Human RNA sequencing

Changes in isoform variants and relative abundancies are an important clue in deciphering how genomic variants drive the phenotypic differences between health and disease. Only long-read sequencing can provide unbiased, direct detection of all the isoforms present in your sample.

Profile transcriptome complexity without assembly

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

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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

Sequencing

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.

Data analysis

Output high-quality, full-length transcript sequences; no assembly required.

Generate highly accurate long reads (HiFi reads) to characterize whole transcriptomes
- Dataset: Universal Human Reference RNA (UHRR)
- Dataset: Alzheimer Brain
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).