Nanopore sequencing

Research sequencing using Oxford Nanopore Technology

Our research sequencing uses the GridION and PromethION machines. We offer standard ligation-based library preparation (LSK109) and ligation based barcoding up to 24 samples (NBD104 and NBD114). We may also be able to accommodate special requests for library preparation.

Please contact us with the details of your request if you would like additional information.

GridION vs PromethION

Currently, we offer  two sequencing platforms. The primary differences between the two are the size of the flow cell and the amount of data that each can generate.

The GridION uses the same flow cell as the MinION, which has up to 512 nanopore channels.

The PromethION machine can run up to 24 flow cells simultaneously, and each flow cell has up to 3000 nanopore channels.

For DNA that is extracted from fresh human blood using the DNeasy Qiagen kit, we routinely get 10-20 Gb of data from a GridION flow cell and 80-120 Gb of data from a PromethION flow cell. The yield from other sample types varies according to the quality and average fragment size. As the quality of the DNA extraction has a significant effect on the outcome, we cannot guarantee any output volumes from our service.

How to request sequencing

We usually aim to respond to your quote request within several working days.

We accept purified DNA with the following quality standards:

  • Average fragment size > 60 kb
  • Qubit concentration > 24 ng/µl
  • Volume > 50 µl
  • Total amount of DNA >1.2 µg
  • 260/280 ~ 1.8
  • 260/230 ~ 2.0

We recommend using the Qiagen DNeasy blood and tissue kit for extractions as it has provided the most consistent quality output. If your 260/280 or 260/230 values do not meet the above quality standards, we recommend a clean up with Ampure or SPRI beads to improve the quality values. The library prep includes quality checks on the tapestation for size, nanodrop for purity, and Qubit for concentration.

If you do not have the equipment to perform the quality checks, we can perform them. If the samples do not meet the quality standards you will be charged for the QC steps only, and we will contact you to regarding how you would like to proceed with the samples.

When you are ready to submit your samples, please download and fill out the sample submission form (XLS, 30KB). Please send the form with your samples, and email a copy to nanopore@garvan.org.au.
Samples from within Australia can be posted to the following address:

Attn: Genomic Technologies Group 
Precinct Loading Dock 
West Street (off Burton Street) 
Darlinghurst NSW 2010 
Australia    

We recommend shipping samples on ice.

If you are sending your samples from outside Australia, please contact us at nanopore@garvan.org.au for the appropriate forms and instructions. Australian customs are very strict and will delay shipments without the appropriate documentation.

1D Ligation (LSK109)  

The 1D ligation library prep kit is the standard ONT sequencing library prep. We have found that this is the best option for the majority of the samples we receive including genomic DNA and cDNA.

If you would like a different library preparation for your sample, please contact us directly for more information at nanopore@garvan.org.au.

Shearing

We can shear your sample to an average fragment size of 8-10 kb using a Covaris tube g-tube (520079). We find that this increases the total yield of data from the flow cell by 30-50% compared to samples that are not sheared to a uniform size. Shearing is recommended for experiments where the amount of data is more important than having longer fragment length.

Short-read depletion

Samples that are degraded from long storage or the DNA extraction method and have a smear of smaller fragments can benefit from depletion of the short fragments.  We can perform the Circulomics short-read eliminator kit (SS-100-101-01) on your sample to remove fragments shorter than 25 Kb. If you choose this option, please supply at least 3.3 ug of total DNA, and more if you can, as a large fraction of the total DNA is often composed of short fragments and is therefore depleted during the process. This is not recommended for samples with an average fragment size below 60 kb.

Barcoding

If you would like to barcode your samples and run multiple samples on the same flow cell, we can do this with the extension kits ligation based barcoding kits NBD104 and NBD114. We can barcode up to 24 samples on a single flow cell. This is ideal for uniform samples that don’t require a significant amount of data per sample, such as PCR products. The average amount of data per barcode is reliant on the quality of each of the samples going into the run. Once the barcodes are added, the ONT adapter is added using the standard 1D ligation kit (LSK109).

The raw fast5 data will be basecalled using the latest version of Guppy available, and your sequencing data will be provided in a .fastq format. You will receive a quality report with your results that will include the total output of your run as well as the average length and quality scores.

You will be able to access your results through cloudstor and will therefore need to create an account. Once your results are ready, you will receive an email with a link to download your results. The results will be available to download from cloudstor for one month from the date they are available to you. The data will be stored locally at the Garvan for three months from the same date (if you would like the data removed from our servers before three months, please let us know).

If you would like to arrange access to the .fast5 files that contain the raw data, please contact us directly for a quote (hard drive and shipping).

Pricing will vary depending on the flow cell, library prep method, and number of samples. The pricing for 1D ligation-based library prep on a GridION or PromethION is shown below. Please submit an estimate request to get the pricing for your experiment.

Library Prep

Nanopore machine

Flow cell chemistry

Price

1D ligation

PromethION

FLO-PRO002

$3800 AUD

1D ligation

GridION

R9.4

$1500 AUD