Evaluation of DNA extraction methods for clinical Mycobacterium tuberculosis primary liquid culture for whole-genome sequencing
Bibliographic record
Abstract
Whole-genome sequencing (WGS) has potential for determining the complete drug-resistance profile of clinical Mycobacterium tuberculosis (Mtb) strains. Cetyltrimethylammonium-bromide (CTAB), the conventional method for Mtb DNA extraction, is labour-intensive and difficult to implement in routine laboratory settings. This study evaluated the performance of commercial kits to extract DNA from clinical primary liquid cultures (CPC). Mtb-positive decontaminated sputum sediments were pooled and used to inoculate mycobacteria-growth-indicator tubes (MGIT). Positive non-contaminated MGIT cultures were pooled and aliquoted to generate 10 technical replicate isolates for DNA extraction by CTAB method with (+) or without (-) RNAse and nine DNA extraction commercial kits (+/-modifications): Zymo-DNA Clean and Concentrator, Zymo-Quick-DNA Fungal/Bacterial (+ lysozyme-digestion), InstaGene (IGM) +/-RiboLyser Homogeniser (RH), GenoLyse (+/-precipitation), FluoroLyse (+/-precipitation), PrepGEM-Bacterial (+/-precipitation), NucleoSpin-Tissue, NucleoMag-Pathogen, and Gene-Xpert buffer (+ precipitation or +purification). Genomic libraries for WGS were generated using the Illumina DNA-Prep Kit and evaluated for quality using NanoDrop, Qubit DNA ds/HS and TapeStation assays. Ten performance parameters were evaluated out of a score of 5: quantity of total dsDNA, DNA purity ratio A260/280 and A260/230, PCR amplifiability (targeting the pncA gene, 615 bp), concentration of genomic libraries (ng/µl), library fragment size (bp), mapped percentage, median coverage, turnaround time and cost. IGM/RH showed the highest overall performance, followed by IGM, CTAB/RNase, GenoLyse+precipitation, and FluoroLyse+precipitation. Commercial methods achieved sequencing results comparable to CTAB while requiring shorter processing times and lower costs. Simplified commercial extraction methods can produce DNA suitable for WGS from CPC samples while reducing processing time and cost compared with CTAB, supporting their use in routine TB workflows.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.005 | 0.009 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.002 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.002 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 0.003 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".