microliters of eluate and transfer it into a clean, 1.5 milliliter microcentrifuge tube. To prepare the flow cell priming solution, mix the given components thoroughly.Open the priming port of the flow cell, aspirate approximately 20 microliters of buffer to remove air bubbles, and load 800 microliters of the priming mix into the port without introducing air bubbles. To prepare the DNA library, thoroughly mix the components shown on the screen. Gently lift the sample port cap of the flow cell, load 200 microliters of priming mix into the priming port without introducing air bubbles.After mixing the prepared DNA library, load 75 microliters of the library dropwise into the sequencing flow cell sample port. Close the flow cell sample port cap, ensuring the bung securely enters the sample port, and then close the priming port tightly. Gel electrophoresis confirmed amplicon integrity across nearly all samples, but lanes corresponding to samples 89 and 136 displayed faint or absent bands.Samples 89 and 136 had insufficient amplicon concentrations, leading to their exclusion from long-read sequencing. Across all targeted genes, the long-read and short-read platforms achieved high coverage depths with the EIS gene showing the highest average values and RRS gene exhibiting the lowest coverage depths. Despite the range in coverage depth, both platforms maintained a high coverage breadth of 99.9%across all resistance-associated genes.Drug resistance variants for ethambutol, fluoroquinolones, kanamycin, amikacin, and capreomycin were detected with 100%concordance across short and long-read sequencing platforms. No resistance-associated mutations were found for linezolid, bedaquiline, and clofazimine using either sequencing approach. Streptomycin resistance detection showed lower concordance between platforms, with only 71.43%agreement.Detection sensitivity for key genes rpoB, katG/fabG1/ahpC/inhA, fabG1/inhA, and pncA ranged from 89.47%to 96.77%between platforms.

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

Research

JoVE Journal

Biochemistry

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

This protocol describes a method for mapping pre-mRNA 3' end processing sites.

3' End Sequencing Library Preparation with A-seq2

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

This protocol describes a workflow from ex vivo or in vitro cell cultures to transcriptomic data pre-processing for cost-effective transcriptome-based drug screening.

Cost-Efficient Transcriptomic-Based Drug Screening

The current study describes a workflow for nanopore sequencing of plasmids when the chromosomal sequence is already known or of no interest. An initial plasmid DNA enrichment step ensures optimal plasmid sequence depth and maximizes the number of samples that can be sequenced per flow cell.

Long-Read Plasmid Sequencing and Assembly Using Nanopore Sequencing-Based Workflows

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Discover portable long-read sequencing for tuberculosis drug-resistance detection using targeted amplicon analysis. This method delivers rapid, accurate diagnostics without requiring extensive bioinformatics expertise.

Amplicon Sequencing using the Long-Read Sequencing Technologies

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