What a month for Iso-Seq and Kinnex.
The June 2025 edition of Powered by PacBio puts the spotlight on a suite of standout studies that tap into the full potential of HiFi long-read RNA sequencing. This month’s roundup showcases just how far Iso-Seq method and Kinnex kits have come, from driving high-confidence isoform quantification to redefining transcriptome annotation in complex tissues and disease models. Across human genomics, proteogenomics, developmental biology, and translational research, researchers are using long-read sequencing to go deeper, see clearer, and ask smarter questions.
Each study featured here pairs long-read sequencing with innovative tools or large-scale datasets, demonstrating what’s possible when you move past the limitations of short reads. Whether it’s characterizing allele-specific splicing with precision, profiling single oocytes, or finding novel transcripts in druggable genes, the story is the same: resolution matters – and this month, Iso-Seq and Kinnex delivered.
Jump to topic:
RNA research | Human genomics research | Human translational research | Proteogenomics
RNA research
In this preprint, researchers from Switzerland, U VA, Germany and PacBio conclude that “PacBio Kinnex is a reliable method for transcriptome profiling”.
Highlights:
- The study features “one of the largest PB lrRNA-seq datasets” and is “sample-matched with Illumina short-read RNA-seq”, allowing for “rigorous benchmarking to characterize quantification accuracy between platforms”
- “PacBio and Illumina quantifications were strongly concordant” and “Pearson correlations exceeding 0.9 at the gene level and approaching 0.9 at the transcript level … These results indicate that Kinnex quantifications closely matched Illumina for transcripts that could be considered as reliably quantified by both platforms”, with “a small to moderate length bias for the Kinnex data, with shorter transcripts showing a somewhat lower correlation”
- “Illumina exhibited substantially higher inferential variability compared to Kinnex”, with “greater replicate-to-replicate fluctuations of estimated transcript abundances from the short-reads …, whereas Kinnex demonstrated consistent quantification for the same transcripts”.
- “Instability caused by inferential variability in Illumina quantifications impacted downstream analyses”. “Illumina’s short-read limitations led to unreliable quantifications for complex genes, manifested either as transcript flips across replicates or transcript division of expression among multiple similar transcripts”
Conclusion:
Kinnex sets a new standard for transcriptomics, offering accurate quantification, reliable discovery, and a scalable, cost-effective workflow for isoform biology. Available on both the Vega and Revio systems, it delivers high-quality transcriptome analysis without the need for multiple platforms, making it easy to combine measurement and discovery in one powerful assay.
Human genomics research
SNP calling, haplotype phasing and allele-specific analysis with long RNA-seq reads
In this preprint, researchers from Dana-Farber and Harvard create a new bfx tool and complete analysis of 202 HPRC Kinnex datasets.
Highlights:
- The study unveils a new longcallR tool for “joint SNP calling, haplotype phasing, and allele-specific analysis” of long-read RNA-seq data.
- Benchmarking on GIAB samples, researchers found that PacBio Kinnex (“MAS”) has significantly higher SNP calling performance than ONT (“Nanopore SNP calling is more challenging due to the higher sequencing error rate”), and ~3x more true positives (TP).
- When applied to 202 HPRC Kinnex datasets, researchers “identified 88 significant allele-specific splicing events per sample on average. 46% of them involved unannotated junctions.”
Conclusion:
Once again, HiFi outperforms ONT, detecting three times more true positive variants and reaffirming its strength for precise allele-specific splicing analysis. This level of resolution helps researchers more confidently link genetic variants to gene function and disease. It’s also a standout showcase for one of the largest Kinnex datasets to date, recently released by the HPRC.
Human translational research
In this preprint, researchers from China produce a study“showcasing the complexity and diversity of RNA isoforms in oocytes”.
Highlights:
- Researchers noted that “Due to the scarcity of human oocyte samples and the short-read limitation of NGS, the overall profiling of oocyte-specific transcripts, as well as the comparative analyses between human and mouse oocytes has likely been underestimated.”
- They found that “the quality of transcripts obtained [by Iso-Seq] was distinctly improved over NGS and GENCODE assemblies”, noting, “Considerable isoform diversity was identified …, with about 40% of which characterized as novel transcripts not annotated according to the GENCODE reference”.
- In addition, “over 25% of NNC [novel-not-in-catalog] isoforms were derived from transposable elements (TEs)” even though “Previous studies on the contribution of TE sequences to the transcriptome have been challenging due to the repetitive nature of TEs and short read lengths of NGS technique.”
- Authors determined “these results demonstrate the high throughput, sensitivity, and integrity of the characterization and quantification of full-length isoform sequencing from single oocyte.”
Conclusion:
Across zebrafish, mouse, and human studies, the Iso-Seq method continues to reveal layers of biology that short reads miss – often nearly half of the isoform landscape. Without a complete view of transcript diversity, understanding development remains incomplete. Iso-Seq is essential for capturing gene isoform expression with the depth and accuracy complex biology demands. PacBio’s Kinnex kits on the Revio system enable high-throughput, full-length RNA sequencing, even from low-input samples like single oocytes, while the Vega platform offers a cost-effective option for labs focused on isoform discovery.
Proteogenomics
TX2P: A proteogenomic tool for comprehensive transcript analysis
In this preprint, researchers from UCL, and U Cambridge created a tool that “integrates transcriptomic and proteomic data to link RNA discoveries with protein function”.
Highlights:
- The new tool “predicts open reading frames (ORFs) from transcript sequences and integrates these predictions with mass spectrometry (MS) data”.
- It “analysed transcript structures derived from long-read sequencing of human brain samples and leveraged brain-relevant mass spectrometry datasets to find evidence for the translation of novel open reading frames (ORFs) in disease-relevant genes.”
- The researchers focused on “556 unique genes causally implicated in early onset and syndromic epilepsy”. Iso-Seq detected a total of 4,341 unique novel transcripts, predicting 1,978 unique ORFs, from which “mass spectrometry datasets identified 514 peptides unique to the predicted amino acid sequences derived from these ORFs, and which did not match those in UniProt”. Mass spec provided “supporting evidence for the translation of 745 out of the 4,341 transcripts (17.2%) with 356 unique ORFs (18.0%)”.
- Remarkably,“All genes included in this study are causally implicated in human disease, and their current annotations serve as the foundation for diagnostic testing. Misannotation or incomplete annotation could therefore overlook clinically relevant isoforms or misannotate variants that affect splicing. Furthermore, many of these genes belong to the druggable genome; KCNQ2 & STXBP1, are currently included in gene therapy trials listed on ClinicalTrials.gov. The identification of abundant, previously unannotated isoforms in such genes underscores the need to refine transcript annotations to improve both diagnostic and therapeutic strategies.”
Conclusion:
Iso-Seq is a foundational tool for proteomics, providing full-length transcripts that short reads can’t resolve, making isoform identification far more accurate. Determining which isoforms are expressed and translated in specific tissues has historically been a challenge, but Iso-Seq offers the high-resolution annotations needed to clarify the picture. Paired with tools like TX2P, which integrates long-read transcript data with proteomics, researchers can now validate findings at scale, positioning Iso-Seq as a key driver in decoding the human proteome and advancing precision medicine.
Ready to make discoveries of your own?
Together, these publications show why Iso-Seq and Kinnex have become the go-to tools for researchers looking to cut through the noise of incomplete annotations and unreliable quantification. This month’s highlights include some of the largest and most rigorous benchmarking efforts to date, spanning over 200 HPRC samples, proteogenomic integration in disease-relevant genes, and direct comparisons to short-read platforms that reveal the advantages of HiFi sequencing at every level.
It’s clear: high-throughput, full-length transcript data isn’t just a nice-to-have, it’s essential for tackling the questions that matter most in biology and medicine. And with scalable, easy-to-run Kinnex kits now available on both Revio and Vega, more labs than ever can bring this level of clarity in-house.
Let’s keep this momentum going.
Ready to see what Iso-Seq and Kinnex can uncover in your own research? We’ve got everything you need to get started.