Physical and Life Sciences, Lawrence Livermore National Laboratory, Livermore, California, United States of America.
PLoS One. 2013 Sep 9;8(9):e73455. doi: 10.1371/journal.pone.0073455. eCollection 2013.
Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy.
炭疽芽孢杆菌是炭疽病的潜在致命病原体，也是生物防御领域的一个重要关注点。有效生物防御策略的基石之一是能够在复杂样本背景下以高灵敏度和特异性检测到感染性病原体。然而，由于与蜡状芽孢杆菌和苏云金芽孢杆菌具有密切的同源性，炭疽芽孢杆菌基因组的性质使得特定检测变得困难。因此，我们选择确定下一代测序分析和微阵列在环境背景下检测炭疽芽孢杆菌的功效。我们应用下一代测序技术，在气溶胶和土壤样本中提取的背景核酸存在的情况下，对炭疽芽孢杆菌的基因组拷贝数进行滴定。我们发现，下一代测序技术能够检测到每纳克背景核酸中低至 10 个基因组当量的炭疽芽孢杆菌 DNA。通过将读取序列映射到定义的参考基因组子集或完整的 GenBank 数据库来实现检测。此外，从炭疽芽孢杆菌获得的序列数据可以可靠地区别于映射到蜡状芽孢杆菌或苏云金芽孢杆菌的序列数据。我们还证明了微生物计数微阵列在检测相同样本中的炭疽芽孢杆菌的功效，这是一种具有成本效益和高通量的方法，与下一代测序互补。我们的结果结合测序提供对复杂和新型生物基因组特征的深入了解的能力，表明这些平台应被视为生物监测策略的重要组成部分。
