Why TGS mRNA Sequencing Matters: The Core Value of Full-Length Transcriptomics

As core tools for in-depth transcriptome research, PacBio and ONT long-read sequencing differ greatly from traditional fragmented NGS sequencing. Without breaking RNA into short fragments, TGS can capture complete intact mRNA molecules directly. Based on massive practical projects, BMKGENE confirms that full-length TGS sequencing greatly improves research depth and data accuracy, and achieves the following core goals:

01. Obtain Complete Transcript Structures

Acquire full-length sequences covering the 5′ terminal region, coding sequence and 3′ poly(A) tail, precisely identify alternative splicing isoforms, novel transcripts and fusion transcripts.

02. Stably Cover Complex Transcript Regions

Reliably sequence ultra-long transcripts, high-GC segments and internal repetitive sequences within mRNA, realizing unbiased and comprehensive transcriptome coverage.

03. Reduce Amplification Preference in Library Preparation

Compared with high-cycle PCR required before adapter ligation in NGS library construction, TGS adopts low-cycle mild amplification to minimize expression quantification deviation.

In general, TGS long-read sequencing supports intact transcript acquisition and native RNA characteristic detection, supporting more precise and systematic mRNA research.

Core Advantages: TGS (ONT/PacBio) vs NGS (Illumina/MGI)

NGS is suitable for large-scale routine gene expression quantification, while ONT and PacBio focus more on transcript structural integrity and native molecular feature analysis. The two TGS platforms have their own strengths and complement each other perfectly.

01. PacBio HiFi: High-Precision Full-Length Transcript Research Solution

Technical Positioning: Preferred platform for accurate full-length transcript identification.

Key Strengths:

• Ultra-high base accuracy over 99.9%, consistent with mainstream short-read data quality.
• Average 10–25 kb long reads realize non-assembly full-length transcript coverage.
• Excellent adaptability to high-GC and repetitive sequences inside transcripts.

Applicable Scenarios: Transcript function annotation, alternative splicing mechanism study, fusion gene screening and high-precision differential transcript analysis.

02. ONT Nanopore: Native RNA Multidimensional Sequencing Platform

Technical Positioning: Core platform for in situ RNA structure and epigenetic research.

Key Strengths:

• Support direct native RNA sequencing, able to identify multiple RNA base modifications.
• Precisely locate transcription start sites and detect poly(A) tail length variation.
• Max read length over 100 kb, suitable for ultra-long transcript detection with flexible cost.

Applicable Scenarios: RNA epigenetic modification analysis, post-transcriptional regulation research, low-abundance sample transcriptome sequencing and exploratory research.

Expert Best Practices for TGS mRNA Sequencing

BMKGENE sorts out standardized operation specifications to maximize TGS sequencing value:

01. Reasonable Platform Selection

• Choose PacBio HiFi for high-precision transcript structure and isoform identification.
• Choose ONT for native RNA modification detection and full-length structural exploration.
• Adopt a TGS combined with NGS strategy to integrate structural advantage and quantitative advantage.

02. Strict RNA Sample Quality Control

Ensure an RNA integrity number (RIN) > 7.0 and avoid genomic DNA contamination; adopt fresh samples or stable cryopreservation to prevent mRNA degradation and fragmentation.

03. Standardized TGS Library Construction

Use special long-read sequencing kits and control purification steps properly to retain full-length transcripts and low-abundance functional transcripts.

04. Common Pitfalls to Avoid

• Do not copy high-cycle pre-adapter PCR amplification protocols of NGS, which will affect the integrity of full-length transcripts.
• Reject severely degraded RNA samples to avoid generating incomplete sequencing reads.
• Transcript structural verification cannot rely only on short-read assembly results.
• Match professional TGS-exclusive bioinformatics analysis pipelines for accurate data interpretation.

Your Reliable TGS Transcriptomics Partner

BMKGENE provides full-set one-stop full-length transcriptome sequencing services based on PacBio or ONT platforms, and these services can be combined with the NGS platform. Our optimized library construction schemes effectively lower PCR amplification bias and eliminate fragment splicing errors, outputting real and reliable full-length mRNA data to support diverse transcriptomics research.

Our Core Services

• Reliable TGS sequencing solutions for various sample types.
• Combined transcriptome analysis of TGS full-length structure and NGS quantitative expression.
• Multi-omics deep analysis including splicing variation, transcript-level quantification and fusion transcript mining.
• Whole-process technical support from experimental design to publication-level data sorting.

BMKGENE: Full-Length Transcriptome Sequencing, Empowering In-Depth RNA Research