Application of Targeted Next-Generation Sequencing Assay on a Portable Sequencing Platform for Culture-Free Detection of Drug-Resistant Tuberculosis from Clinical Samples.
Application of Targeted Next-Generation Sequencing Assay on a Portable Sequencing Platform for Culture-Free Detection of Drug-Resistant Tuberculosis from Clinical Samples.
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DOI：
10.1128/jcm.00632-20
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发表时间：
2020-09-22
影响因子：
9.4
通讯作者：
Cirillo DM
中科院分区：
文献类型：
--
作者：
Cabibbe AM;Spitaleri A;Battaglia S;Colman RE;Suresh A;Uplekar S;Rodwell TC;Cirillo DM
靶向新一代测序（tNGS）已成为一种全面替代现有方法，从患者痰样本中进行结核分枝杆菌药敏试验（DST），用于耐药结核病（DR-TB）的临床诊断。然而，测序平台的复杂性限制了它们在低资源环境中的应用。本研究的目的是评估基于tngs的DST解决方案Genoscreen Deeplex Myc-TB在紧凑、低成本Oxford Nanopore Technologies MinION测序仪上的使用。靶向新一代测序（tNGS）已成为一种全面替代现有方法，从患者痰样本中进行结核分枝杆菌药敏试验（DST），用于耐药结核病（DR-TB）的临床诊断。然而，测序平台的复杂性限制了它们在低资源环境中的应用。本研究的目的是评估基于tngs的DST解决方案Genoscreen Deeplex Myc-TB在紧凑、低成本Oxford Nanopore Technologies MinION测序仪上的使用。从涂片阳性痰沉积物中提取的104个DNA样本，先前使用Illumina MiniSeq上的Deeplex测定测序，在应用定制文库制备后，在MinION上重新测序。miniion读取质量、映射统计信息和变量调用使用内部管道计算，并与参考minieq数据进行比较。MinION读取到H37RV参考基因组的平均百分比为90.8%，而minieq为99.5%。MinION和MiniSeq的平均覆盖深度分别为4151 x和4177 x，目标基因间存在异质性分布。两个平台的综合参考覆盖宽度均为0.99%。我们观察到报告临床相关耐药标记的技术完全一致，包括完整的基因缺失。总之，我们证明了从miniion上的Deeplex获得的工作流程和测序数据与minieq相当，尽管miniion上的原始错误率更高，但miniion的便携性、多功能性和低资本成本具有额外的优势。MinION上的靶向NGS是一种有前景的DST解决方案，可快速提供临床相关数据，以管理复杂的耐药结核病病例。
Targeted next-generation sequencing (tNGS) has emerged as a comprehensive alternative to existing methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis from patient sputum samples for clinical diagnosis of drug-resistant tuberculosis (DR-TB). However, the complexity of sequencing platforms has limited their uptake in low-resource settings. The goal of this study was to evaluate the use of the tNGS-based DST solution Genoscreen Deeplex Myc-TB, for use on the compact, low-cost Oxford Nanopore Technologies MinION sequencer. Targeted next-generation sequencing (tNGS) has emerged as a comprehensive alternative to existing methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis from patient sputum samples for clinical diagnosis of drug-resistant tuberculosis (DR-TB). However, the complexity of sequencing platforms has limited their uptake in low-resource settings. The goal of this study was to evaluate the use of the tNGS-based DST solution Genoscreen Deeplex Myc-TB, for use on the compact, low-cost Oxford Nanopore Technologies MinION sequencer. One hundred four DNA samples extracted from smear-positive sputum sediments, previously sequenced using the Deeplex assay on an Illumina MiniSeq, were resequenced on MinION after applying a custom library preparation. MinION read quality, mapping statistics, and variant calling were computed using an in-house pipeline and compared to the reference MiniSeq data. The average percentage of MinION reads mapped to an H37RV reference genome was 90.8%, versus 99.5% on MiniSeq. The mean depths of coverage were 4,151× and 4,177× on MinION and MiniSeq, respectively, with heterogeneous distribution across targeted genes. Composite reference coverage breadth was >99% for both platforms. We observed full concordance between technologies in reporting the clinically relevant drug-resistant markers, including full gene deletions. In conclusion, we demonstrated that the workflow and sequencing data obtained from Deeplex on MinION are comparable to those for the MiniSeq, despite the higher raw error rates on MinION, with the added advantage of MinION’s portability, versatility, and low capital costs. Targeted NGS on MinION is a promising DST solution for rapidly providing clinically relevant data to manage complex DR-TB cases.