Genomic Sequencing from Sputum for Tuberculosis Disease Diagnosis, Lineage Determination, and Drug Susceptibility Prediction

Abstract

Universal access to drug susceptibility testing for newly diagnosed tuberculosis patients is recommended. Access to culture-based diagnostics remains limited, and targeted molecular assays are vulnerable to emerging resistance mutations. Improved protocols for direct-from-sputum Mycobacterium tuberculosis sequencing would accelerate access to comprehensive drug susceptibility testing and molecular typing. We assessed a thermo-protection buffer-based direct-from-sample M. tuberculosis whole-genome sequencing protocol. We prospectively analyzed 60 acid-fast bacilli smear-positive clinical sputum samples in India and Madagascar. A diversity of semiquantitative smear positivity-level samples were included. Sequencing was performed using Illumina and MinION (monoplex and multiplex) technologies. We measured the impact of bacterial inoculum and sequencing platforms on genomic read depth, drug susceptibility prediction performance, and typing accuracy. M. tuberculosis was identified by direct sputum sequencing in 45/51 samples using Illumina, 34/38 were identified using MinION-monoplex sequencing, and 20/24 were identified using MinION-multiplex sequencing. The fraction of M. tuberculosis reads from MinION sequencing was lower than from Illumina, but monoplexing grade 3+ samples on MinION produced higher read depth than Illumina (P < 0.05) and MinION multiplexing (P < 0.01). No significant differences in sensitivity and specificity of drug susceptibility predictions were seen across sequencing modalities or within each technology when stratified by smear grade. Illumina sequencing from sputum accurately identified 1/8 (rifampin) and 6/12 (isoniazid) resistant samples, compared to 2/3 (rifampin) and 3/6 (isoniazid) accurately identified with Nanopore monoplex. Lineage agreement levels between direct and culture-based sequencing were 85% (MinION-monoplex), 88% (Illumina), and 100% (MinION-multiplex). M. tuberculosis direct-from-sample whole-genome sequencing remains challenging. Improved and affordable sample treatment protocols are needed prior to clinical deployment.

Keywords: antimicrobial resistance; methods development; molecular epidemiology; tuberculosis; whole-genome sequencing.

FIG 1

Drug susceptibility testing accuracy of Illumina-based direct-from-sputum sequencing using culture-based WGS on Illumina results as the reference standard. Note that only those samples with culture-based Illumina sequencing are shown. FN, false negative; FP, false positive; TP, true positive; TN, true negative; NA, data not available.

FIG 2

Drug susceptibility testing accuracy of nanopore MinION-based direct-from-sputum sequencing using culture-based WGS on Illumina results as the reference standard. For each sample, predictions from monoplexing and multiplexing (triangles with dots) are presented when available. Note that only those samples with culture-based Illumina sequencing are shown. FN, false negative; FP, false positive; TP, true positive; TN, true negative; NA, data not available.

FIG 3

DNA concentration (y axis) for each smear grade (x axis). Each point represents a single sample. Annotated P values were calculated with a Wilcoxon rank-sum test. ns, not significant.

FIG 4

Relationship between mean read depth on the M. tuberculosis genome (y axis) and the concentration of DNA extracted from the isolate (x axis) for all three sequencing approaches. The shaded areas represent the 95% confidence intervals. Each point represents a sample-sequencing strategy pair, and so some samples appear multiple times.

FIG 5

Mean read depth on the M. tuberculosis genome (y axis), stratified by smear grade (x axis) and sequencing strategy (colors). Each point represents a single sample. Annotated P values were calculated with a Wilcoxon rank-sum test.

FIG 6

Fraction of reads (y axis) from sequencing strategies that aligned to given organisms (colors). Each bar represents a single sample. Bars are vertically aligned such that the same isolate has the same place on the x axis across the sequencing strategies. Where a bar is missing, that isolate was not sequenced with that strategy. Bars to the left of the vertical black dashed line are negative controls. Note that most sputum isolates have TB reads, but the fraction is so low it is sometimes not possible to see it. NTM, nontuberculous mycobateria; TB, Mycobacterium tuberculosis; NA, sequencing not available.

FIG 7

Impact of mean read depth (x axis) on predicting species (top panel), lineage (middle panel), and DST result (bottom panel). Samples are stratified by whether the prediction was correct (blue) or not (red). Note that for DST each point is a sample-drug pair and hence why it has more points than species and lineage. Annotated P values were calculated with a Wilcoxon rank-sum test.