Kinnex full length RNA-seq
Kinnex Full-Length RNA-Seq Service — Scalable Isoform Sequencing with PacBio HiFi
CD Genomics delivers Kinnex full-length RNA sequencing, PacBio's next-generation upgrade to the Iso-Seq method. Using MAS-Seq (Multiplexed Array Sequencing) chemistry to concatenate 8 cDNA molecules into single ~10–15 kb arrays, Kinnex achieves an 8-fold throughput increase over standard Iso-Seq and a 16-fold increase vs. Sequel II — yielding up to 40 million full-length HiFi reads per Revio SMRT Cell, at >99.9% base accuracy.
Why researchers choose our Kinnex full-length RNA-seq service
- Up to 40M full-length reads per Revio SMRT Cell — 16× vs. standard Iso-Seq on Sequel II
- Full-length isoforms up to 10 kb, no assembly required
- Bulk transcriptome and single-cell (10x Chromium) modes available
- 12-plex sample multiplexing per SMRT Cell for cost-effective multi-sample studies
What Is Kinnex Full-Length RNA Sequencing
Kinnex is PacBio's commercial implementation of MAS-Seq (Multiplexed Array Sequencing), announced in October 2023. It builds directly on the Iso-Seq method — using the same Iso-Seq Express 2.0 kit for full-length cDNA generation — and adds a downstream concatenation step that joins 8 cDNA molecules end-to-end into a single ~10–15 kb array. Each array is sequenced as one PacBio HiFi read; after sequencing, a segmentation algorithm splits the array back into its 8 constituent full-length transcripts.
The result is a dramatic throughput gain with no loss of accuracy or transcript integrity. Where the original Iso-Seq method on a Sequel II system produces approximately 2–3 million full-length reads per SMRT Cell 8M, Kinnex on the PacBio Revio system delivers up to 40 million full-length reads per 25M SMRT Cell — a 16-fold increase. This shifts the cost and feasibility calculus for transcriptome-scale isoform studies: sample multiplexing becomes practical, deeper per-gene isoform coverage becomes affordable, and rare isoform discovery that was previously impractical becomes routine.
Kinnex preserves everything that makes the Iso-Seq method scientifically valuable: full-length reads from the 5' cap to the 3' poly(A) tail, no assembly required, and HiFi base accuracy exceeding 99.9%. It adds throughput, multiplexing, and cost efficiency — making it the preferred mode for all new full-length RNA sequencing projects on PacBio instruments. Unlike standard short-read RNA-seq, Kinnex directly sequences full-length transcript structures without computational reconstruction.
Kinnex vs. Iso-Seq vs. Short-Read RNA-Seq
Kinnex combines the accuracy and full-transcript coverage of Iso-Seq with throughput that approaches short-read scales — making it the most complete solution for isoform-level transcriptomics.
| Feature | Short-Read RNA-Seq | Iso-Seq (Sequel II) | Kinnex (PacBio Revio) |
| Read length | 150 bp paired-end | Full-length (up to 10 kb) | Full-length (up to 10 kb) |
| Reads per SMRT Cell / Flow Cell | 400M–600M (Illumina NovaSeq) | ~2–3M full-length reads | Up to 40M full-length reads |
| Throughput vs. Iso-Seq on Sequel II | N/A | 1× (baseline) | 16× increase |
| Base accuracy | >99.9% (Illumina) | >99.9% HiFi | >99.9% HiFi |
| Full-length isoform detection | ✗ — requires computational assembly | ✓ — direct full-length reads | ✓ — direct full-length reads |
| Alternative splicing resolution | Limited — junction-based inference | Complete — all AS events per transcript | Complete — all AS events per transcript |
| Novel isoform discovery | Difficult — assembly artifacts | ✓ | ✓ — at higher depth |
| Sample multiplexing per run | Up to 96+ samples | Up to 12 barcoded samples | Up to 12 barcoded samples |
| Single-cell isoform mode | Short-read scRNA-seq (no isoform) | MAS-Seq single-cell (limited) | Kinnex single-cell RNA kit (10x Chromium) |
| Cost per full-length isoform read | N/A (no full-length equivalent) | High | ~16× lower than Iso-Seq on Sequel II |
Service Modes
CD Genomics offers three Kinnex RNA sequencing modes, each optimized for distinct experimental designs and biological questions.
Mode 1: Kinnex Bulk Full-Length RNA
- Input: total RNA or poly(A)-selected RNA from bulk tissue or cell lysate
- Iso-Seq Express 2.0 cDNA → MAS-Seq concatenation → PacBio Revio HiFi sequencing
- Up to 12 barcoded samples per SMRT Cell
- Applications: isoform atlas construction, alternative splicing profiling, novel transcript discovery, APA analysis, NMD substrate identification
- Recommended for: transcriptome annotation, comparative isoform studies, cancer splice variant discovery
Mode 2: Kinnex Single-Cell RNA
- Input: 10x Genomics Chromium 3' or 5' full-length single-cell cDNA library
- Kinnex single-cell RNA kit concatenates and sequences cDNA for full-length isoform resolution at cell level
- Deconvolute cell-type-specific isoform usage invisible to short-read single-cell RNA sequencing
- Applications: single-cell splice isoform heterogeneity, fusion gene detection, allele-specific expression per cell type
- Recommended for: tumor microenvironment studies, neuronal isoform diversity, developmental cell atlases
Mode 3: Kinnex Targeted Enrichment
- Combines Kinnex full-length RNA library with IDT xGen Hybridization target enrichment
- Enriches for rare isoforms of selected gene panels — oncogenes, splicing factors, neurodegenerative disease genes
- Enables deep isoform sequencing of low-abundance transcripts without whole-transcriptome depth requirements
- Applications: clinical mutation detection in RNA, rare isoform characterization, allele-specific splicing in targeted gene sets
- Compatible with long amplicon analysis workflows for amplicon-based enrichment
Service Workflow
End-to-end from RNA to publication-ready isoform catalog — all modes follow a validated 5-step pipeline.
Step 1 — Sample QC & RNA Input Verification: Total RNA samples undergo fluorometric quantification (Qubit) and quality assessment (Bioanalyzer or TapeStation, RIN ≥ 7 recommended). Poly(A) enrichment or ribosomal RNA depletion is performed as appropriate for the sample type. For single-cell mode, input is 10x Genomics full-length cDNA library; input QC parameters differ from bulk RNA.
Step 2 — Full-Length cDNA Synthesis (Iso-Seq Express 2.0): Full-length cDNA is generated using the Iso-Seq Express 2.0 kit, which employs template-switching chemistry to capture transcripts from the 5' cap to the 3' poly(A) tail in a single reverse transcription reaction. Barcoded cDNA primers enable up to 12-plex multiplexing at this stage. Long-range PCR amplification enriches full-length cDNA molecules.
Step 3 — MAS-Seq Concatenation & PacBio Revio HiFi Sequencing: 8 full-length cDNA molecules are joined end-to-end using Kinnex array adapters, forming ~10–15 kb concatemers. SMRTbell library adaptors are ligated and libraries are sequenced on PacBio Revio using CCS/HiFi mode — yielding up to 40 million array reads per 25M SMRT Cell, each with >99.9% base accuracy.
Step 4 — Segmentation & Isoform Bioinformatics: CCS reads are segmented using the SMRT Link Read Segmentation module, splitting each array into 8 individual full-length reads. Segmented reads are processed through the Iso-Seq pipeline (lima → refine → cluster) and classified by SQANTI3 for isoform annotation. Alternative splicing events, novel transcripts, and APA sites are cataloged.
Step 5 — Results Delivery: You receive raw CCS and segmented HiFi FASTQ files, per-sample QC metrics, a classified isoform FASTA, SQANTI3 classification table, splice junction annotation GFF, alternative splicing event summary, differential isoform expression results (if multi-condition), and a consultation session to discuss findings.
Key Applications
Kinnex full-length RNA-seq resolves biological questions that short-read RNA-seq fundamentally cannot answer — at a scale now competitive with short-read workflows.
Alternative Splicing Profiling & Isoform Quantification
Every Kinnex read is a complete transcript from 5' cap to 3' poly(A) tail — no splice junction inference, no assembly ambiguity. Exon skipping, alternative 5'/3' splice sites, intron retention, and alternative polyadenylation are all captured in their full genomic context. Differential isoform analysis between conditions identifies disease-associated splicing switches with confidence that short reads cannot achieve.
Novel Transcript & Gene Discovery
Kinnex identifies full-length transcripts not present in existing annotations — novel lncRNAs, unannotated protein-coding isoforms, readthrough transcripts, and antisense RNAs — directly from reads without assembly. Particularly valuable for non-model organisms and tissue types with limited transcriptome annotation. For comparison with nanopore-based full-length approaches, see our Nanopore Full-Length Transcripts Sequencing service.
Single-Cell Isoform Landscape
Combined with 10x Chromium cDNA, Kinnex single-cell RNA mode resolves which splice isoforms are expressed in which cell types — linking isoform identity to cell identity for the first time at scale. This reveals cell-type-specific splicing programs, fusion gene expression in tumor subclones, and allele-specific isoform biases invisible to standard short-read scRNA-seq.
Cancer Fusion Gene & Splice Variant Detection
Full-length reads spanning entire fusion transcript structures identify breakpoint sequences and expressed fusion isoforms in a single read — more accurately than short-read-based fusion callers that reconstruct chimeric sequences from junction reads. Splice variants in oncogenes, tumor suppressors, and RNA-binding proteins are catalogued at full structural resolution.
Genome Annotation & Reference Transcriptome Construction
Kinnex provides the depth needed to support high-confidence genome annotation — essential for non-model organisms, polyploid plant genomes, and species with highly heterozygous transcriptomes. Full-length reads define precise exon-intron boundaries, UTR extents, and poly(A) site positions without assembly ambiguity, complementing PacBio SMRT sequencing-based genome projects.
Sample Requirements
RNA quality is the primary determinant of Kinnex library quality. Please review requirements carefully before sample preparation and collection. Contact us if your sample type is non-standard.
| Sample Type | Mode | Recommended Input | Minimum Input | Notes |
| Total RNA | Bulk Kinnex | 500 ng – 1 µg | 100 ng | RIN ≥ 7 recommended; RIN ≥ 8 preferred for long-read library |
| Poly(A)-enriched RNA | Bulk Kinnex | 100–500 ng | 50 ng | Pre-selected; specify enrichment method used |
| 10x Chromium full-length cDNA | Kinnex single-cell | ≥200 ng amplified cDNA | 100 ng | From 3' or 5' Chromium workflow; Agilent TapeStation QC required |
| Pre-amplified cDNA (Iso-Seq Express 2.0) | Bulk or targeted | ≥100 ng | 50 ng | Contact team for protocol compatibility check before submission |
| FFPE-extracted RNA | Bulk Kinnex | ≥1 µg | 500 ng | DV200 ≥ 30% required; highly degraded FFPE may produce shorter transcripts |
- RNA quantification: Use Qubit fluorometric method (not NanoDrop alone). OD A260/A280 ≥ 1.8 and A260/230 ≥ 1.8 required. Ship dissolved in RNase-free water or 10 mM Tris-HCl pH 8.0 on dry ice.
- Multiplexing: Up to 12 samples per SMRT Cell using barcoded Iso-Seq Express cDNA primers. Multiplexing reduces per-sample cost but proportionally reduces per-sample read depth. We advise on optimal multiplexing strategy during consultation.
- Organisms: Compatible with eukaryotic RNA from any organism. For prokaryotic full-length RNA-seq, contact our scientific team for protocol adaptation.
Bioinformatics Analysis & Deliverables
Our Kinnex bioinformatics pipeline follows the PacBio SMRT Link–validated Iso-Seq analysis workflow with additional steps for MAS-Seq segmentation, extended isoform classification, and differential analysis. All outputs are formatted for direct use in downstream analysis and publication.
- Raw Data & QC: Raw HiFi CCS FASTQ + segmented read FASTQ; per-sample QC metrics including CCS pass rate, subread coverage, ZMW yield, and post-segmentation read count.
- Full-Length Isoform Catalog: FASTA of all non-redundant full-length isoforms; GFF3 annotation of transcript structures against reference genome; SQANTI3 classification (FSM, ISM, NIC, NNC, novel gene categories).
- Alternative Splicing Event Analysis: Per-transcript annotation of exon skipping, alternative 5'/3' splice sites, intron retention, and alternative polyadenylation events; differential event analysis across conditions using DEXSeq or SUPPA2.
- Isoform Quantification: Normalized expression levels (TPM) per isoform per sample; differential isoform usage analysis (edgeR/DESeq2 adapted for isoform-level counts).
- Single-Cell Outputs (Mode 2): Cell-barcode demultiplexed isoform assignment; UMAP/t-SNE projected isoform diversity scores; cell-type-specific isoform usage heatmaps.
Integration with complementary data types — short-read RNA-seq, ATAC-seq, genome assembly — is available as a custom analysis option. For projects comparing Kinnex results to existing short-read transcriptomes, we provide automated concordance reporting between datasets.
Case Study: Full-Length RNA Sequencing Reveals PTBP1-Dependent Splicing in Leukemia
Customer Publication Highlight
The Splicing Factor PTBP1 Interacts with RUNX1 and Is Required for Leukemia Cell Survival
Journal: Leukemia | Impact Factor: 11.4 | Published: November 10, 2025
Service Used: Nanopore Long-Read RNA Sequencing (full-length isoform profiling)
Background
RUNX1 (Runt-related Transcription Factor 1) is among the most frequently mutated genes in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Understanding how RUNX1 regulates gene expression at the RNA processing level — including alternative splicing — requires full-length transcript data that short-read RNA-seq cannot provide. Dhir, Ethell, and colleagues at the University of Nebraska Medical Center used long-read RNA sequencing alongside proteomics and chromatin profiling to identify novel RUNX1 interacting partners and decode their role in RNA splicing regulation in leukemia cells.
Results
- Proteomics identified PTBP1 (Polypyrimidine Tract Binding Protein 1) as a novel RUNX1-interacting partner in both AML and ALL cells, with the interaction dependent on HDAC1 — revealing an unexpected non-histone role for HDAC1 in the RUNX1 regulatory complex. Chromatin profiling showed PTBP1 co-occupies thousands of active gene promoters together with RUNX1, establishing a genome-wide co-regulatory role.
- Full-length RNA sequencing identified specific alternative splicing events in RUNX1-regulated genes that are disrupted upon PTBP1 knockdown — events that short-read RNA-seq could not resolve at the isoform level. PTBP1 knockdown reduced leukemia cell survival across multiple AML and ALL model lines, confirming its functional requirement in leukemia maintenance.
Long-read RNA sequencing identified PTBP1-dependent isoform regulation in leukemia cells — full-length isoform coverage revealed splicing events invisible to short-read approaches. (Dhir A et al., Leukemia, 2025)
Conclusion
This study demonstrates how full-length RNA sequencing is essential for decoding the isoform-level consequences of RNA-binding protein perturbation in cancer cells. The PTBP1–RUNX1 interaction and its splicing-regulatory consequences would have remained invisible without long-read transcript data spanning complete exon-intron structures. Kinnex full-length RNA-seq extends this capability further — providing the same full-length isoform resolution at 16× higher throughput, enabling multi-sample, multi-condition splicing studies in cancer biology at a scale previously impractical with standard Iso-Seq.
Demo Results
1. Throughput comparison: Kinnex on PacBio Revio (40M full-length reads / 25M SMRT Cell) vs. standard Iso-Seq on Sequel II (~2.5M reads / 8M SMRT Cell)
2. SQANTI3 isoform classification from Kinnex full-length RNA-seq of Universal Human Reference RNA (UHRR)
References
- Al'Khafaji AM, Smith JT, Garimella KV, et al. High-throughput RNA isoform sequencing using programmed cDNA concatenation. Nat Biotechnol. 2023;42:582–586. https://doi.org/10.1038/s41587-023-01815-7
- Dhir A, Ethell A, Watkins R, et al. The splicing factor PTBP1 interacts with RUNX1 and is required for leukemia cell survival. Leukemia. 2025;40:138–151. https://doi.org/10.1038/s41375-025-02799-w
- Pardo-Palacios FJ, Wang D, Reese F, et al. Systematic assessment of long-read RNA-seq methods for transcript identification and quantification. Nat Methods. 2024;21:1349–1363. https://doi.org/10.1038/s41592-024-02298-3
FAQs
Q: What is the relationship between Kinnex and Iso-Seq — are they the same service?
Kinnex and Iso-Seq are closely related but distinct. Iso-Seq refers to PacBio's full-length RNA sequencing method using SMRT sequencing — the underlying biology (template-switching cDNA generation, full-length read production) is the same. Kinnex is PacBio's commercial kit that adds MAS-Seq array concatenation on top of the Iso-Seq Express 2.0 cDNA workflow, multiplying throughput 8-fold per SMRT Cell. When run on Revio versus Sequel II, the combined throughput gain reaches 16-fold. CD Genomics offers both services: our existing Iso-Seq service for projects with legacy Sequel II data requirements, and Kinnex for all new projects where throughput and cost efficiency are priorities.
Q: Can Kinnex be used for non-polyadenylated RNA or prokaryotic transcripts?
The standard Kinnex full-length RNA kit uses oligo(dT) priming and is optimized for eukaryotic polyadenylated RNA. Non-poly(A) transcripts, bacterial mRNAs, and rRNA-depleted total RNA from prokaryotes require protocol adaptation. CD Genomics can advise on modified cDNA generation strategies (random hexamer priming, Cappable-seq for primary transcript capture) compatible with downstream Kinnex array preparation — contact our scientific team to discuss your specific application.
Q: How does Kinnex single-cell mode compare to short-read 10x Genomics scRNA-seq?
Both methods use the same 10x Chromium cDNA input, so cell barcode assignment and UMI deduplication are handled equivalently. The critical difference is read length: short-read 10x scRNA-seq generates 150 bp reads that provide gene-level expression counts but cannot resolve splice isoforms. Kinnex single-cell mode sequences full-length cDNA molecules — identifying which specific isoform of each gene is expressed in each cell. This reveals cell-type-specific splicing programs, fusion transcripts in subclonal tumor populations, and allele-specific expression patterns that short-read scRNA-seq cannot detect.
Q: What sequencing depth is needed for robust isoform discovery?
For transcriptome-wide isoform profiling in well-annotated organisms (human, mouse), 5–10 million full-length reads per sample provides robust detection of medium-to-high abundance isoforms. For deep rare isoform discovery, novel gene identification in non-model organisms, or single-cell applications, 15–40 million reads per sample or condition is recommended. With Kinnex on Revio, a single 25M SMRT Cell can generate sufficient reads for up to 12 multiplexed samples at standard depth — making multi-sample studies economically practical for the first time.
Q: Does Kinnex work with FFPE samples?
Yes, but with important caveats. FFPE RNA is chemically degraded and fragmented, which reduces full-length transcript capture efficiency and biases recovery toward shorter transcripts. We require DV200 ≥ 30% as a minimum quality threshold for FFPE RNA Kinnex projects, and we strongly recommend fresh-frozen tissue wherever possible. For clinical archived FFPE samples, contact our team to discuss protocol optimization and expected output characteristics before submitting samples.
For research purposes only, not intended for personal diagnosis, clinical testing, or health assessment