Culturing | DNA extraction | DNA quantification | Library preparation | Data analysis | Laboratory Manual | Costs

*NEW* The MedRxiv manuscript is now available to read here!

These pages are designed to help you build a pragmatic pipeline for whole genome sequencing (WGS) for sites with little/no access to larger scale Illumina sequencing platforms. The pipeline is primarily designed to identify antimicrobial resistances (e.g. for hard to treat cases) and lineages (e.g. to identify relapse vs reinfection or non-tuberculous mycobacteria. This is likely to be small-scale (at least at first), to be used in house as an alternative to the potentially expensive or logistically difficult Illumina platform.

Mycobacterium tuberculosis (MTB) drug resistance can be seen phenotypically, by undertaking drug sensitivity tests (DSTs), but there are occasions when a patient’s response to a drug differs from what is expected. WGS can be used to try and identify whether there are genetic reasons for this, which may help to inform the clinical decision making for that patient. WGS can also be used to establish reinfection vs relapse, by identifying MTB strains. 

Whole genome sequencing (WGS) is the process of determining the complete DNA sequence of an organism's genome at a single time. This can be done using a number of platforms, but a small-scale approach is to use the Oxford Nanopore Technologies (ONT) MinION device. A nanopore is a nano-scale hole. In its devices, ONT passes an ionic current through nanopores and measures the changes in current as biological molecules (the DNA) pass through the nanopore or near it. The information about the change in current can be used to identify that molecule (e.g. A, T, G or C). The overlapping identical sections of these strings of bases can then be lined up, so that the whole genome can be predicted. This can then be compared to a reference genome and differences mapped.

An overview of the practicalities of WGS laboratory set up can be found on the PANDORA-ID-NET TGHN sequencing set up hub page.

Please note that MTB culturing and DNA extraction must be done in a biological safety level 3 (BSL3) laboratory. Once the DNA has been extracted, it can be quantified outside of the BSL3 facilities.

Overview presentation of the TB ONT sequencing pipeline

This presentation by Dr Linzy Elton from the Centre for Clinical Microbiology, UCL, UK, gives an overview of the recently published Oxford Nanopore Technologies tuberculosis drug resistance and lineage diagnostics pipeline. This video covers each of the steps needed to optimise sequencing of M. tuberculosis:

  • Culture methods
  • DNA extraction
  • DNA quantification (and the importance of good DNA)
  • Library preparation kits
  • Basecalling algorithms
  • Analysis programmes
  • Logistical considerations for LMICs

This video was recorded as part of training provided for the the National Institute for Medical Research Tanzania (NIMR), permission for recording was obtained from participants. For image credits and links from the slides, or for further questions on the pipeline, please email pandora.id.net@gmail.com.

Workshop presentations on TB sequencing pipeline steps

These presentations were recorded during the 'ONT for tuberculosis sequencing' workshop in Lusaka, Zambia in February 2023. You can find more information on the workshop on the training page here. 

Use of genomics for tuberculosis research, surveillance and diagnosis in Zambia

This presentation by Dr Kabengele Keith Siame of the Tropical Disease Research Centre in Ndola, Zambia looks at the current diagnosis tools for tuberculosis in Zambia and why they are inadequate for tracking drug resistance and transmission patterns. Dr Siame discusses how sequencing, especially whole genome sequencing, can be used to fill these information gaps and improve diagnostics and surveillance of tuberculosis. 

Introduction to sequencing and Oxford Nanopore Technologies

This presentation by Dr John Tembo of HerpeZ, Zambia explains the basics and brief history of sequencing and describes and compares how Sanger, Illumina and Oxford Nanopore sequencing works. He then discusses in further detail Oxford Nanopore's range of devices and kits, as well as the basic sequencing workflow (which is then described in greater detail across the rest of the presentations).

Introduction to the tuberculosis sequencing pipeline (setting up a sequencing workshop and issues with Mycobacteria)

This presentation by Dr Linzy Elton from the Centre for Clinical Microbiology, UCL (UK) firstly describes the infrastucture and logistics that must be considered before setting up a sequencing laboratory (with emphasis on resource poor settings) and then discusses some of the difficulties of extracting DNA from, and then sequencing the Mycobacteria using Oxford Nanopore Technologies.

The CTAB DNA extraction method for use in sequencing 

In this presentation Caren Kabanda from HerpeZ, Zambia gives an overview of the CTAB DNA extraction method commonly used for extracting DNA from M. tuberculosis.

DNA quality analysis

This presentation by Dr John Tembo looks at ways of quantifying and identifying the quality of extracted DNA. He explains why quality and quantity of DNA is so important for long read sequencing and which methods provide the most accurate results.

Library preparation with the rapid barcoding 96 kit (SQK-RBK110.96)

In this presentation, Dr Linzy Elton from the Centre for Clinical Microbiology, UCL (UK) explains how to prepare a DNA library for the Rapid Barcoding 96 Kit. Note that the RBK110.96 is an update of the original RBK004 kit, which we have previously used (this kit still uses kit 10 chemistry). 

Basic bioinformatics for Oxford Nanopore sequencing data analysis

This presentation, led by Dr John Tembo from HerpeZ, Zambia demonstrates how to basecall using Guppy (processing raw fast5 files into fastq files) on Mac and Windows computers, how to concatenate (combine) these files into a single fastq for downstream processing, and how to use FastQC to analyse the quality of your fastq files.

Using TB-Profiler to analyse tuberculosis sequencing data

This presentation, led by Dr Linzy Elton from the Centre for Clinical Microbiology at UCL in the UK, looks at some common processed file types you might come across (e.g. .sam, .bam, .fasta and .vcf) as well as how to use the server-based version of the data analysis programme TB-Profiler to identify drug resistance SNPs and lineage information.

The resources on these pages have been developed by Dr Linzy Elton (UCL), Professor Neil Stoker (UCL) and Dr Sylvia Rofael (UCL)