NanoAmpli-Seq: A workflow for amplicon sequencing for mixed microbial communities on the nanopore sequencing platform
NanoAmpli-Seq: A workflow for amplicon sequencing for mixed microbial communities on the nanopore sequencing platform
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NanoAmpli-Seq：纳米孔测序平台上混合微生物群落的扩增子测序工作流程
DOI：
10.1101/244517
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
Calus S
中科院分区：
文献类型：
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作者：
Calus S
背景 Illumina 测序平台上的扩增子测序利用其深度测序和多重分析能力，但由于读长较短，遗传分辨率受到限制。虽然 Oxford Nanopore 或 Pacific Biosciences 测序平台克服了这一限制，但由于错误率较高或数据输出较低，其应用受到限制。结果在本研究中，我们引入了一种扩增子测序工作流程，即 NanoAmpli-Seq，它建立在分子内连接纳米孔一致性测序 (INC-Seq) 方法的基础上，并展示了其在全长 16S rRNA 基因测序中的应用。 NanoAmpli-Seq 对 INC-Seq 协议进行了重要改进，可减少样本处理时间，同时显着提高序列准确性。开发的协议包括用于 INC-Seq 一致读段的片段化和读段方向校正的 ChopSeq 软件，而 nanoClust 算法设计用于基于读段分区的从头聚类和簇内一致调用，以获得准确的全长 16S rRNA 基因序列。 结论 NanoAmpli-Seq 准确估计了测试模拟群落的多样性，在纳米孔测序平台上进行 2D 和 1D2 测序时，平均一致序列准确度为 99.5%。 NanoAmpli-Seq 序列中几乎所有残留错误均源自均聚物区域的缺失，这表明均聚物感知碱基检出或纠错可以实现与短读长测序平台相当的测序精度。
BackgroundAmplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity but is limited in genetic resolution due to short read lengths. While Oxford Nanopore or Pacific Biosciences sequencing platforms overcome this limitation, their application has been limited due to higher error rates or lower data output.ResultsIn this study, we introduce an amplicon sequencing workflow, i.e., NanoAmpli-Seq, that builds on the intramolecular-ligated nanopore consensus sequencing (INC-Seq) approach and demonstrate its application for full-length 16S rRNA gene sequencing. NanoAmpli-Seq includes vital improvements to the INC-Seq protocol that reduces sample processing time while significantly improving sequence accuracy. The developed protocol includes chopSeq software for fragmentation and read orientation correction of INC-Seq consensus reads while nanoClust algorithm was designed for read partitioning-basedde novoclustering and within cluster consensus calling to obtain accurate full-length 16S rRNA gene sequences.ConclusionsNanoAmpli-Seq accurately estimates the diversity of tested mock communities with average consensus sequence accuracy of 99.5% for 2D and 1D2sequencing on the nanopore sequencing platform. Nearly all residual errors in NanoAmpli-Seq sequences originate from deletions in homopolymer regions, indicating that homopolymer aware base calling or error correction may allow for sequencing accuracy comparable to short-read sequencing platforms.