Vector Transcript Splicing & Expression
Vector Transcript Splicing & Expression
Built on high-depth strand-specific Illumina short-read RNA-seq, this service delivers single-base-resolution interpretation of splice junctions and independent quantification of expression levels (TPM) for vector-related transcripts. It serves as paired orthogonal verification of full-length transcript conclusions from PacBio Iso-Seq long-read sequencing.
1. Background
Analogous to the situation at the DNA level, transcript-level analysis benefits from a complementary relationship between long reads (which reveal structure) and short reads (which provide depth and per-base accuracy). Long-read Iso-Seq produces complete isoform structure and fusion junctions from 5′ to polyA in a single read, excelling at full-picture views; short-read RNA-seq, at single-base resolution, precisely interprets splice junctions, sensitively detects low-abundance aberrant transcripts, and provides independent quantification (TPM) at high depth. Used together, structure and quantification cross-corroborate, and the evidence is more robust.
Expression level (e.g., TPM) is a common metric for evaluating transgene expression level and consistency. Providing independent TPM measurements from a mechanistically different high-depth platform and cross-corroborating them with full-length structural conclusions substantially strengthens the credibility of transcript-level evidence. When conclusions are to be incorporated into a regulatory submission data package or used to inform critical decisions, paired long- and short-read verification carries greater persuasiveness.
Figure 1. Schematic of the complementary relationship between long-read Iso-Seq and short-read RNA-seq.
From a regulatory standpoint, the relevant guidance applies equally: ICH Q6B requires quality specifications and acceptance criteria including identity; the FDA guidance "Considerations for the Development of CAR T Cell Products" (Final, 2024) requires evaluation of identity, potency, and safety; and China NMPA/CDE's "Technical Guideline for Pharmaceutical Research and Evaluation of Immune Cell Therapy Products (Trial)" (2022) sets explicit requirements for the pharmaceutical research and safety of CAR-T and other products. This service serves as orthogonal confirmation of transcript-level conclusions and is used in conjunction with the Full-Length Vector Transcript Analysis.
2. Technical Principle
This service is built on strand-specific Illumina short-read RNA-seq, uses parallel samples to the Full-Length Vector Transcript Analysis, and produces results that are compared item by item. Strand-specific libraries discriminate transcriptional direction, supporting interpretation of vector transcription and splicing events. High-depth sequencing ensures stable detection and quantification of low-abundance events and provides single-base resolution at splice sites. The core workflow is as follows:
(1) Sample receipt and QC
Receive cell pellets or extracted RNA; assess concentration, purity, and integrity. Submission within the same batch as the Full-Length Vector Transcript Analysis is recommended to facilitate cross-validation.
(2) Strand-specific library preparation
Strand-specific RNA-seq libraries are prepared to discriminate transcriptional direction and improve the directional reliability of splice-site interpretation.
(3) High-depth short-read sequencing
Sequencing is performed on the Illumina platform to high depth, ensuring sensitive detection and stable quantification of low-abundance transcripts and splice sites.
(4) Bioinformatic analysis and consistency cross-validation
Alignment is performed against the customer's vector sequence and the human reference genome as custom references. Splice junctions are called, expression is quantified (TPM), and results are cross-compared item by item against Iso-Seq full-length findings; a cross-validation report is then issued.
Figure 2. Workflow for strand-specific RNA-seq and Iso-Seq consistency cross-validation.
3. Technical Features and Advantages
(1) Single-base resolution at splice junctions
High-depth short reads provide single-base resolution interpretation of junction positions — a clear strength for confirming splice-event locations and exon boundaries.
(2) Sensitive detection of low-abundance events
High depth combined with strand-specific library construction enables stable detection and quantification of low-abundance aberrant transcripts and rare splicing events.
(3) Independent expression quantification
Provides TPM measurements independently on a platform of a different mechanistic principle from long reads, offering corroborating evidence on expression integrity and consistency.
(4) Orthogonal cross-validation with the long-read platform
Using parallel samples, junction/PSI/isoform consistency is compared item by item against the Full-Length Vector Transcript Analysis (Iso-Seq), building a complete evidence base of structure + per-base accuracy + quantification.
(5) Custom alignment to the client's vector
Read-by-read alignment is performed against the customer-provided full vector sequence as a custom reference, with conclusions that are quantitative, locus-resolved, and comparable across runs.
4. Applications
Orthogonal verification of critical transcript conclusions: Independent platform confirmation of Iso-Seq full-length structure and fusion-event findings.
Precise localization of splice sites: Single-base resolution re-examination when alternative splicing or aberrant junctions have already been identified.
Independent expression quantification: TPM-level evidence for transgene expression level and consistency.
Strengthening of submission data packages: Significantly increase data credibility at IND/BLA milestones through paired verification.
5. Report and Deliverables
The report is centered on orthogonal cross-validation. Core contents include:
·Splice junction catalog and exon usage (PSI).
·Independent expression quantification (TPM) of vector-related transcripts.
·Cross-validation report against Iso-Seq long-read findings, clearly indicating which isoforms and junctions are confirmed by both platforms and which require particular attention.
·Supplementary sensitive detection of aberrant or low-abundance transcripts.
·Data deliverables: Complete analysis report (PDF), junction/PSI/TPM result files, and raw sequencing data.
6. Service Workflow
Service Step | Description |
Project consultation and study design | Strand-specific short-read sequencing plan designed according to vector type and paired-verification needs |
Sample receipt and QC | Assessment of RNA concentration, purity, and integrity; submission in the same batch as the Iso-Seq sample is recommended |
Library preparation | Preparation of strand-specific RNA-seq libraries |
High-throughput sequencing | Sequencing on the Illumina platform to the target high depth |
Bioinformatic analysis | Call junctions/PSI/TPM and perform item-by-item consistency comparison against Iso-Seq results |
Report delivery and technical support | Complete cross-validation report (PDF), result files, and raw data, plus follow-up technical consultation |
* Standard turnaround: 30–35 business days; if performed in combination with Iso-Seq, simultaneous initiation is recommended.
7. Sample Requirements
Item | Submission Requirement |
Sample type | Cell pellets or extracted RNA; submission as part of the same batch as the Full-Length Vector Transcript Analysis is recommended to facilitate cross-validation. |
Recommended input | Total RNA ≥500 ng recommended (refer to the latest Sample Submission Guide; submit sufficient overhead above the minimum input). |
Concentration and quality | RNA concentration ≥20 ng/μL recommended; OD260/280 ≈ 1.8–2.1; RIN ≥7 or DV200 ≥70%; no significant degradation. |
Storage and shipping | Store at −80 °C; ship on dry ice with continuous cold-chain. |
* The latest Sample Submission Guide takes precedence. This service is not applicable to severely degraded samples. Please schedule and confirm the study plan before sample submission.
8. Technical Specifications
Parameter | Description |
Sequencing platform | Illumina high-throughput platform (short-read). |
Library type | Strand-specific RNA-seq library. |
Read strategy | Paired-end sequencing; read length selected as required by the project. |
Applicable sample | Cell pellets or extracted RNA. |
Alignment reference | Customer-provided full vector sequence and the human reference genome as custom references. |
Detection capability | Splice junctions, exon usage (PSI), expression quantification (TPM), and low-abundance aberrant transcripts. |
Sequencing depth | Set per target sensitivity; detection and quantification accuracy of low-abundance events scales with depth. |
Cross-validation strategy | Item-by-item comparison with the Full-Length Vector Transcript Analysis (PacBio Iso-Seq) using parallel samples. |
Method status | IND: fit-for-purpose method qualification; BLA: full validation per ICH Q2(R2). |
Species supported | Primarily human cells; other species accommodated using the customer's vector sequence. |
9. References
[1] ICH. Q6B: Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. Current Step 4 version, 10 March 1999.
[2] ICH. Q2(R2): Validation of Analytical Procedures. Step 4 version, adopted 1 November 2023.
[3] U.S. Food and Drug Administration (FDA), Center for Biologics Evaluation and Research (CBER). Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products. Final, January 2024. (Docket No. FDA-2021-D-0404)
[4] Center for Drug Evaluation, National Medical Products Administration of China (NMPA-CDE). Technical Guideline for Pharmaceutical Research and Evaluation of Immune Cell Therapy Products (Trial) [in Chinese]. Notice No. 11 of 2022, issued 2022.
