颜林林 (2022-06-17 22:10):
#paper doi:10.1101/2022.06.12.495839 bioRxiv, 2022, Accurate Estimation of Molecular Counts from Amplicon Sequence Data with Unique Molecular Identifiers. 高通量测序数据中充满由PCR扩增和测序过程导致的错误,为解决此问题,人们通常会引入分子标签(UMI)技术,即用一段随机序列来标记出哪些序列来自同一原始模板分子,而哪些不是。很多工具在处理UMI时,都简单粗暴地将相同UMI的序列直接进行合并,而由于UMI序列本身也存在突变,会导致还原样本中原始模板分子信息的过程被误判。这个过程在扩增子测序(amplicon-seq)中尤为明显。本文通过构建一个单步隐马科夫模型(one step HMM),来处理PCR和测序过程中的错误,并用C语言实现了一套EM算法,对UMI测序数据的真实原始模板分子数进行估算。在模拟数据和真实数据中,分别进行了评测,对比既往其他类似工具,本文开发的工具(DAUMI),能有效识别出UMI冲突(UMI collision),表现出更优异的性能。
Accurate Estimation of Molecular Counts from Amplicon Sequence Data with Unique Molecular Identifiers
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Abstract:
Motivation: Amplicon sequencing is widely applied to explore heterogeneity and rare variants in genetic populations. Resolving true biological variants and quantifying their abundance is crucial for downstream analyses, but measured abundances are distorted by stochasticity and bias in amplification, plus errors during Polymerase Chain Reaction (PCR) and sequencing. One solution attaches Unique Molecular Identifiers (UMIs) to sample sequences before amplification eliminating amplification bias by clustering reads on UMI and counting clusters to quantify abundance. While modern methods improve over naive clustering by UMI identity, most do not account for UMI reuse, or collision, and they do not adequately model PCR and sequencing errors in the UMIs and sample sequences. Results: We introduce Deduplication and accurate Abundance estimation with UMIs (DAUMI), a probabilistic framework to detect true biological sequences and accurately estimate their deduplicated abundance from amplicon sequence data. DAUMI recognizes UMI collision, even on highly similar sequences, and detects and corrects most PCR and sequencing errors in the UMI and sampled sequences. We demonstrate DAUMI performs better on simulated and real data compared to other UMI-aware clustering methods. Availability: Source code is available at https://github.com/xiyupeng/AmpliCI-UMI.
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