Full-length Transcriptome Sequencing

Full-length Transcriptome Sequencing is a modern transcriptome analysis method using long-read sequencing technologies, enabling direct reading of complete mRNA molecules from the 5' to the 3' end. Unlike conventional short-read RNA-Seq technology, Full-length Transcriptome Sequencing identifies complete transcript isoforms without requiring bioinformatic reconstruction.

Using Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT)platforms, it is possible to accurately detect alternative splicing, alternative polyadenylation sites, novel genes, long non-coding RNAs (lncRNAs) and other complex regulatory elements of gene expression.

The technology is used in basic research and translational projects, functional genomics, developmental biology, plant biology, microbiology and medicine.

Full-length Transcriptome Seq workflow

Applications of Full-length Transcriptome Seq

Full-length transcript analysis is used in many areas of biology and genomics:

Technical specifications of Full-length Transcriptome Seq

• Sequencing mode: long reads
• Library preparation: PolyA enrichment to capture mRNA transcripts.
• Sequencers: PacBio platform (Revio and PacBio Sequel II); ONT (Nanopore PromethION 48)
• Recommended amount of data per sample for PacBio:
  • 1M HiFi reads
  • 5M HiFi reads
  • 10M HiFi reads
  • Other configurations are also available depending on the client's needs.
• Recommended data output per sample for ONT:
  • 6Gb
  • 12 Gb
• Data quality:
  • PacBio: 99.8 - 99.99%
  • ONT: Full-length ratio >70%
• Recommended Sample Requirements - DNA/RNA:
  • Sample type: total RNA
  • Amount: >1.0 μg
  • Concentration (NanoDrop): >100 ng/μL
  • Volume: >15 μL
  • Purity:
    • D260/280=1.7-2.5
    • OD260/230=0.5-2.5
    • Little or no protein or DNA contamination on the gel
  • RIN (PacBio)
    • For plants: RIN ≥ 7.5;
    • For animals: RIN ≥ 8.0;
    • 5.0 ≥ 28S/18S ≥ 1.0;
  • RIN (ONT):
    • For plants: RIN ≥ 7.0;
    • For animals: RIN ≥ 7.5;
    • 5.0 ≥ 28S/18S ≥ 1.0;
• RNA isolation from the supplied samples is also available. Details here.

Full-length Transcriptome Seq bioinformatics analysis

Comprehensive bioinformatics analysis of Full-length Transcriptome Sequencing covers transcript structure characterisation as well as functional and quantitative analysis.

• Full-length transcript assessment and redundancy removal

• Full-length Transcript Assessment: this stage identifies complete transcripts containing:
  • complete 5' UTR regions,
  • CDS,
  • 3' UTR regions,
  • polyadenylation signals.

Transcript Dereplication: repeated transcripts are grouped and consolidated to obtain a set of unique isoforms used in further analyses.

• lncRNA identification and target-gene analysis, including:
  • identification of lncRNA candidates,
  • lncRNA classification,
  • prediction of target genes,
  • analysis of potential regulatory mechanisms.
• Analysis of functional transcript elements:
  • SSR (Simple Sequence Repeats)
  • CDS (Coding Sequence)
  • TF (Transcription Factors)
  • AS (Alternative Splicing)
  • APA (Alternative Polyadenylation)
• Gene expression analysis
• Functional annotation and enrichment analysis