Rui LIU, Product Manager, Novogene AMEA

10 June 2024

Traditional bulk RNA-seq often faces challenges in resolving the complete isoform structure due to the complexities of alternative splicing. PacBio’s Iso-Seq method, which leverages long-read sequencing and HiFi data (base accuracy>99.9%), now enables the direct capture of full-length transcripts from the 5’ end to the 3’ poly A tail. This breakthrough enables more accurate isoform discoveries and intricate structural analyses.

The new Kinnex Full-Length RNA approach enhances the original Iso-Seq method by optimizing the balance between cost and sequencing depth, significantly improving research efficiency. Utilizing the MAS-Seq method, the kit concatenates 8 cDNA molecules into longer fragments¹, leading to an impressive 8-fold increase in throughput. Coupled with the PacBio Revio system, the data output per Cell exhibits a remarkable 16-fold increase compared to the regular Iso-Seq method on the Sequel II system (Figure 1). This substantial boost in throughput not only makes transcriptome data more accessible but also reduces research costs.

The Kinnex Full-Length RNA approach demonstrates remarkable sensitivity in transcripts capture. As illustrated by the saturation curve, approximately 80% of known isoforms can be detected with 10Mb HiFi reads (Figure 2), indicating that this amount is sufficient for detecting the majority of known isoforms. Increasing the sequencing depth further allows for the detection of more rare isoforms, providing even more comprehensive transcriptome coverage.

Figure 1. Comparison of Yield per Cell for Universal Human Reference RNA (UHRR) Sample: Regular Iso-Seq Library on Sequel II System vs. Kinnex Library on Revio System

Figure 2. Saturation analysis of known isoforms for different Kinnex samples.

1.Yield performance and data quality

An 8-plex human library sequenced in a single REVIO SMRT cell, yield a total of 39Mb HiFi reads, with each sample producing approximately 5M HiFi reads, illustrating exceptional uniformity across the dataset (Table 1). The data exhibited superior quality, with average read quality indices surpassing Q35 (Figure 3).

Table 1. Data output and read length status.

Figure 3. Read quality distribution.

2.Transcript structural analysis

From a single human tissue sample, we obtained a remarkable total of 4.96Mb full-length non-chimeric (FLNC) sequences. These sequences were then utilized for transcript structural analysis. Using SQANTI3², we identified an impressive total of 106,012 unique full-length transcripts. Notably, 36% of the transcripts categorized in this study are novel transcripts (NIC+NNC) (Figure 4).

Figure 4. Transcript classification. Note: FSM: Matches all SJ perfectly ISM: Matches the reference SJs partially NiC: Novel isoform with a new combination of known splice sites NNC: Novel isoform with at least a new splicing site.

Why choose Novogene? 1.Increased Data Delivery

Novogene introduces a new delivery method tailored to the diverse needs of researchers. We now offer two packages—5M HiFi reads per sample and 10M HiFi reads per sample—and will deliver the HiFi data directly, designed to meet varied research requirements.

Table 2. Kinnex full-length RNA applications use cases.

2.Enhanced Analysis Workflow

We utilize SQANTI3 software for precise isoform classification, complemented by IsoQuant3.3³ for in-depth analysis of gene and transcript expression levels. The combination of these two software packages enables researchers to uncover valuable insights from their transcriptome data.

Highlights of SQANTI3 software:

• Specifically designed for long-read RNA sequencing data to characterize transcript structures.
• Incorporates multiple indicators to characterize the structural properties of transcripts, including transcription start and end sites, splicing junctions, and other structural features, allowing for the filtering of potential false positives and other errors.
• Includes a Rescue module which prioritizes high-confidence annotations, preserving crucial gene and transcript information during analysis. It ensures retention of well-annotated transcripts by comparing novel transcripts against reference databases.

References

1. Al’Khafaji AM, Smith JT, Garimella KV, Babadi M, Popic V, Sade-Feldman M, et al. High-throughput RNA isoform sequencing using programmed cDNA concatenation. Nat Biotechnol. 2023. doi: 10.1038/s41587-023-01815-7
2. Pardo-Palacios FJ, Arzalluz-Luque A, Kondratova L, Salguero P, Mestre-Tomás J, Amorín R, et al. SQANTI3: curation of long-read transcriptomes for accurate identification of known and novel isoforms. Nat Methods. 2024 May 1;21(5):793-797. doi: 10.1038/s41592-024-02229-2.
3. Prjibelski A, Pfeil R, Mikheenko A, Webber J, Tomescu A, Tilgner H. IsoQuant: A tool for isoform assignment and quantification for long and barcoded reads [Internet]. University of Helsinki and Saint Petersburg State University; 2022-2024. Available from: https://github.com/ablab/IsoQuant.