previously established as having a greater resolution than MIRUVNTR, and its usefulness in defining outbreaks is confirmed;
[9, 10] however, these were retrospective studies, and using WGS
in a diagnostic setting would allow for this information to act on in
a patient-centric fashion. RNA bait enrichment allows for the selection of target nucleic acids from a sample, which would potentially
contain human and also normal flora RNA and DNA, as well as the
pathogen of interest. This would allow for pathogens to be wholegenome sequenced directly from patient samples, removing the
need for culturing, which would shorten the time taken by sample
for data acquisition, and has been previously described for the
sequencing of viruses from clinical samples [11].
Here, we describe the use of an RNA bait enrichment-based
system (SureSelect
XT
, Agilent) for the sequencing of M. tuberculosis
samples, obtained from sputum. Our previously published work for
M. tuberculosis WGS [12] shows that using these method sequence
data obtained identified the presence of mutations associated with
resistance to rifampicin, isoniazid, and ethambutol, which correlates with the resistant phenotype observed from the strains tested;
this was confirmed using Sanger sequencing, validating the usefulness in terms of specificity and selectivity of the RNA bait approach
in a clinical setting for identifying SNPs. Our data also showed
improved coverage over the M. tuberculosis genome, in particular,
across the notoriously difficult to sequence PE/PPE genes [13],
again demonstrating the robustness of the bait enrichment
approach.
The method described in this chapter utilizes the SureSelect
XT
Target-Enrichment protocol (Agilent Technologies), which uses
custom-designed 120-mer RNA oligonucleotides. These RNA
‘baits’ span the entire genome and can recover (by hybridization)
low copy number pathogens from clinical samples with sufficiently
high sensitivity and specificity to enable ultra-deep whole-genome
sequencing. Using this method, we were able to obtain full-length
M. tuberculosis genomes (>95–100% coverage of a reference
genome). We have successfully used this approach on not just
M. tuberculosis [12] but also herpesviruses (including cytomegalovirus (CMV)), C. trachomatis, [14–16] hepatitis B, hepatitis C,
human immunodeficiency virus (HIV), influenza A, and
norovirus [17].
2 Materials
In addition to the materials listed below, sterile, nuclease-free,
aerosol barrier pipette tips should be used for all liquid manipulations, and standard molecular biology protocol, such as wearing of
gloves, taking care to not cross contaminate samples, etc., should
be performed. Figure 1 shows a schematic of the steps involved in
the library preparation described here.
Direct WGS of M. tb’
461
[9, 10] however, these were retrospective studies, and using WGS
in a diagnostic setting would allow for this information to act on in
a patient-centric fashion. RNA bait enrichment allows for the selection of target nucleic acids from a sample, which would potentially
contain human and also normal flora RNA and DNA, as well as the
pathogen of interest. This would allow for pathogens to be wholegenome sequenced directly from patient samples, removing the
need for culturing, which would shorten the time taken by sample
for data acquisition, and has been previously described for the
sequencing of viruses from clinical samples [11].
Here, we describe the use of an RNA bait enrichment-based
system (SureSelect
XT
, Agilent) for the sequencing of M. tuberculosis
samples, obtained from sputum. Our previously published work for
M. tuberculosis WGS [12] shows that using these method sequence
data obtained identified the presence of mutations associated with
resistance to rifampicin, isoniazid, and ethambutol, which correlates with the resistant phenotype observed from the strains tested;
this was confirmed using Sanger sequencing, validating the usefulness in terms of specificity and selectivity of the RNA bait approach
in a clinical setting for identifying SNPs. Our data also showed
improved coverage over the M. tuberculosis genome, in particular,
across the notoriously difficult to sequence PE/PPE genes [13],
again demonstrating the robustness of the bait enrichment
approach.
The method described in this chapter utilizes the SureSelect
XT
Target-Enrichment protocol (Agilent Technologies), which uses
custom-designed 120-mer RNA oligonucleotides. These RNA
‘baits’ span the entire genome and can recover (by hybridization)
low copy number pathogens from clinical samples with sufficiently
high sensitivity and specificity to enable ultra-deep whole-genome
sequencing. Using this method, we were able to obtain full-length
M. tuberculosis genomes (>95–100% coverage of a reference
genome). We have successfully used this approach on not just
M. tuberculosis [12] but also herpesviruses (including cytomegalovirus (CMV)), C. trachomatis, [14–16] hepatitis B, hepatitis C,
human immunodeficiency virus (HIV), influenza A, and
norovirus [17].
2 Materials
In addition to the materials listed below, sterile, nuclease-free,
aerosol barrier pipette tips should be used for all liquid manipulations, and standard molecular biology protocol, such as wearing of
gloves, taking care to not cross contaminate samples, etc., should
be performed. Figure 1 shows a schematic of the steps involved in
the library preparation described here.
Direct WGS of M. tb’
461