Single-cell isoform RNA sequencing (ScISOr-Seq) across thousands of cells reveals isoforms of cerebellar cell types

三代平台喜获单细胞测序技能

Methods

• we employ microfluidics to produce amplified full-length cDNAs barcoded for their cell of origin
• one pool for 3’ sequencing to measure gene expression(Pacific, Nanopore)
• another pool for long-read sequencing and isoform expression(10xGenomics)

Results

a.expression

• We sequence a mean of 17,885 reads per cell
• After filtering cells and considering only reads confidently mapped to genes, we have 3,875 unique molecular identifiers (UMIs) and 1,448 genes per cell during 3’end sequencing
• 6,627 cells were clustered into 17 groups
• To assess stability of clusters, we tripled Illumina sequencing depth for rep2

b.Full-length cDNAs

• generate ~5.2 million PacBio circular consensus reads
• cellular barcodes are located close to the polyA-tail, we first searched for polyA-tails
• chimeras, which were removed from further analysis
• for 58.0% (compared to 74.0% for 10x-3’seq) of the polyA-tail-containing CCS, we identified a perfect-match 16mer cellular barcode

c.ScISOr-Seq using Nanopore sequencing

• Using 1µg of barcoded cDNA
• ~31.4% (1D) and ~35.2% (passed 1D2) of Nanopore reads have a 30bp window with >=25 Ts
• nanopore 需要比 pacbio 更高的样品起始量，nanopore 数据错误率更高

Question

1. 三代数据仍然只是用于转录本组装、注释，需要二代数据定量。分析逻辑约等于三代注释，cell特异转录本，外加二代scRNAseq
2. single cell full-length cDNAs 带 barcode 扩增原理？？？