Amplicon Sequencing using the Long-Read Sequencing Technologies

Summary

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.

Abstract

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.

Introduction

Tuberculosis (TB) continues to be a major cause of mortality globally and presents an ongoing public health challenge, especially in low- and middle-income countries^1,2. 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 techniques^3,4,5. While molecular diagnostic methods like line probe assays (LPAs) and automated nucleic acid amplification tests (NAATs) have sped up the identifi....

Protocol

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

1. Heat inactivation
   1. Incubat....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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