Rapid and economical drug resistance profiling with Nanopore MinION for clinical specimens with low bacillary burden of Mycobacterium tuberculosis.
Rapid and economical drug resistance profiling with Nanopore MinION for clinical specimens with low bacillary burden of Mycobacterium tuberculosis.
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DOI：
10.1186/s13104-020-05287-9
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发表时间：
2020-09-18
影响因子：
1.8
通讯作者：
Tang BSF
中科院分区：
文献类型：
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作者：
Chan WS;Au CH;Chung Y;Leung HCM;Ho DN;Wong EYL;Lam TW;Chan TL;Ma ESK;Tang BSF
我们设计并测试了用于直接结核病耐药性分析的纳米孔测序板。该组靶向10个耐药相关基因座。我们评估了从23份低细菌负荷的临床标本中扩增和测序这些基因座的可行性。至少有8个位点被成功扩增，从大多数预测一线和二线耐药（14/23，60.87%），并产生所有10个目标的12个标本测序与Nanopore MinION和Illumina MiSeq。通过Nanopolish校正MinION测序数据，并过滤复发变体。分析了所有共有序列中的总共67，082个碱基，其中67，019个碱基被MinION和MiSeq称为野生型。在MiSeq以100%的变异等位基因频率（VAF）识别的41个单核苷酸变异（SNV）中，MinION识别了39个（95.1%）。对于MiSeq调用的22个混合碱基，MinION调用具有最高VAF（70%）的SNV。由于测定时间短，试剂成本合理，以及不断改进的测序化学和信号校正管道，这种纳米孔方法可以在不久的将来成为直接结核病耐药性分析的可行选择。
We designed and tested a Nanopore sequencing panel for direct tuberculosis drug resistance profiling. The panel targeted 10 resistance-associated loci. We assessed the feasibility of amplifying and sequencing these loci from 23 clinical specimens with low bacillary burden. At least 8 loci were successfully amplified from the majority for predicting first- and second-line drug resistance (14/23, 60.87%), and the 12 specimens yielding all 10 targets were sequenced with Nanopore MinION and Illumina MiSeq. MinION sequencing data was corrected by Nanopolish and recurrent variants were filtered. A total of 67,082 bases across all consensus sequences were analyzed, with 67,019 bases called by both MinION and MiSeq as wildtype. For the 41 single nucleotide variants (SNVs) called by MiSeq with 100% variant allelic frequency (VAF), 39 (95.1%) were called by MinION. For the 22 mixed bases called by MiSeq, a SNV with the highest VAF (70%) was called by MinION. With short assay time, reasonable reagent cost as well as continuously improving sequencing chemistry and signal correction pipelines, this Nanopore method can be a viable option for direct tuberculosis drug resistance profiling in the near future.