Single-cell transcriptomics with Oxford Nanopore: Getting Started
During this Knowledge Exchange, Carly Tyer provided an overview of sample and library preparation for single-cell transcriptome sequencing with nanopore technology. Carly focused on the benefits of single-cell analysis with Oxford Nanopore sequencing, including the ability to generate full-length cDNA sequences for exploring splice isoforms, obtaining quantitative expression data for comparing transcriptome variation at the single-cell level, and the identification of novel transcripts. Single-cell nanopore sequencing has been used in applications ranging from characterising aberrant splicing in cancer, to immune system profiling of full-length antibody sequences.
Carly presented a brief overview of the 10x Genomics Next GEM single cell 3’ preparation kit and how to get started with the “Single-cell transcriptomics with cDNA prepared using 10X Genomics” protocol that enables the generation of full-length cDNA reads. Carly explained how this protocol maximises yield by incorporating an enrichment step to remove PCR artifacts. The protocol generates up to 150 M reads per library per PromethION Flow Cell, where approximately 90% have the correct transcript structure, and 70% are successfully assigned a 10X cell barcode.
Viewers will learn:
- Considerations for wet lab and library prep for 10x Genomics single-cell transcriptome sequencing with Oxford Nanopore
- What to expect from a single-cell library run on PromethION
- How to identify and quantify full-length transcripts from long nanopore reads
- How to explore splice variants and isoforms with single-cell data
Meet the speaker
Carly Tyer is an Applications Scientist on the Genomic Applications team at Oxford Nanopore Technologies, where she specialises in wet lab sequencing protocol development, including chromatin conformation capture using Pore-C, genomic characterisation in cancer and immunology, and single-cell sequencing of full-length human transcriptomes. Prior to joining Oxford Nanopore Technologies, she completed a B.S. in biotechnology at the University of California, Davis, and later worked as a research scientist for an agricultural microbiology company, helping to develop microbial sequencing practices and pipelines. She joined Oxford Nanopore Technologies 3.5 years ago in San Francisco, where she applies a variety of sequencing protocols to biological samples for projects showcasing the unique aspects of Oxford Nanopore technology.