Nanopore Long-Reads Sequencing
Oxford Nanopore sequencing is a third-generation, single-molecule sequencing platform. The length of sequencing reads it produces is typically 10-100 kb for long reads sequencing mode and 100-300 kb for ultra-long reads sequencing. The longest reads achieved so far is 4 Mb. These long reads are needed for a list of applications (see below) that short-reads sequencing struggles with. As illustrated in the YouTube video below, it detects the order of nucleotides off a strand of DNA (or RNA) using measurement of ionic current change when the molecule passes through a protein nanopore.
How nanopore sequencing works.
With continuous technology development, the error rate of Nanopore sequencing raw reads is at 1% achieving 99% (Q20) accuracy. Consensus accuracy can reach Q47 at 60x coverage for human DNA through combining multiple raw reads from a genomic region to form a single consensus sequence. The most common sequencing error in nanopore sequencing occurs in homopolymeric regions.
Oxford Nanopore currently offers three main devices at different data throughput levels, i.e., MinION, GridION, and PromethION. MinION/GridION use the same flow cell type which typically produces 10-20 Gb data (30 Gb maximum). The flow cell used on the PromethION has a throughput of 50-100 Gb (170 Gb maximum).
Below are some of the major applications for Oxford Nanopore sequencing:
- Structural variation detection
- Single nucleotide variant phasing
- Full-length transcript sequencing and splicing isoform detection
- Detection of fusion transcripts
- De novo genome assembly
- Direct detection of epigenetic base modifications on DNA and RNA
Nanopore Sequencing Pricing
Sequencing library prep: $350 per sample for Northwestern/CBC users
Long-reads sequencing: $950 per MinION flow cell (Northwestern/CBC); $1,750 per PromethION flow cell (Northwestern/CBC)
Please check the Core Pricing page for external rates.
Nanopore Sequencing
Read Length and Sequencing Output
Long-reads sequencing mode: 10-100 kb in length. Good for:
- Full-length transcript sequencing and splicing isoform detection
- Detection of fusion transcripts
- Direct base modification on DNA and RNA
Ultra-long reads sequencing mode: 100-300 kb in length. Good for:
- De novo genome assembly
- Haplotype phasing
- Structural variants detection
Total data output: 10-20 Gb per MinION flow cell; up to 200 Gb per PromethION flow cell
Service Request
Project consultation is provided free-of-charge. Nanopore sequencing services can be requested through NUcore.
If you need more data than a MinION flow cell can provide, please contact us for an upgrade to PromethION.
Sample Submission
NUSeq takes extracted total RNA for RNA-seq or enriched poly(A) mRNA for direct RNA modification sequencing. The quality of RNA is the single most important factor that determines final outcome. After sample dropoff, core staff conducts sample QC, which includes Qubit concentration measurement and Bioanalyzer-based RNA Integrity Number (RIN) generation, prior to library construction. A RIN of 7 is required to proceed with mRNA-seq library construction. Submitted RNA samples also need to be DNA-free and we suggest to always include a DNase treatment step during RNA extraction. Presence of genomic DNA contamination is visible on Bioanalyzer traces in the range of 4-10 kb.
For DNA sample used for genome assembly, DNA purity and length (in Mb) are critical to obtain high quality data. Please consult with NUSeq core for your project.
Bioinformatics
Data analysis is provided upon request.
Frequently Asked Questions
What kind of base modification(s) can be detected using Nanopore sequencing?
5mC, 5hmC, 6mA, and BrdU in DNA, and m6A in RNA.
What is the length of transcripts that can be detected?
We can detect the full-length of each transcript (such as 10-20 kb) as long as the input RNA or cDNA is intact.
How much data do I need for my project?
For genome assembly and amplicon sequencing, at least 60X coverage is recommended. For whole genome RNA-seq, at least 30 million reads is recommended.