Nanopore Long Read Sequencing

The ATGC now offers Long Read sequencing on the Oxford Nanopore PromethION 24 platform. This unique, scalable, third-generation sequencing technology enables the direct (amplification-free) sequencing of DNA and RNA and can directly read base modifications including methylation.

Technology Overview

The Oxford Nanopore PromethION works by monitoring changes to an electrical current as nucleic acids are passed through a protein nanopore.

The Instrument

On the instrument, flow cells house an array of protein nanopores, embedded in an electrically resistant polymer membrane. During sequencing, changes in electrical currents are captured as nucleic acids are passed through nanopores. The signal generated is decoded to provide the specific DNA or RNA sequence.

For more information on the Nanopore Sequencing: https://nanoporetech.com/support/how-it-works

Technology Highlights 

Oxford Nanopore Technology (ONT) long read sequencing can be used to:

• Resolve complex structural variants and repetitive regions
• Simplify de novo genome assembly
• Study linkage and resolve phasing
• Enhance metagenomic identification of closely related species
• Sequence entire microbes in single read
• Explore epigenetic modifications using direct, long-read DNA sequencing

Applications:

• Whole genome sequencing (Available)
• Targeted sequencing (adaptive sampling- Available)
• Whole transcriptome sequencing (coming soon)
• Single Cell cDNA-sequencing (Available)
• Amplicon sequencing (pool up to 96 different samples in one sequencing experiment)
• Metagenomics sequencing (coming soon)
• Custom applications (coming soon)

Whole Genome Sequencing

Due to the sample quality and purity, the N50 varies from 5 to 50+ kb for WGS.

N50 is related to the median and mean length of a set of sequences. Its value represents the length of the shortest read in the group of longest sequences that together represent (at least) 50% of the nucleotides in the set of sequences.

Targeted Sequencing

• Adaptive sampling (no library enrichment required)

• Bulk cDNA sequencing
• Single cell cDNA sequencing

Amplicon DNA

• Pool up to 96 different samples in one sequencing experiment)

Flow Cell Output

Depending on the sample quality and library type, the PromethION can generate up to 100+Gb of sequence data flow cell, however actual output is highly variable.

In general, flow cell yield is inversely related to sequence length (shorter fragments yield more data) and will vary widely depending on the sample. We therefore cannot guarantee flow cell output.

Submitting samples for Nanopore sequencing

1. Nanopore sequencing is sold by the flow cell (72 hours+)
2. We do not accept investigator-prepared libraries for this service. All prices include library preparation and sequencing.

Submission Workflow

Project Consultation

We strongly recommend a consultation meeting before your first Nanopore sequencing project. Consultations are free for investigators utilizing the ATGC’s Nanopore sequencing service. To request a meeting, please contact Erika Thompson (ejthomps@mdanderson.org).

Sample Submission

To submit samples, please complete a Nanopore Sequencing Service Request Form and email the completed form to NanoporeSubmissions@mdanderson.org All samples should be accompanied by a completed submission form.

Sample requirements

Samples submission requirements (minimum quantity and recommended length) vary based on the application and sample type.

WGS and Targeted Sequencing

Investigators have the option of submitting extracted DNA or cells in suspension. Note: There is an additional cost for DNA extraction by the ATGC.

For investigator extracted DNA:

• DNA purity (measured using Nanodrop): OD 260/280 >1.8-2.0, OD 260/230: 2.0–2.2
• Investigator extracted DNA should be accompanied by a gel picture that includes a ladder with a marker >48kb
• No detergents or surfactants in the buffer
• DNA must be RNA-free
• DNA Dissolved in 10mM TRIS (PH 8.0) or Qiagen EB Buffer
• Concentrations must be measured using 3 times using a fluorescence-based method

Please note: The length of the reads depends on the fragmentation size of the DNA and on its quality. 

Recommended Extraction Kits:

For N50 5kb: 30kb most column-based extraction kits are acceptable

For N50 30kb-50kb: NanoBind CBB kit, Monarch HMW DNA extraction kits

For N50 >50kb: Wizard HMW, Monarch HMW DNA extraction kits 

Nanopore Sample Requirements 

Note: The N50 application selected determines the kit used to process your sample. It is not a guarantee of actual N50.

Sample workflow 

1. Quantification, purity check, and pulse-field gel analysis
2. Library preparation
3. Nanopore sequencing
4. Report of all sequences generated and sent to customer 

Data Delivery

ATGC will provide sequencing data in fastq formats. The size of each file can be large (hundreds of gigabytes to terabytes). Please be sure to check computer to ensure adequate storage. 

Data Analysis

Director of Bioinformatics

Xiaoping Su, Ph.D. 

Contact information

Erika Thompson
Email: ejthomps@mdanderson.org