PacBio RNA Sequencing — Full-Length Transcript Isoform Discovery, Alternative Splicing & Fusion Gene Detection by SMRT Sequencing on the Revio Platform
Alternative splicing generates transcript diversity that short-read RNA sequencing systematically undercounts. Every human gene produces an average of 6–7 transcript isoforms, yet standard short-read RNA-seq detects fewer than 30% of expressed isoforms in a typical experiment — and for genes with more than 10 annotated isoforms, the detection rate falls below 15%. Fusion genes, circular RNAs, transcript isoforms with retained introns, and novel splice junctions in non-model organisms are even more severely underrepresented because short reads cannot span complete splice junctions, cannot distinguish isoforms that share exon boundaries, and cannot assemble full-length transcript structures without ambiguity.
PacBio RNA sequencing by Iso-Seq (Isoform Sequencing) eliminates these ambiguities by sequencing full-length cDNA molecules end-to-end on the Single Molecule, Real-Time (SMRT) platform. Every read is a complete transcript — from 5' end to poly(A) tail — captured as a single contiguous sequence without computational assembly or inference. The PacBio Revio system, with HiFi read accuracy ≥ Q30 (99.9%) and Kinnex chemistry throughput of up to 60 million reads per SMRT Cell, produces isoform-resolved transcriptomes at a scale that was unattainable with earlier long-read platforms. For reference-free transcript discovery in non-model organisms, full-length isoform quantification in cancer transcriptomes, and single-cell isoform profiling at single-cell resolution, PacBio RNA sequencing provides the definitive single-molecule ground truth for transcript isoform biology.
Why PacBio RNA Sequencing — Service Highlights
- Full-length transcripts without assembly — Every read spans the entire transcript from 5' cap to poly(A) tail. No fragmentation, no computational transcript reconstruction, no isoform ambiguity. The transcript is the read, and the read is the transcript
- HiFi accuracy at transcript scale — PacBio Revio HiFi reads achieve ≥ Q30 (99.9%) consensus accuracy across the full transcript length (typically 1–10 kb with Kinnex, up to 10+ kb with standard Iso-Seq), enabling confident single-nucleotide variant detection within isoform sequences and unambiguous isoform assignment at the gene level
- Comprehensive isoform discovery across all transcript classes — PacBio RNA sequencing detects known and novel isoforms, fusion transcripts, circular RNAs (circRNAs), and chimeric transcripts in a single experiment. The full-length read structure provides unambiguous evidence for exon connectivity, splice junction usage, and transcript start/end boundaries that short-read methods cannot resolve
- Scalable throughput with Kinnex chemistry — Kinnex full-length RNA kits concatenate cDNA amplicons to achieve up to 60 million long reads per Revio SMRT Cell 8M, enabling population-scale isoform profiling, multi-sample multiplexing, and detection of low-abundance isoforms that were previously accessible only through targeted approaches
- Single-cell isoform profiling capability — Our single-cell Iso-Seq workflow combines 10x Genomics single-cell capture with PacBio full-length sequencing to deliver isoform-level expression data at single-cell resolution — the only method that simultaneously captures cellular heterogeneity and full-length isoform structure
- Multi-species applicability — PacBio RNA sequencing works across all organisms, including non-model species without reference genomes. De novo isoform clustering, functional annotation, and coding sequence prediction are performed directly from full-length reads without requiring an existing genome assembly or transcript annotation
Why PacBio RNA Sequencing — and Why Full-Length Transcripts Change the Question
Transcriptome analysis by short-read RNA sequencing has been the standard approach for gene expression quantification and differential expression analysis for nearly two decades, and it remains the most cost-effective method for measuring gene-level expression changes across large sample cohorts. But when the question moves beyond "which genes are expressed at different levels?" to "which transcript isoforms mediate the functional difference?" or "what are the full-length sequences of expressed transcripts in a non-model organism?" or "which fusion transcripts are present in this tumor?", short-read methods encounter fundamental limitations that no increase in sequencing depth can overcome.
Short-read RNA-seq (50–300 bp reads) cannot span complete splice junctions in multi-exon genes, cannot distinguish transcript isoforms that share the same initial and terminal exons but differ in internal exon composition, and cannot resolve phasing between splice junctions separated by more than a read length. Every isoform-level analysis from short-read data depends on computational inference — transcript quantification tools like Salmon or RSEM estimate isoform abundances by deconvoluting read counts across a known isoform catalog, but they cannot discover novel isoforms or resolve isoform structures de novo. Assembly-based methods like Trinity or rnaSPAdes reconstruct transcripts from overlapping short reads, but assemblies routinely produce chimeric transcripts, miss low-abundance isoforms, and fail to resolve transcripts with highly similar isoforms. The problem is inherent: you cannot reliably determine the full-length structure of a 3 kb transcript from 150 bp fragments.
PacBio RNA sequencing by Iso-Seq solves this problem at the source. By sequencing full-length cDNA molecules end-to-end on the SMRT platform, every read captures a complete transcript — 5' UTR, coding sequence, 3' UTR, and poly(A) tail — in a single contiguous sequence. With HiFi accuracy (≥ Q30), these reads are accurate enough for confident variant detection within isoform sequences, and with Kinnex throughput (up to 60 million reads per SMRT Cell), the method is now scalable to whole-transcriptome isoform discovery across multiple samples and conditions. This is not a replacement for short-read RNA-seq in all applications — for gene-level differential expression in large cohorts, short-read methods remain more economical — but for every question that requires unambiguous isoform-level information, PacBio RNA sequencing provides the definitive single-molecule answer. Our Full-Length Transcript Sequencing (Iso-Seq) service page provides detailed technical information on the standard Iso-Seq workflow and data analysis options.
What Is PacBio RNA Sequencing (Iso-Seq)?
PacBio RNA sequencing — commonly referred to as Iso-Seq (Isoform Sequencing) — is a long-read RNA sequencing method that captures full-length transcript sequences by converting RNA to cDNA, sequencing the complete cDNA molecules on the PacBio SMRT platform, and producing high-accuracy consensus reads that represent individual transcript isoforms.
Standard Iso-Seq workflow: PolyA⁺ RNA is enriched and reverse-transcribed using an oligo-dT primer that captures the 3' end of each transcript. A template-switching oligo at the 5' end captures complete 5' termini, ensuring full-length coverage from the 5' cap to the poly(A) tail. The resulting full-length cDNA is size-fractionated (typically 1–2 kb, 2–3 kb, and 3–6 kb fractions; up to 10+ kb with optimized protocols) to optimize sequencing efficiency across the transcript size range. After SMRTbell library preparation, each cDNA molecule is sequenced on the PacBio Revio system, where circular consensus sequencing (CCS) produces a HiFi read — a single consensus sequence with ≥ Q30 accuracy derived from multiple passes of the polymerase around the circular template. Each HiFi read represents one full-length transcript isoform, complete with 5' UTR, CDS, 3' UTR, and poly(A) tail. Depending on the library preparation method, the Kinnex full-length RNA kit concatenates multiple cDNA fragments into a single SMRTbell template, increasing throughput to up to 60 million reads per SMRT Cell 8M by sequencing multiple transcripts per CCS circle.
Key data products from a single Iso-Seq experiment: Iso-Seq data simultaneously produces (1) full-length transcript sequences with isoform structures (exon–intron boundaries defined by alignment to a reference genome if available, or de novo by isoform clustering if no reference exists); (2) isoform-level expression quantification based on read counts per isoform; (3) detection of alternative splicing events (exon skipping, intron retention, alternative 5'/3' splice sites, mutually exclusive exons) and novel isoform discovery; (4) fusion transcript identification when reads span across-gene junctions; (5) coding sequence prediction and functional annotation of all detected isoforms through in silico translation and domain analysis; and (6) lncRNA and novel RNA species identification. The breadth of information obtainable from a single Iso-Seq library makes it the most information-rich single-assay transcriptome profiling method available.
Distinction from other long-read RNA methods: Unlike Nanopore direct RNA sequencing (which sequences native RNA and detects base modifications but produces lower per-read accuracy), PacBio Iso-Seq sequences cDNA copies of the transcriptome at Q30+ accuracy, making it the platform of choice for applications requiring highest-confidence isoform sequences — novel isoform validation, fusion transcript confirmation, single-nucleotide variant detection in transcripts, and cross-species comparative transcriptomics where alignment accuracy at single-nucleotide resolution is essential. Unlike standard short-read RNA-seq, Iso-Seq produces complete transcript structures without computational assembly, eliminating the isoform ambiguity inherent to fragment-based approaches.
Key Advantages of PacBio RNA Sequencing
Scientific Advantages
- Unambiguous full-length isoform structures
Every PacBio RNA read captures a complete transcript from 5' end to poly(A) tail. Alternative splicing events, novel exon junctions, retained introns, and fusion boundaries are observed directly in the read rather than inferred from fragment alignments. Isoform-level structural information — including complete 5' UTR and 3' UTR sequences that are systematically truncated in short-read assemblies — is recovered for every detected transcript.
- Simultaneous discovery and quantification
Iso-Seq discovers novel isoforms and quantifies their abundance in the same experiment. The read count per isoform provides direct digital expression measurements, and the full-length sequence provides the isoform's complete structure — eliminating the "discovery vs. quantification" trade-off that separates short-read RNA-seq (discovery from assembly) from expression arrays or targeted qPCR (quantification of known targets).
- Fusion transcript identification from single reads
Fusion transcripts — genes formed by the juxtaposition of sequences from two different genes, frequently in cancer genomes — are identified by single reads that span the fusion junction and extend into both partner genes. A single PacBio read covering the full fusion transcript provides unambiguous evidence of the fusion structure, including the exact breakpoint and the complete isoform context, whereas short-read fusion detection depends on computational prediction from paired-end reads that may not span the full junction.
Business & Project Advantages
- Scalable throughput for population-scale transcriptomics
Kinnex chemistry delivers up to 60 million full-length reads per Revio SMRT Cell — a >10-fold increase over standard Iso-Seq — enabling multiplexed sample analysis, low-abundance isoform detection, and multi-condition experimental designs at a per-sample cost that is competitive with short-read approaches for isoform-level studies.
- Integrated RNA sequencing service modules covering the full PacBio RNA analysis spectrum
Our PacBio RNA sequencing service integrates with complementary long-read transcriptomics services — Single-Cell Full-Length Transcriptome for isoform discovery at single-cell resolution, and Full-Length Transcriptome Profiling for combined long-read and short-read integrative analysis — providing a comprehensive, multi-scale transcriptome analysis capability from a single service provider.
- End-to-end bioinformatics with validated isoform analysis pipelines
Our computational team deploys industry-standard PacBio RNA bioinformatics tools: SMRT Link and Iso-Seq Analysis for read clustering and isoform consensus calling, SQANTI3 for isoform classification and quality control, TAMA and Cupcake for isoform-level analysis, and custom pipelines for fusion detection, differential isoform expression, and functional annotation. Deliverables include isoform sequences, expression tables, splice junction annotations, and integrated analysis reports.
- Reference-free analysis for non-model organisms
For non-model species without reference genomes or transcript annotations, our bioinformatics pipeline performs de novo isoform clustering from full-length reads, followed by functional annotation through homology search, CDS prediction, lncRNA identification, and GO/KEGG pathway assignment. This makes PacBio RNA sequencing directly applicable to any organism without requiring pre-existing genomic resources.
PacBio Revio & Kinnex Chemistry — Full-Length Transcript Sequencing Technology
PacBio RNA sequencing is performed on the Revio system, PacBio's third-generation long-read sequencing platform, using the Kinnex full-length RNA kit for high-throughput isoform profiling or standard Iso-Seq for longer-read applications. The combination of circular consensus sequencing (CCS) accuracy and concatenation-based throughput scaling makes this platform uniquely suited for comprehensive transcriptome analysis.
| Feature | PacBio Revio — Kinnex RNA | PacBio Revio — Standard Iso-Seq |
| Library method | cDNA concatenation (MAS-Seq/Kinnex) — multiple cDNA fragments ligated per SMRTbell | Single cDNA fragment per SMRTbell (size-fractionated) |
| Read throughput per SMRT Cell 8M | Up to 60 million full-length reads | 2–5 million full-length reads |
| Read accuracy | ≥ Q30 (99.9% HiFi consensus accuracy) | ≥ Q30 (99.9% HiFi consensus accuracy) |
| Transcript length range | 1–5 kb (optimal); up to 10 kb with size selection | 1–10+ kb (with optimized protocols) |
| Typical reads per sample (multiplexed) | 5–15 million (4–12 samples per SMRT Cell) | 0.5–1 million (4–8 samples per SMRT Cell) |
| Low-abundance isoform detection | Superior — higher read depth captures rare isoforms | Moderate — limited by total read throughput |
| Single-cell compatibility | ✓ (10x Genomics cell barcodes preserved) | ✓ (10x Genomics cell barcodes preserved) |
| Best suited for | Whole-transcriptome isoform profiling, multi-sample studies, rare isoform discovery, single-cell Iso-Seq | Ultra-long transcript (>5 kb) characterization, targeted isoform validation, low-input RNA |
Integrated RNA Sequencing Service Modules
Our PacBio RNA sequencing service extends beyond standard bulk Iso-Seq. We offer a suite of integrated service modules that address specific experimental designs — from single-cell transcriptome profiling at isoform resolution to combined long-read and short-read integrative analysis — enabling researchers to match the sequencing strategy to their biological question and sample type.
Full-Length Transcript Sequencing (Iso-Seq) — Core Isoform Discovery Service
Core service: Standard PacBio Iso-Seq from polyA⁺ RNA with size-fractionated library preparation, SMRTbell library construction, Revio HiFi sequencing, and full bioinformatics analysis. Suitable for projects where the primary goal is comprehensive isoform discovery in a small number of samples — including novel transcript identification, alternative splicing characterization, and fusion gene detection. Deliverables include full-length isoform sequences (FASTA), isoform expression counts, splice junction annotations, and functional annotation reports. The Full-Length Transcript Sequencing (Iso-Seq) service page provides detailed information on standard Iso-Seq protocols, library preparation options, and data analysis packages.
Single-Cell Full-Length Transcriptome — Isoform Discovery at Single-Cell Resolution
Single-Cell Full-Length Transcriptome Sequencing combines 10x Genomics single-cell capture (3' v3.1, v4, or 5' kits) with PacBio Revio full-length sequencing to deliver isoform-level transcriptome data at single-cell resolution. Each read is tagged with a cell barcode and molecular barcode from the 10x system, enabling isoform discovery, alternative splicing analysis, and fusion detection within individual cell populations while simultaneously capturing cellular heterogeneity. This approach resolves the fundamental ambiguity of "pseudo-bulk" isoform analysis — are two isoforms of the same gene expressed in the same cell or in different cells? — by preserving single-cell resolution throughout the full-length sequencing workflow. Applications include cell-type-specific isoform discovery in complex tissues, rare cell population transcriptomics, and developmental lineage tracing at isoform resolution.
Full-Length Transcriptome Profiling — Integrated Long-Read and Short-Read Analysis
Full-Length Transcriptome Profiling combines PacBio Iso-Seq (full-length isoform discovery and annotation) with Illumina short-read RNA-seq (deep gene-level expression quantification in the same samples) in an integrated analysis workflow. The PacBio data provides a comprehensive, sample-matched isoform catalog including novel isoforms and non-coding transcripts; the Illumina data provides deep, cost-effective expression quantification across all detected isoforms at a sequencing depth that would be cost-prohibitive with long-reads alone. This hybrid approach is particularly valuable for projects requiring both comprehensive isoform discovery and robust statistical power for differential isoform expression analysis across multiple conditions or time points.
Kinnex High-Throughput Iso-Seq — Multiplexed Population-Scale Isoform Profiling
For projects requiring isoform analysis across multiple samples, conditions, or time points, our Kinnex-based high-throughput Iso-Seq service enables multiplexed analysis of 4–12 samples per SMRT Cell 8M, with up to 60 million total reads per run. Sample multiplexing is achieved through barcoded cDNA primers during the reverse transcription step, with demultiplexing performed during bioinformatics analysis. This service module is suitable for population-scale transcriptome studies, multi-condition experimental designs (e.g., developmental time series, drug treatment panels), and comparative transcriptomics across species or genotypes. The Kinnex throughput also improves detection of low-abundance isoforms that may fall below the detection threshold in standard Iso-Seq experiments.
Applications of PacBio RNA Sequencing
Cancer Transcriptomics and Fusion Gene Discovery
- Full-length isoform profiling of tumor transcriptomes to identify cancer-specific isoforms, fusion transcripts (gene fusions, read-through transcripts, and intergenic fusions), and alternative splicing events associated with oncogene activation or tumor suppressor inactivation — all detected from single reads that span complete transcript structures without computational ambiguity
- Isoform-level expression analysis of immune checkpoint genes, neoepitope-encoding transcripts, and therapy resistance-associated isoforms in cancer cell lines, patient-derived xenografts, and clinical tumor samples. The single-molecule resolution of PacBio Iso-Seq enables detection of isoform switches — changes in the dominant isoform of a gene between normal and tumor tissue — that are invisible to gene-level short-read quantification
Non-Model Organism Transcriptomics
- De novo transcriptome assembly and annotation for any species without a reference genome, including plants, animals, fungi, and microbial eukaryotes. PacBio Iso-Seq produces complete transcript sequences with full 5' and 3' UTR coverage, enabling accurate CDS prediction, functional annotation, and comparative transcriptomics across species — capabilities that depend on full-length transcript information and cannot be achieved from short-read assemblies alone
- lncRNA identification and characterization, including multi-exonic lncRNAs, antisense transcripts, and intergenic non-coding RNAs, whose full-length structures are systematically disrupted in short-read assemblies
Neurobiology and Developmental Transcriptomics
- Isoform-level characterization of neural transcriptomes, where genes involved in synaptic plasticity, axon guidance, and neuronal signaling generate complex alternative splicing patterns with hundreds of isoforms per gene. Long neural transcripts (>5 kb) that carry essential regulatory information but are underrepresented in fragmented short-read data are captured and characterized in full by PacBio Iso-Seq
- Single-cell isoform analysis of developing tissues and organoids, where cell-type-specific isoform usage determines cell fate decisions and functional specialization. Our single-cell Iso-Seq module preserves the linkage between isoform expression and cell identity
Agricultural and Plant Transcriptomics
- Full-length transcriptome analysis of crop species to annotate alternatively spliced transcripts associated with agronomic traits — stress tolerance, yield components, disease resistance — and to generate comprehensive isoform catalogs for genome annotation improvement in polyploid crop species where short-read isoform analysis is confounded by homeologous gene copies
- Fusion transcript discovery in plant genomes, where chromosomal rearrangements and transposable element activity generate transcript fusions that may contribute to phenotypic variation and environmental adaptation
Bioinformatics Analysis
| Analysis Feature | Standard Package | Advanced Package |
| Read processing, HiFi consensus calling (SMRT Link CCS), demultiplexing, and QC | ✓ | ✓ |
| Full-length isoform clustering, consensus generation, and polishing (Iso-Seq Analysis, isoseq3) | ✓ | ✓ |
| Reference genome alignment and isoform classification (SQANTI3) — full-length, novel in catalog, novel not in catalog, fusion, etc. | ✓ | ✓ |
| Isoform-level expression quantification (read count per isoform, TPM normalization) | ✓ | ✓ |
| Alternative splicing analysis (exon skipping, intron retention, alternative 5'/3' splice sites, mutually exclusive exons) | ✓ | ✓ |
| Fusion transcript detection and validation | — | ✓ |
| Differential isoform expression analysis between conditions (DEXSeq, IsoformSwitchAnalyzer) | — | ✓ |
| De novo isoform clustering (reference-free) and functional annotation (BLAST, InterProScan, GO, KEGG) | — | ✓ |
| lncRNA identification and classification (CPC2, CNCI, PLEK) | — | ✓ |
| Single-cell isoform analysis — cell barcode assignment, molecular barcode deduplication, cell-type-specific isoform detection | — | ✓ |
| Custom downstream analysis and publication-ready figures | — | ✓ |
Sample Requirements for PacBio RNA Sequencing
| Category | Requirement | Notes |
| Sample type | Total RNA or polyA⁺-enriched RNA from tissue, cells, or purified RNA | High-integrity RNA essential for full-length cDNA synthesis |
| Minimum input (standard Iso-Seq) | 1–5 µg total RNA; 200 ng–1 µg polyA⁺ RNA | Kinnex protocol requires ≥ 1 µg total RNA or ≥ 100 ng polyA⁺ RNA |
| Minimum input (single-cell Iso-Seq) | 10,000–100,000 cells (10x Genomics capture); RNA input per the 10x protocol | Cell viability ≥ 80% required for 10x capture efficiency |
| RNA quality | RIN ≥ 7 (standard); RIN ≥ 8 (recommended for Kinnex); A260/280 ≥ 1.9 | Assessed by Bioanalyzer or TapeStation; degraded RNA reduces full-length cDNA yield and biases against longer transcripts |
| Coverage recommendation | 5–15 million reads per sample (Kinnex whole-transcriptome); 0.5–1 million reads (standard Iso-Seq whole-transcriptome); 100,000–500,000 (targeted/focused) | Higher coverage enables detection of low-abundance isoforms and rare splicing events |
| Shipping conditions | Overnight on dry ice (tissue/cell pellet); liquid nitrogen (purified RNA) | See our sample submission guidelines for detailed instructions |
Why Choose CD Genomics
Dedicated PacBio Revio capacity with Kinnex-enabled high-throughput Iso-Seq.
CD Genomics operates in-house PacBio Revio systems configured specifically for RNA sequencing applications, with validated Kinnex full-length RNA and standard Iso-Seq protocols. Our dedicated Revio capacity means your Iso-Seq project is sequenced on our own instruments with optimized run parameters — not subcontracted or run on a shared platform with competing prioritization.
End-to-end Iso-Seq expertise — from RNA QC to isoform annotation.
Our team has extensive experience with every stage of the PacBio RNA sequencing workflow, from RNA integrity assessment and size-fractionated library preparation through HiFi CCS processing, isoform clustering, reference alignment, and functional annotation. We understand the technical decisions that affect Iso-Seq data quality — including optimal size selection ranges, cDNA amplification cycles, and bioinformatics parameter choices — and apply this experience to every project.
Multi-sample multiplexing for cost-effective population-scale studies.
With Kinnex chemistry delivering up to 60 million reads per SMRT Cell, we routinely multiplex 4–12 samples per run using barcoded cDNA primers, bringing the per-sample cost of full-length isoform sequencing to levels compatible with multi-condition experimental designs, biological replicates, and population-scale surveys. For single-cell Iso-Seq, our multiplexing strategy accommodates pooled single-cell libraries from multiple samples in a single SMRT Cell run.
Proven track record across diverse applications.
Our PacBio RNA sequencing services have supported published studies across cancer transcriptomics (fusion gene discovery, isoform switches in drug resistance), plant transcriptomics (isoform discovery in polyploid crops), non-model organism transcriptome annotation, and single-cell isoform profiling. Our customer publication library includes peer-reviewed studies where our Iso-Seq data was used for transcript discovery and isoform-level analysis.
Case Study: Full-Length Isoform Concatenation Sequencing Resolves Cancer Transcriptome Complexity in Pediatric Diffuse Midline Glioma
Wijeratne S, Subramanian SL, Warden C, et al. Full-length isoform concatenation sequencing to resolve cancer transcriptome complexity. BMC Genomics. 2024;25:1. (CC BY 4.0)
1. Background
Cancer transcriptomes are characterized by complex isoform dysregulation — alternative splicing, fusion transcripts, and novel isoform expression — that are systematically under-detected by standard short-read RNA sequencing because the reads cannot span complete transcript structures. Wijeratne and colleagues developed PB_FLIC-Seq (PacBio Full-Length Isoform Concatemer Sequencing), a concatenation-based PacBio Iso-Seq workflow designed to increase full-length transcript read throughput while maintaining HiFi accuracy, and applied it to characterize the transcriptome of pediatric diffuse midline glioma (DMG) — an aggressive brain tumor with limited treatment options and poorly characterized isoform-level transcriptomic alterations.
2. Methods
Full-length cDNA was synthesized from total RNA extracted from pediatric DMG tumor tissue and adjacent non-malignant brain tissue. The PB_FLIC-Seq method concatenated full-length cDNA amplicons before SMRTbell library preparation, enabling multiple transcript fragments to be sequenced in a single CCS circle and producing a 3.4-fold increase in full-length read output per SMRT Cell compared to standard Iso-Seq. Sequencing was performed on the PacBio Sequel IIe system. HiFi reads were processed through the standard Iso-Seq Analysis pipeline for isoform clustering and consensus calling. Isoform classification, fusion detection, and differential expression analysis were performed against the reference genome and transcript annotation. SQANTI3 was used for isoform classification and quality assessment.
3. Results
Figure 3. PacBio PB_FLIC-Seq analysis of pediatric DMG transcriptome. (A) PB_FLIC-Seq workflow showing cDNA concatenation, SMRTbell library preparation, and HiFi CCS sequencing. (B) Isoform classification results comparing tumor vs. non-malignant tissue. (C) Validation of SPARC isoform overexpression in tumor. From Wijeratne et al. (2024, BMC Genomics, CC BY 4.0).
Key Findings
- PB_FLIC-Seq achieved a 3.4-fold increase in full-length read output per SMRT Cell compared with standard Iso-Seq from the same amount of input RNA, demonstrating that concatenation-based library preparation substantially improves the cost-effectiveness of PacBio full-length transcript sequencing without compromising per-read HiFi accuracy
- An isoform of the SPARC gene (secreted protein acidic and rich in cysteine) was expressed 11,676-fold higher in tumor tissue compared to adjacent non-malignant brain — the largest single-isoform expression difference identified in the study, and a finding that would be challenging to resolve at isoform resolution from short-read RNA-seq data alone due to the presence of multiple SPARC isoforms with shared exon structures
- Multiple cancer-specific novel isoforms were identified in the tumor transcriptome that were not represented in reference transcript annotations, including isoforms with retained introns, alternative promoter usage, and previously unannotated exon junctions in genes with established roles in glioma biology
- Increased expression of extracellular matrix (ECM) genes was observed at the isoform level in the DMG tumor, with specific ECM isoforms showing differential expression that was not detectable at the gene level — revealing isoform-level regulation of the tumor microenvironment that was invisible to conventional gene-level expression analysis
- Fusion transcript candidates were identified from individual full-length reads spanning across-gene junctions, with the complete fusion transcript structure — including breakpoint coordinates, fused exon boundaries, and preserved open reading frames — determined from single reads without computational prediction
4. Conclusions
This study provides direct experimental validation that PacBio full-length isoform sequencing — particularly the concatenation-based PB_FLIC-Seq approach — delivers unambiguous, clinically informative transcript isoform data from cancer transcriptomes, including the detection of isoform-level expression differences, cancer-specific novel isoforms, and fusion transcripts that are structurally inaccessible to short-read methods. The 11,676-fold SPARC isoform overexpression identifies a specific transcript isoform as a potential DMG biomarker and therapeutic target that would not have been discovered through gene-level expression analysis. These findings validate our PacBio RNA sequencing service design: for cancer transcriptome projects requiring definitive isoform-level information — including fusion transcript validation, isoform switch discovery, and cancer-specific novel isoform characterization — full-length Iso-Seq provides the single-molecule resolution that short-read methods cannot match, and Kinnex-based concatenation methods make this resolution accessible at throughput scales compatible with clinical and translational research.
FAQs
Q: How does PacBio Iso-Seq compare with short-read RNA-seq for isoform-level analysis?
PacBio Iso-Seq and short-read RNA-seq serve complementary roles. Short-read RNA-seq remains the most cost-effective method for gene-level differential expression analysis across large sample cohorts, detecting the majority of expressed genes at high statistical power. However, short-read methods detect fewer than 30% of expressed isoforms in a typical experiment because they cannot span complete splice junctions and depend on computational inference for isoform-level analysis. PacBio Iso-Seq detects and sequences every expressed isoform as a complete, unambiguous full-length read, providing definitive isoform structures and accurate isoform-level quantification. For projects that require isoform-level answers — novel isoform discovery, fusion transcript validation, isoform-specific expression changes, or full-length transcript catalog construction — Iso-Seq provides information that short-read data fundamentally cannot. For large-scale gene-level screening, short-read RNA-seq is more economical.
Q: What is the difference between Kinnex high-throughput Iso-Seq and standard Iso-Seq?
The primary difference is throughput. Kinnex chemistry concatenates multiple cDNA fragments into a single SMRTbell template, enabling up to 60 million full-length reads per SMRT Cell 8M — a 10- to 30-fold increase over standard Iso-Seq (2–5 million reads per cell). Both methods produce HiFi reads at ≥ Q30 accuracy. Kinnex is better suited for whole-transcriptome isoform profiling, multi-sample multiplexed studies, and detection of low-abundance isoforms. Standard Iso-Seq supports longer transcript detection (up to 10+ kb) and is preferred for projects focused on ultra-long transcripts or targeted isoform validation with lower throughput requirements.
Q: Can PacBio Iso-Seq detect fusion transcripts and circular RNAs?
Yes — one of the key strengths of full-length PacBio RNA sequencing is the ability to detect fusion transcripts and circular RNAs (circRNAs) from single reads. Fusion transcripts are identified when a single full-length read contains sequences from two different genes with an unambiguous breakpoint at single-nucleotide resolution, providing definitive evidence of the fusion structure including the complete isoform context. Circular RNAs are detected through the characteristic back-splice junction spanning the 3'-5' circularization point within a single read. The full-length read structure eliminates the false positives associated with short-read fusion detection algorithms and provides complete fusion isoform sequences that cannot be reconstructed from fragmented data.
Q: Can PacBio RNA sequencing be performed on non-model organisms without a reference genome?
Yes — this is one of the most important applications of PacBio Iso-Seq. Because every read is a complete transcript, de novo isoform clustering can be performed directly from the full-length reads without requiring a reference genome or transcript annotation. Our bioinformatics pipeline clusters full-length reads by sequence similarity, generates consensus isoform sequences, and performs functional annotation through homology search (BLAST against NCBI NR, UniProt), coding sequence prediction, and GO/KEGG pathway assignment. This makes PacBio RNA sequencing directly applicable to any organism — including non-model species, endangered species, and organisms with unsequenced or poorly assembled genomes — providing a comprehensive full-length transcript catalog without requiring pre-existing genomic resources.
Q: Do you offer single-cell PacBio RNA sequencing?
Yes — our Single-Cell Full-Length Transcriptome service combines 10x Genomics single-cell capture with PacBio Revio full-length sequencing to deliver isoform-level transcriptome data at single-cell resolution. Cell barcodes and molecular barcodes from the 10x system are preserved throughout library preparation and sequencing, enabling isoform discovery, alternative splicing analysis, and fusion detection within individual cell populations while simultaneously capturing cellular heterogeneity. This service is available for both the Kinnex high-throughput Iso-Seq workflow (for whole-transcriptome single-cell isoform profiling) and standard Iso-Seq (for longer-read single-cell applications). Minimum input is 10,000–100,000 cells with viability ≥ 80%.
Sample Deliverables
1. Full-length isoform sequences (FASTA) with isoform classification codes (full-length splice match, novel in catalog, novel not in catalog, fusion, etc.) as defined by SQANTI3
2. Isoform-level expression quantification table (read counts, TPM, FPKM) for all detected isoforms across all samples, annotated with gene symbols, genomic coordinates, and isoform structural features
3. Alternative splicing event report with categorized events (exon skipping, intron retention, alternative 5'/3' splice sites, mutually exclusive exons) and associated isoform abundance changes
4. Fusion transcript report with breakpoint coordinates, fusion junction sequences, and predicted functional consequences (preserved ORF, domain architecture) for each detected fusion candidate
5. Optional: integrated report combining isoform-level expression, splicing analysis, and functional annotation with publication-ready figures (transcript structure diagrams, isoform expression heatmaps, splicing event classification plots) and single-cell isoform UMAP visualizations for sc-Iso-Seq projects
References
- Full-length isoform concatenation sequencing to resolve cancer transcriptome complexity. Wijeratne S, Subramanian SL, Warden C, et al. BMC Genomics. 2024;25:1.
- High-throughput single-molecule long-read RNA sequencing analysis of tissue-specific genes and isoforms in lettuce. Shi Z, Xiang Q, Zhao Y, et al. Communications Biology. 2024;7:920.
- Full-length transcriptomes of 25 grassland plant species. Dong X, et al. Scientific Data. 2025. (CC BY 4.0)
For research use only. Not for use in diagnostic procedures.