The current ecosystem of single-cell RNA-seq platforms is rapidly expanding, but robust solutions for single-cell and single-molecule full-length RNA sequencing are virtually absent. A high-throughput solution that covers all aspects is necessary to study the complex life of mRNA on the single-cell level. The Nanopore platform offers long read sequencing and can be integrated with the popular single-cell sequencing method on the 10× Chromium platform. However, the high error-rate of Nanopore reads poses a challenge in downstream processing (e.g., for cell barcode assignment). We propose a solution to this particular problem by using a hybrid sequencing approach on Nanopore and Illumina platforms. Our software ScNapBar enables cell barcode assignment with high accuracy, especially if sequencing saturation is low. ScNapBar uses unique molecular identifier (UMI) or Naïve Bayes probabilistic approaches in the barcode assignment, depending on the available Illumina sequencing depth. We have benchmarked the two approaches on simulated and real Nanopore data sets. We further applied ScNapBar to pools of cells with an active or a silenced nonsense-mediated RNA decay pathway. Our Nanopore read assignment distinguishes the respective cell populations and reveals characteristic nonsense-mediated mRNA decay events depending on cell status.

中文翻译：

---

使用 ScNapBar 在 Nanopore 平台上进行单细胞转录组测序

当前的单细胞 RNA-seq 平台生态系统正在迅速扩展，但几乎不存在用于单细胞和单分子全长 RNA 测序的强大解决方案。要在单细胞水平上研究 mRNA 的复杂生命，需要一个涵盖所有方面的高通量解决方案。Nanopore 平台提供长读长测序，可以与 10× Chromium 平台上流行的单细胞测序方法集成。然而，纳米孔读取的高错误率给下游处理（例如，细胞条形码分配）带来了挑战。我们通过在 Nanopore 和 Illumina 平台上使用混合测序方法提出了针对这一特定问题的解决方案。我们的软件 ScNapBar 可以高精度分配细胞条形码，尤其是在测序饱和度较低的情况下。ScNapBar 在条形码分配中使用唯一分子标识符 (UMI) 或朴素贝叶斯概率方法，具体取决于可用的 Illumina 测序深度。我们在模拟和真实的纳米孔数据集上对这两种方法进行了基准测试。我们进一步将 ScNapBar 应用于具有活性或沉默的无义介导的 RNA 衰变途径的细胞池。我们的 Nanopore read 分配区分了各自的细胞群，并根据细胞状态揭示了特征性的无义介导的 mRNA 衰减事件。