Single Molecule, Real-Time (SMRT) sequencing delivers the long reads needed to capture intact isoform information without assembly or complicated algorithms. Use the Iso-Seq method to:
- Profile whole transcriptomes exhaustively at the isoform level
- Characterize alternative start and polyadenylation sites as well as exon skipping events
- Detect isoforms as potential biomarkers
- Target genes of interest using synthetic probes with sample multiplexing to query isoforms in a cost-effective manner
Workflow: from RNA to full-length transcripts
Sample + library preparation
Prepare full-length transcripts with flexible sample and library workflows
- Prepare full-length cDNA from 300 ng of total RNA using the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module kit
- Use the SMRTbell express template prep kit 3.0 to prepare libraries in one day
- Multiplex up to 12 samples
Simplify sample prep workflow and reduce project costs by using the Sequel II and IIe systems
Scale throughput on Sequel systems
- Use the Sequel II and IIe systems to generate up to 4 million* full-length reads per SMRT Cell 8M*
- Or use the Sequel II and IIe systems to generate up to 500,000* FLNC reads per SMRT Cell 1M
* Read lengths, number of reads, data per SMRT Cell, and other sequencing performance results vary based on sample quality/type and insert size, among other factors.
Output high-quality, full-length transcript sequences; no assembly required.
- Generate highly accurate long reads (HiFi reads) to characterize whole transcriptomes
- Use the Iso-Seq analysis in SMRT Link to output high-quality, full-length transcript FASTA sequences, with no assembly required, to characterize transcripts and splice variants
- Run Iso-Seq analysis with or without a reference genome. Follow up with community tools for different analyses such as SQANTI3 (transcript classification), tappAS (differential expression analysis), and TAMA (annotation).
IDENTIFY NOVEL FULL-LENGTH ISOFORMS IN BREAST CANCER CLINICAL AND CELL LINE SAMPLES LINKED TO SURVIVAL OUTCOMES
Scientists used the Iso-Seq method to identify novel isoforms uniquely associated with breast cancer subtypes and survival outcomes.
Veiga et al., “A comprehensive long-read isoform analysis platform and sequencing resource for breast cancer“, Science Advances (2022)