TABLE 2.

Summary of studies exploring DNA extraction techniques directly from sputum samples applying next generation sequencing technology.

Extraction Technique	Extraction Methodology	Performance of extraction technique and sequencing outcome	Sequencing Platform	Reference
Mechanical bead-beating	Sputum samples (n = 43) were centrifuged (16,200 × g, 30 min) and pelleted; ribolysis with 50 μl of glass beads (425–600 μm) in FastPrep-24 Classic Instrument (45 s, 6.4 m/s). After adding extraction buffer, proteinase K, vortexing and incubating (56°C, 10 min), DNA was extracted on the LIAISON IXT/Arrow (DiaSorin) automated system utilizing magnetic bead chemistry.	74% of sputum samples generated whole genomes at >85% coverage (majority were smear 3+). Required 5 days from sputum.	WGS on Illumina MiSeq and NextSeq	Doyle et al., 2018
Lysis of human/eukaryotic cells	Smear positive sputum samples (n = 40) were decontaminated with NAC-PAC RED (Alphatec, United States) and heat killed (95°C, 30 min). DNA extraction by saline wash, MolYsis Basic5 (Molzym Germany) eukaryotic cell lysis, bead beating, ethanol precipitation and GlycoBlue Co-Precipitant for MiSeq sequencing. For MiniSeq and ONT MinION sequencing, samples were saline washed, bead beaten, ethanol precipitated and cleaned with AMPure beads (BC, United Kingdom).	83% of sputum samples yielded sufficient DNA and 65% yielded sequencing data for resistance prediction at >3× coverage. Samples sequenced on the MinION gave >95% genome coverage and >5× depth of coverage. 54% of sputum samples had >12× depth and recovered >90% of the Mtb genome.	WGS on Illumina MiSeq, MiniSeq and ONT Minion	Votintseva et al., 2017a
	Smear positive sputum samples (n = 8) were NALC/NaOH decontaminated before differential osmotic lysis of human cells in water and digestion with RNase-Free DNase Set (Qiagen). Heat treatment (75°C, 10 min), DNA extraction with NucleoSpin Tissue-Kit (Macherey-Nagel, Germany).	20–99% mapped to the human genome (high contamination) and poor average depth of coverage (adc) (0.002X–0.07X). No samples had sufficient sequencing data. Cost < $69.34 per sample.	Shotgun sequencing on Illumina MiSeq	Doughty et al., 2014
Thermolysis	Smear positive sputum samples (n = 24) were NALC/NaOH decontaminated, pelleted, resuspended in PBS then in TE before adding 0.1 mm glass beads. Samples were heat killed (80°C, 50 min), freeze/thawed, vortexed and treated with DNeasy blood and tissue DNA extraction kit (Qiagen).	83% of sputum samples had high quality sequencing data (>20× depth, >90% genome covered). Kit costs $350 per sample. Total lab time of 50–96 h.	WGS on Illumina MiSeq	Brown et al., 2015
	Non-decontaminated sputum samples (n = 33) were heat killed (95°C, 1 h), ribolysed and purified with LIAISON Ixt (DiaSorin, Italy) or DNeasy blood and tissue kit (Qiagen, Germany).	At least 85% genome coverage at 20× and average depth of coverage (adc) of 60×.	WGS on Illumina NextSeq	Nimmo et al., 2019
Organic/enzymatic-based methods including CTAB	Smear 3+ sputum samples (n = 40; stored at −20°C) were NALC/NaOH decontaminated, boiled (85°C, 10 min) in TE buffer and frozen immediately (−20°C, 15 min). Incubation with lysozyme, proteinase K, SDS and 10% CTAB (1% final concentration). DNA extraction by alcohol precipitation and elution in TE buffer. This was compared to the PrimeXtract DNA extraction kit (Longhorn Vaccines and Diagnostics, United States).	The PrimeXtract DNA extraction kit had higher yields of Mtb DNA than CTAB method. DNA yield still too low (<2 μg) for SMRT sequencing.	PacBio sequencing	Ndhlovu et al., 2018
Sure SelectXT Target enrichment (AgiIent)	DNA extracted as described in Brown et al. (2015) above. Mtb DNA was enriched with Sure SelectXT Target enrichment (AgiIent) using 120-mer biotinylated RNA baits spanning the entire positive strand of H37Rv.	83% of sputum samples had high quality sequencing data (>20× depth, >90% genome covered). Requires 2–3 days and high-cost ($281.52 per sample).	WGS on Illumina MiSeq	Brown et al., 2015
	DNA extracted as described in Doyle et al. (2018) above. Sure SelectXT Target enrichment (AgiIent) was similarly followed as that of Brown et al. (2015) above including pull-down with streptavidin coated beads. Another reduced set of 120-mer biotinylated RNA baits, designed by Agilent, was used to capture only DR genes (35,960 bp) for MDR-TB sputum samples (n = 3) with the ‘Fast’ hybridization buffer (1 h, 65°C).	74% of sputum samples generated whole genomes at >85% coverage (majority were smear 3+) and >2000× average depth of coverage (adc). Required 5 days from sputum and 3 days using a reduced bait set. Streptavidin-coated beads are expensive.	WGS on Illumina MiSeq and NextSeq	Doyle et al., 2018
Deeplex®-MycTB assay (Geno Screen, Lille, France)	200 μL of spot sputum samples (n = 1494) preserved in 96% ethanol, were digested with proteinase K. Semi-automated Maxwell 16 FFPE DNA purification kit with a Maxwell machine (Promega, United States) was used. The Deeplex®d-MycTB assay (GenoScreen, France) amplified PCR products were purified with Agencourt AMPure XP magnetic beads (BC, United States) and DNA quantified by Qubit dsDNA BR assay (Life Technologies, United Kingdom).	Average depth of coverage (adc) of 1349×. 80% of Xpert positive samples were Mtb positive by Deeplex. 89% of Deeplex samples had a spoligotype, 49% were assigned lineages and 27% had an incomplete drug susceptibility assessment across target regions due to low bacterial load. 4.7% cases of mixed infections and 4.9% cases of NTMs were detected.	Targeted sequencing (18 genes/15 drugs) on Illumina MiSeq	Kayomo et al., 2020
	Smear positive sputum samples (n = 104) were liquefied in OMNIgene sputum reagent (DNA Genotek, Canada) before heat killing (95°C, 30 min) and lysis at 60°C with Maxwell method, as similarly performed by Kayomo et al. (2020)	90.8% of MinION reads while 99.5% of MiniSeq reads mapped to H37Rv. The average depth of coverage (adc) was 4,151× on MinION and 4,177× on MiniSeq. Deeplex amplicons are short for optimal processing on MinION long-read sequencer, therefore higher raw error rates (∼9%). Costs $138.68 per sample for both platforms.	Targeted sequencing (18 genes/15 drugs) on ONT MinION (previously sequenced on Illumina MiSeq)	Cabibbe et al., 2020
Next Generation Rapid DST Assay	Sputum samples (n = 12) were NALC/NaOH decontaminated and DNA extracted with GenoLyse®(Hain Lifescience, Germany) for the Next Generation rapid DST Assay (Translational Genomics Research Institute, AZ, United States).	Sputum samples could be examined at all 6 gene targets to at least 50,000× coverage in 72 h at ∼$30 per sample for reagent costs.	Targeted sequencing on Illumina MiSeq	Colman et al., 2015

Mtb, Mycobacterium tuberculosis; adc, average depth of coverage; h, hours; mins, minutes; secs, seconds; Bp, base pairs; Kb, kilo base pairs; Ds, double stranded; BR, broad range; HS, high sensitivity; FFPE, formalin-fixed paraffin-embedded; NALC, N-acetyl-L-cysteine; NaOH, Sodium hydroxide; PBS, Phosphate buffer saline; TE, Tris-Ethylenediamine tetra-acetic acid; SDS, sodium dodecyl sulfate; CTAB, Cetyltrimethylammonium bromide; BC, Beckman Coulter; USA, United States of America; UK, United Kingdom; DR, drug resistant; MDR, multidrug resistant; NTM, non-tuberculosis mycobacteria; DST, drug susceptibility testing; DNA, deoxyribo-nucleic acid; RNA, ribonucleic acid; WGS, whole genome sequencing; PCR, polymerase chain reaction; ONT, Oxford Nanopore Technologies; SMRT, Single-Molecule Real Time.