Mycobacterium tuberculosis is the leading cause of death from bacterial infection. Improved rapid diagnosis and antimicrobial resistance determination, such as by whole-genome sequencing, are required. Our aim was to develop a simple, low-cost method of preparing DNA for sequencing direct from M. tuberculosis-positive clinical samples (without culture). Simultaneous sputum liquefaction, bacteria heat inactivation (99°C/30 min), and enrichment for mycobacteria DNA were achieved using an equal volume of thermo-protection buffer (4 M KCl, 0.05 M HEPES buffer, pH 7.5, 0.1% dithiothreitol [DTT]). The buffer emulated intracellular conditions found in hyperthermophiles, thus protecting DNA from rapid thermodegradation, which renders it a poor template for sequencing. Initial validation experiments employed mycobacteria DNA, either extracted or intracellular. Next, mock clinical samples (infection-negative human sputum spiked with 0 to 10⁵ Mycobacterium bovis BCG cells/ml) underwent liquefaction in thermo-protection buffer and heat inactivation. DNA was extracted and sequenced. Human DNA degraded faster than mycobacteria DNA, resulting in target enrichment. Four replicate experiments achieved M. tuberculosis detection at 10¹ BCG cells/ml, with 31 to 59 M. tuberculosis complex reads. Maximal genome coverage (>97% at 5× depth) occurred at 10⁴ BCG cells/ml; >91% coverage (1× depth) occurred at 10³ BCG cells/ml. Final validation employed M. tuberculosis-positive clinical samples (n = 20), revealing that initial sample volumes of ≥1 ml typically yielded higher mean depths of M. tuberculosis genome coverage, with an overall range of 0.55 to 81.02. A mean depth of 3 gave >96% 1-fold tuberculosis (TB) genome coverage (in 15/20 clinical samples). A mean depth of 15 achieved >99% 5-fold genome coverage (in 9/20 clinical samples). In summary, direct-from-sample sequencing of M. tuberculosis genomes was facilitated by a low-cost thermo-protection buffer.

中文翻译：

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DNA热保护促进直接从临床样品中分枝杆菌的全基因组测序。

结核分枝杆菌是细菌感染导致死亡的主要原因。需要改进的快速诊断和抗菌素耐药性测定，例如通过全基因组测序。我们的目标是开发一种简单，低成本的方法，直接从结核分枝杆菌中制备用于测序的DNA阳性临床样本（无培养物）。使用等体积的热保护缓冲液（4 M KCl，0.05 M HEPES缓冲液，pH 7.5、0.1％二硫苏糖醇[DTT]同时进行痰液化，细菌热灭活（99°C / 30分钟）和分枝杆菌DNA富集。 ]）。该缓冲液可模拟嗜热菌中的细胞内情况，从而保护DNA免受快速热降解的影响，从而使其成为测序的不良模板。最初的验证实验采用了分枝杆菌DNA，无论是提取的还是细胞内的。接下来，模拟临床样本（感染阴性的人类痰中掺入0至10 ⁵ 牛分枝杆菌BCG细胞/ ml）在热保护缓冲液中液化并热失活。提取DNA并测序。人类DNA的降解速度比分枝杆菌DNA降解快，从而导致靶标富集。四个重复实验以10 ¹ BCG细胞/ ml实现了结核分枝杆菌的检测，具有31至59个结核分枝杆菌复合物读数。10 ⁴ BCG细胞/ ml时最大基因组覆盖率（在5x深度处> 97％）；在10 ³ BCG细胞/ ml时，发生了> 91％的覆盖率（1倍深度）。最终验证采用结核分枝杆菌阳性的临床样本（n = 20），表明初始样本量≥1ml通常产生较高的结核分枝杆菌平均深度基因组覆盖范围为0.55至81.02。平均深度为3时，结核菌（TB）基因组覆盖率> 96％（在15/20临床样品中）。15的平均深度实现了> 99％的5倍基因组覆盖率（在9/20临床样品中）。总而言之，低成本的热保护缓冲液有助于结核分枝杆菌基因组的直接从样品测序。