Chapter 20
Whole-Genome Sequencing of Mycobacterium tuberculosis
Directly from Sputum Samples
Amanda Claire Brown
Abstract
Whole-genome sequencing is a powerful, high-resolution tool that can be used to generate accurate data on
bacterial population structure, phylogeography, and mutations associated with antimicrobial resistance.
The ability to sequence pathogen genomes directly from clinical specimens, without the requirement for
in vitro culturing, is attractive in terms of time- and labor-saving, especially in the case of slow growing
pathogens, such as Mycobacterium tuberculosis. However, clinical samples typically contain too low levels of
pathogen nucleic acid, plus relatively high levels of human and natural microbiota DNA/RNA, to make this
a viable option. Using a combination of whole-genome enrichment and deep sequencing, which has been
proven to be a nonmutagenic approach, we can capture all known variations found within M. tuberculosis
genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of
M. tuberculosis directly from clinical samples and has the potential to be adapted to other pathogens with a
similar clonal nature.
Key words Whole-genome enrichment, Whole-genome sequencing, Mycobacterium tuberculosis,
Clinical samples, SureSelect
XT
, TB
1 Introduction
The introduction of relatively affordable, desktop next-generation
sequencing (NGS) machines, such as Illumina’s MiSeq and NextSeq, and Life Technologies’ Ion PGM and Ion Torrent, has revolutionized the ability to perform whole-genome sequencing (WGS)
on small sized genomes, such as those of bacterial and viral pathogens. It is now possible to introduce WGS into diagnostic microbiology laboratories [1–3]. However, clinical samples typically
contain too low levels of pathogen nucleic acid, plus relatively
high levels of human DNA/RNA as well as DNA/RNA from the
natural microbiota, to allow sequencing without an in vitro culture.
Adding a culturing step can add significant delay to data acquisition, especially in the case of organisms that are slow-growing, such
as Mycobacterium tuberculosis, or those that are difficult to culture,
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_20, © Springer Science+Business Media, LLC, part of Springer Nature 2021
459
Whole-Genome Sequencing of Mycobacterium tuberculosis
Directly from Sputum Samples
Amanda Claire Brown
Abstract
Whole-genome sequencing is a powerful, high-resolution tool that can be used to generate accurate data on
bacterial population structure, phylogeography, and mutations associated with antimicrobial resistance.
The ability to sequence pathogen genomes directly from clinical specimens, without the requirement for
in vitro culturing, is attractive in terms of time- and labor-saving, especially in the case of slow growing
pathogens, such as Mycobacterium tuberculosis. However, clinical samples typically contain too low levels of
pathogen nucleic acid, plus relatively high levels of human and natural microbiota DNA/RNA, to make this
a viable option. Using a combination of whole-genome enrichment and deep sequencing, which has been
proven to be a nonmutagenic approach, we can capture all known variations found within M. tuberculosis
genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of
M. tuberculosis directly from clinical samples and has the potential to be adapted to other pathogens with a
similar clonal nature.
Key words Whole-genome enrichment, Whole-genome sequencing, Mycobacterium tuberculosis,
Clinical samples, SureSelect
XT
, TB
1 Introduction
The introduction of relatively affordable, desktop next-generation
sequencing (NGS) machines, such as Illumina’s MiSeq and NextSeq, and Life Technologies’ Ion PGM and Ion Torrent, has revolutionized the ability to perform whole-genome sequencing (WGS)
on small sized genomes, such as those of bacterial and viral pathogens. It is now possible to introduce WGS into diagnostic microbiology laboratories [1–3]. However, clinical samples typically
contain too low levels of pathogen nucleic acid, plus relatively
high levels of human DNA/RNA as well as DNA/RNA from the
natural microbiota, to allow sequencing without an in vitro culture.
Adding a culturing step can add significant delay to data acquisition, especially in the case of organisms that are slow-growing, such
as Mycobacterium tuberculosis, or those that are difficult to culture,
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_20, © Springer Science+Business Media, LLC, part of Springer Nature 2021
459