Direct rnA sequencing
About
Nanopore sequencing is a unique, scalable technology that enables direct, real-time analysis of long DNA or RNA fragments. It works by monitoring changes to an electrical current as nucleic acids are passed through a protein nanopore. The resulting signal is decoded to provide the specific DNA or RNA sequence.
About
Nanopore sequencing is a unique, scalable technology that enables direct, real-time analysis of long DNA or RNA fragments. It works by monitoring changes to an electrical current as nucleic acids are passed through a protein nanopore. The resulting signal is decoded to provide the specific DNA or RNA sequence.
Oxford Nanopore Technologies (ONT) enables real-time, long-read sequencing of native DNA and RNA, eliminating PCR bias. The technology feeds single-stranded DNA or RNA molecules through a protein nanopore to detects changes in electrical current. This method preserves strand methylation and enables direct detection of modifications without the need for chemical conversion. Long reads improve the resolution of repeat regions, aiding in structural variant detection, isoform identification, and complete genome assemblies.
Samples are sequenced using the PromethION P24, followed by quality filtering, assembly, annotation, and analysis using ONT’s EPI2ME software and a data analysis pipelines developed by Poochon Scientific.
+ R101: Direct RNA Sequencing
Direct RNA Sequencing by Oxford Nanopore Technology
Turnaround Time: 7 to 15 business days
Service Description:
Construction of an amplification-free long-read sequencing library using the newest ONT v14 library prep chemistry and data acquisition by ONT PromethION P24 (bases up to 20 GB, coverage up to 20X, N50 read length from ~1.5 Kb)
Deliverables:
Raw data files
1) Fasta.gz – a compressed file of all the raw ONT sequencing reads
2) BAM (upon request)
Analysis Report Files
1) Reports (html) – Alignment/Transcriptome
2) .fasta = all transcripts (Transcriptome)
3) VCF files
4) TSV files
5) Other QC files
Suitable Sample Type:
≥ 5 µg of RNA from any species: OD260/OD280 = ~2.0; OD260/OD230 = ~2.0-2.2; average size ~1.5 Kb; concentration ≥ 200 ng/µl; buffer: ddH2O, EB, or low TE (< 0.1 mM EDTA)
Or, cell pellet samples from any species (≥5 million cells per sample)
• Identification of proteins from protein gel bands or gel spots
• Up to 2,000 proteins identified from one gel band
• Workflow: Sample preparation, in-gel enzymatic digestion, peptide extraction, LC-MS/MS analysis, data analysis, and results report
• Protein Identification Report
• List of identified proteins
• List of identified proteins with matched peptides sequences
• Raw data files, upon request
• Gel band or spot, no larger than 3mm (l) x 3.5mm (h) x 1.5mm (w) in size (Note: additional charge applied for larger size)
Sample Preparation Guidelines
Sample Type
RNA (≥ 5 µg) or cell pellet (≥5 million cells ) from any species
Size
Average size ~1.5 Kb
OD Ratio
OD 260/280 = 2.0
2.0 ≤ OD 260/230 ≤ 2.2
Concentration
≥ 200 ng/µl
Buffer
ddH2O, EB, or low TE (< 0.1 mM EDTA)
Advantages of Direct RNA Sequencing by Nanopore Technology
- Direct sequencing of full-length RNA transcripts
- Detection of isoforms, structural variations, and RNA modifications (e.g., methylation)
- Accurate measurement of poly-A tail lengths
- Isoform-level expression analysis, enabling insights into alternative splicing
