Long Read Sequencing (LRS) based transcriptomics offers the advantage of capturing isoforms and fusion transcripts without the need for bioinformatics transcript/transcriptome assembly required for short-read-sequencing transcriptome data. LRS applications include cDNA sequencing of full length transcripts, direct RNA sequencing of full length transcripts along with detection of RNA modifications, and assessing single cell full length transcripts. In the CGI, two platforms are supported for these applications: Oxford Nanopore Technologies and Pacific Biosciences. These options are ideal if you aim to:
- characterize alternative splicing (AS) events (alternative start sites, end sites, intron retention, and exon-skipping)
- detect and quantify transcribed gene fusions
- detect differentially expressed isoforms and isoform-switching events, and
- predict the functional impact of novel isoforms through open reading frame (ORF) prediction.
Oxford Nanopore Technologies RNA applications
Direct RNA (dRNA) and cDNA can be sequenced on PromethION, GridION or MinION systems in the CGI. Oxford Nanopore technology long read platforms deliver high outputs of reads that span full-length transcripts, resulting in unambiguous isoform detection and comprehensive transcriptome characterization. Nanopore reads enable differential isoform expression analysis and allele-specific expression analysis and can identify fusion transcripts that cannot be captured in full with short-read approaches.
Direct RNA (dRNA)-seq
dRNA-seq is an ONT method that directly sequences native RNA molecules by passing them through a nanopore, bypassing reverse transcription and amplification. This application enables real-time detection of RNA modifications (m6A, m5C, pseudouridine, m1A, ac4C, m7G, and 2′-O-methylation(Nm)) and provides full-length transcript characterization, making it ideal for epi-transcriptomics and isoform analysis.
Sample requirement: 300 ng of poly(A) tailed RNA or 1 µg of total RNA in 8 µl (RIN score >8)
Instrument and flow cell choice depends on your sample type and transcriptome size.
cDNA-seq
After extraction, polyadenylation enrichment can be performed on total RNA, either using the Invitrogen Dynabeads mRNA Purification Kit to isolate poly-A-tailed RNA or the Invitrogen RiboMinus Eukaryote Kit v2 to selectively deplete ribosomal RNA. To prepare your extracted RNA for sequencing, we recommend the cDNA-PCR Sequencing Kit, which is optimized for generating high outputs of full-length transcripts and for isoform identification and quantification. With the cDNA-PCR Barcoding Kit, up to 24 samples can be run in parallel (depending on your sample type and transcriptome size).
During library preparation, the RNA undergoes reverse transcription and strand switching to prepare full-length cDNA. Full-length cDNA molecules are then selected for and amplified using PCR before the cDNA library is sequenced.
PacBio cDNA applications
Iso-Seq/Kinnex
ThePacBio Iso-Seq method sequences entire cDNA molecules without the need for bioinformatics transcript assembly. Using full-length cDNA from the Iso-Seq express 2.0 kit as input, we prepare Kinnex Iso-seq libraries, which result in ~8-fold throughput increase over Iso-seq libraries, for sequencing on the PacBio Revio using SPRQ chemisty.
We prepare libraries using the Kinnex Kinnex full-length RNA kit and recommend barcoding 4-6 samples for a 3Gb genome and 7 or more samples for smaller genomes. Read depth can be assessed empirically from a trial run if you have a large project planned.
Users can perform Read Segmentation & Iso-Seq analysis in SMRT® Link software for isoform classification. Moreover, there are many 3rd party analysis tools available for isoform classification and annotation, fusion transcript discovery, variant calling, transcript assembly, differential transcript analysis, differential splicing usage, and transcript visualization; our Computational Biology Core is available to assist or perform these analyses for you.
Sample requirements: at least 400ng total RNA that is DNA-free with a RIN score of at least 8.
MAS-seq/Kinnex single-cell RNA-seq
The PacBio Kinnex single-cell RNA kit utilizes MAS-Seq, an array concatenation method for increasing throughput by joining cDNA molecules from single cell RNA-seq library preparation workflows like 10x Genomics, into longer concatenated fragments bioinformatically. PacBio HiFi reads generated from single cell cDNA library preparation products can then be bioinformatically parsed to retrieve the original cell-specific molecules all maintaining synthetic long reads.
The Kinnex kit is compatible with cDNA generated from most single-cell platforms including PIP-seq (i.e. Illumina single cell sequencing) (offered in the CGI LINK OUT HERE, or user prepared), and other single cell cDNAs prepared by users with Parse WT and WT mini kits, and 10x Chromium Next GEM Universal 3’ (v3.1), Next GEM Universal 5’ (v2), GEM-X Universal 3’ (v4), and GEM-X Universal 5’ (v3).
Sample requirements: 15–75 ng of cDNA.
Please see LINK for additional information and applications supporting single cell/nuclei RNA-seq in the CGI.