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Highly accurate barcode and UMI error correction using dual nucleotide dimer blocks allows direct single-cell nanopore transcriptome sequencing
bioRxiv - Genomics Pub Date : 2021-01-19 , DOI: 10.1101/2021.01.18.427145
Martin Philpott , Jonathan Watson , Anjan Thakurta , Tom Brown , Tom Brown , Udo Oppermann , Adam P Cribbs

Droplet-based single-cell sequencing techniques have provided unprecedented insight into cellular heterogeneities within tissues. However, these approaches only allow for the measurement of the distal parts of a transcript following short-read sequencing. Therefore, splicing and sequence diversity information is lost for the majority of the transcript. The application of long-read Nanopore sequencing to droplet-based methods is challenging because of the low base-calling accuracy currently associated with Nanopore sequencing. Although several approaches that use additional short-read sequencing to error-correct the barcode and UMI sequences have been developed, these techniques are limited by the requirement to sequence a library using both short- and long-read sequencing. Here we introduce a novel approach termed single-cell Barcode UMI Correction sequencing (scBUC-seq) to efficiently error-correct barcode and UMI oligonucleotide sequences synthesized by using blocks of dimeric nucleotides. The method can be applied to correct either short-read or long-read sequencing, thereby allowing users to recover more reads per cell and permits direct single-cell Nanopore sequencing for the first time. We illustrate our method by using species-mixing experiments to evaluate barcode assignment accuracy and evaluate differential isoform usage and fusion transcripts using myeloma and sarcoma cell line models.

中文翻译:

使用双核苷酸二聚体模块的高精度条形码和UMI纠错功能可直接进行单细胞纳米孔转录组测序

基于液滴的单细胞测序技术为组织内的细胞异质性提供了前所未有的见识。但是,这些方法仅允许在短读测序后测量转录本的远端部分。因此,大部分转录物的剪接和序列多样性信息丢失。由于目前与Nanopore测序相关的碱基检出准确性较低,因此将长读取Nanopore测序应用于基于液滴的方法具有挑战性。尽管已经开发了几种使用附加的短读测序来纠错条形码和UMI序列的方法,但是这些技术受到使用短读和长读测序对文库进行测序的要求所限制。在这里,我们介绍一种称为单细胞条形码UMI校正测序(scBUC-seq)的新颖方法,以有效地纠错条形码和使用二聚核苷酸区块合成的UMI寡核苷酸序列。该方法可用于校正短读或长读测序,从而使用户能够每个细胞回收更多的读物,并首次允许直接单细胞纳米孔测序。我们通过使用物种混合实验来评估条形码分配准确性并使用骨髓瘤和肉瘤细胞系模型评估差异同工型使用和融合转录本来说明我们的方法。该方法可用于校正短读或长读测序,从而使用户能够每个细胞回收更多的读物,并首次允许直接单细胞纳米孔测序。我们通过使用物种混合实验来评估条形码分配准确性并使用骨髓瘤和肉瘤细胞系模型评估差异同工型使用和融合转录本来说明我们的方法。该方法可用于校正短读或长读测序,从而使用户能够每个细胞回收更多的读物,并首次允许直接单细胞纳米孔测序。我们通过使用物种混合实验来评估条形码分配准确性并使用骨髓瘤和肉瘤细胞系模型评估差异同工型使用和融合转录本来说明我们的方法。
更新日期:2021-01-20
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