Introduction | ONT sequencing | Costs | Laboratory set up | Training | Computing | References

Introduction

This guide does not aim to go over how sequencing technologies work, or the actual protocols etc. required to do it, as there are already many resources available online (we have linked to some of them within this page). This guide primarily aims to help smaller, or poorly resourced sites identify what they would need, in terms of infrastructure, computing and costs, to set up a sequencing platform in their laboratories.

Despite the cost of sequencing reducing, it is still not commonly used in Low and Middle Income Countries (LMICs) due to the price, although the COVID-19 pendemic has expanded the number of sites performing sequencing. There are many companies that offer sequencing platforms, from the earlier Sanger sequencing to next generation sequencing (NGS) platforms with different technologies. This site lists a number of sequencing companies.

Whilst Illumina appears to be the leading sequencing platform, the initial cost of equipment for Illumina sequencing can be prohibitively expensive (a MiSeq machine costs over $100,000). The advances in Oxford Nanopore Technology’s (ONT) MinION (for which starter kits cost around $1,000), and other devices, may be a more cost effective option for smaller, or less well resourced laboratories.

ONT and Illumina sequencing technology is different, and whilst Illumina sequencing is acknowledged to currently have greater accuracy than ONT (parts of their range are FDA-approved), ever-improving technologies and good validation practices make ONT platforms an attractive, cost effective option, especially for low-throughput laboratories.

Choosing a sequencing platform will depend on a number of factors, and these must be taken into account before you purchase a system. Things you may wish to consider include:

  • How much you wish to spend (up front and over time)
  • The organisms you plan to sequence (e.g. humans, viruses, bacteria etc.)
  • What you wish to sequence (e.g. amplicons, specific regions, metagenomics, whole prokaryotic or eukaryotic genomes)
  • Sample throughput (both the number of samples at once, as well as how many you plan to sequence over time)
  • What you wish to use the data for (e.g. building phylogenetic trees, identifying SNPs, speciation, resistance genes etc.)

Whilst much of the information in this guide is universal to all sequencing platforms, it is aimed at the set up requirements for ONT platforms, as they are likely to be the most accessible to smaller or less well resourced laboratories.

 

ONT sequencing

According to ONT, their sequencing platforms have been designed to “enable the analysis of any living thing, by any person, in any environment”. Information on how ONT sequencing works can be found on their website.

There are also many excellent videos on their YouTube channel.

Depending on your needs and throughput, ONT has a number of devices, including:

  • MinION – small and portable, the MinION is the most widely used device, which can sequence around 12, but up to 96 samples at once
  • Flongle - an adaptor for the MinION, which allows more cost effective sequencing of a smaller number of samples
  • Mk1C - an ‘all in one’ version of the MinION, with screen and computer attached
  • VolTRAX - an automated sample preparation system
  • GridION and PromethION - larger scale versions of the MinION, that can process multiple flow cells at once

Before you begin, it is highly recommended that you sign up to the Nanopore Community as a lot of the documentation found in this guide requires a login to access. You can sign up for free, but to get full access, you will need to buy a starter kit.

 

Costs

When considering setting up a sequencing platform, it is important not only to identify a set up cost, but also the ongoing running costs over time. Depending on the type of kit(s) you plan to use, there may be third party reagents you will need to purchase, so this must also be factored into the overall cost. Some things you may need to factor into your budget include:

  • Initial sequencing platform set up cost – this will include the cost of the sequencing platform ‘starter’ kit (e.g. sequencing device, flow cells, reagents etc.)
  • Per sample costs – generally the more samples you run at once, the cheaper it will be per sample. The exact cost depends on a number of factors, including the sample type, extraction/sample preparation methods and type of sequencing preparation kits used
  • Any extra reagents and equipment required – some sequencing library preparation kits require third party reagents and equipment
  • Sustainability – How long do you plan to use the platform for? Do you have enough money to cover the cost of sequencing for the length of time you plan to use it? The Chatham House Sustainable Laboratories Initiative documentation may help you more generally to create a sustainable laboratory system
  • Size of project – How many samples do you plan to sequence, both overall and at one time? If you are planning a large scale sequencing project, or plan to set up a sequencing hub, Illumina, or ONT’s GridION or PromethION may be a better option. If you are only sequencing a few samples a week, for example, a MinION would be a better option
  • Staffing costs – How many people are going to be employed to run the sequencing at your laboratory? You must consider not just the sequencing time, but the sample preparation and post-sequencing analysis time
  • Training costs – If you are setting up from scratch, you may want to consider training your staff. Whilst there are plenty of free resources online (ONT provide plenty as part of their Nanopore Community), an intensive training workshop may be beneficial, for both the laboratory and bioinformatics part of sequencing.
  • Bioinformatics and computing costs – This will depend on how much data you will produce and what you plan to do with it, but things to consider will include the cost of a compatible computer or laptop, storing your data (which can quickly become terabites), the cost of a bioinformatician (or training for your own staff), internet costs and potentially generator costs etc.
  • Manufacturer servicing/calibration/warranty etc. – consider the long term costs of keeping your sequencing (and other) equipment calibrated and serviced.

 

Laboratory set up

Pre-sequencing

Before you can prepare sequencing libraries, you will need to extract the genetic material that you wish to sequence. The types of extraction kits will depend on the organisms you want to sequence e.g. human, virus, bacteria, fungi, protozoons, helminths etc.

If you are extracting nucleic acid from ‘host’ samples e.g. human blood, sputum etc. it is important to deplete the DNA of the host (if you are looking for pathogens e.g. viruses or bacteria). If you do not remove the genetic material of the host, you will sequence it alongside the nucleic acid you do wish to sequence, which will leave less sequencing availability for the organisms you are trying to sequence. Host DNA can be removed from a sample bioinformatically, but it is probably better to do so in the laboratory. A protocol for human host DNA depletion can be found here. Depletion of human host genetic material is especially important due to the ethical considerations.

Reagents and consumables

Storage

  • Most of the kit reagents need a transportation cold chain and must be kept in a fridge/freezer in the laboratory
  • ONT have created a field sequencing kit that does not require cold packaging for transportation, or refrigeration, when used within a month. It enables library preparation from genomic DNA in only ten minutes. It contains lyophilised sequencing chemistry and needs minimal laboratory equipment to prepare a library, with users having successfully prepared samples with a pipette and a cup of hot coffee. The kit can be stored for up to one month at up to 30°C unopened, or at 2-8° C for three months unopened, and matches the newly released and improved packaging of MinION flow cells
  • MinION flow cells can be stored (unopened) at room temperature for 4 weeks (from delivery date)
  • They can be stored for 12 weeks (unopened) at 2-8°C
  • Flow cells should be QC checked (see our tutorial here) before library preparation (within 3 months of purchase) as part of the warranty. If there are less than 800 active pores, ONT should replace them
  • Some kits and equipment have a lead time (this is especially the case with newer items). Check the manufacturer’s website (it often says the lead time on ONT’s website), or contact customer support before planning your experiments
  • For ONT items, the website suggests allowing a working week for orders to arrive for ‘rest of world’
  • You should also consider your country’s customs process. You may need to request documentation (e.g. certificates of origin) before the items ship, to ensure that they do not get stuck in a warehouse, potentially at the wrong temperature.
  • If few samples are being processed at a time, or you are looking at amplicons, smaller genomes or target regions, the ONT Flongle might be more useful
  • ONT Flongles are only guaranteed for 4 weeks after the shipment date (rather than 12 for a MinION flow cells)
  • If you are planning to run tens or hundreds of samples at a time, over long periods, you may wish to consider an ONT GridION, or one of the Illumina platforms.

ONT Flow cells

  • MinION flow cells can be stored (unopened) at room temperature for 4 weeks (from delivery date)
  • They can be stored for 12 weeks (unopened) at 2-8°C
  • Flow cells should be QC checked (see our tutorial here) before library preparation (within 3 months of purchase) as part of the warranty. If there are less than 800 active pores, ONT should replace them

Lead times

  • Some kits and equipment have a lead time (this is especially the case with newer items). Check the manufacturer’s website (it often says the lead time on ONT’s website), or contact customer support before planning your experiments
  • For ONT items, the website suggests allowing a working week for orders to arrive for ‘rest of world’
  • You should also consider your country’s customs process. You may need to request documentation (e.g. certificates of origin) before the items ship, to ensure that they do not get stuck in a warehouse, potentially at the wrong temperature

Sample throughput

  • If few samples are being processed at a time, or you are looking at amplicons, smaller genomes or target regions, the ONT Flongle might be more useful
  • ONT Flongles are only guaranteed for 4 weeks after the shipment date (rather than 12 for a MinION flow cells)
  • If you are planning to run tens or hundreds of samples at a time, over long periods, you may wish to consider an ONT GridION, or one of the Illumina platforms.

Equipment

Whilst ONT sequencing does not require too many extra pieces of equipment or reagents, you will need basic laboratory supplies for all library preparations:

  • Nanodrop, Qubit or similar (to identify the concentration (and quality) of your nucleic acid)
  • Tapestation, Bioanalyser, gel electrophoresis equipment or similar (to identify the average size (and quality) of your nucleic acid)
  • Pipettes and pipette tips
  • Plasticware (e.g. microfuge tubes, tube racks)
  • Microcentrifuge
  • Vortex
  • Nuclease free water
  • Elution buffer

Some kits will require extra reagents and equipment, so you will need to check the protocols to identify what you need before you begin. Common additional items include:

  • PCR machine
  • PCR master mix
  • Magnetic beads and rack
  • Ethanol

 

Training

It is likely that you and your colleagues will need to be trained in order to undertake sequencing, on whichever platform you choose. Sequencing platform websites are often an excellent source of information, both about how the technology works, as well as how to use it. Depending on your aims, you will likely need training for both the laboratory/practical side of sequencing, as well as the bioinformatic/analysis part.

Practical training support

  • Sign up to ONT community for documentation, forums and software advice
  • When you purchase your first starter pack, your access to the community will be upgraded to ‘full member’ and ONT provide guidance and access to webinars
  • Sequencing platforms offer technical support teams, who should be able to answer your questions (bear in mind that you should check the community/forums/websites first, as it is likely someone has already asked the same question!)
  • ONT YouTube channel
  • Illumina YouTube channel
  • PacBio YouTube channel

Bioinformatics

For wet laboratory scientists with little computer experience, learning the bioinformatics associated with sequencing (or in general) can be daunting. To help you begin to navigate through sequencing analysis and bioinformatics more generally, please visit the bioinformatics page.

Search the internet for online tutorials, ask colleagues who may have experience and consult online forums such as Stack OverflowBiostars and the programme development site GitHub for sources of help

Specific to ONT sequencing, the Nanopore Community has forums, protocols and documentation about bioinformatics, and the excellent EPI2ME labs is a great way to get familiar with some of the programmes you are likely to need for processing raw sequencing data

 

Computing

Computing requirements

  • ONT IT requirements can be found here 
  • As ONT (and general) sequencing improves, it will require more power and more memory to run and store data, so buy the most powerful computer you can afford
  • Some bioinformatics programmes e.g. Flye, require a lot of RAM (usually at least 16 GB) to assemble one (bacterial genome)
  • If you are processing large amounts of sequencing data, you may need to consider obtaining access to a computing server to run high-powered programmes or pipelines for you

Data storage systems

  • An ONT MinION flow cell can generate as much as 30 Gb of data, so storage may become an issue if you are doing multiple runs
  • Running out of disk space during a sequencing run will stop your sequencing experiment (although data already collected will be saved). It is recommended that you save your run data to the local drive on the computer, then transfer it to cloud storage/a hard drive etc. once it has completed. Storing run data directly to a removable drive is slower and may have connection issues with may interrupt your run, so is not advised
  • Users operating the MinION on a lower specification machine (e.g. laptop) will find that MinKNOW will basecall as much as possible in real time and then analyse the remaining files at the end of the sequencing experiment; the computer will need to be left on for this to happen. Unless your machine is running on a GPU, basecalling is unlikely to be in ‘real time’ and, depending on the power of a computer, can take several hours-days to complete once the run has finished. This also depends on how much data you have created from your sequencing run
  • The Mk1C (a MinION device with built in computer) has 1 TB SSD storage and 8 GB RAM
  • Laboratories must consider the general data protection regulations (GDPR) of their data, especially when sequencing directly from a human sample. Consider the security of your data storage (e.g. is it on a secure server, does it have password protection?)

Power issues

You will need power for the duration of your sequencing run, which can be up to 72 hours (plus subsequent basecalling, if you leave it running). If the power cuts out, it will save data up until the power cut. Laboratories with intermittent power supplies should consider the following:

  • A laptop with a reliable rechargeable battery may be more useful than a mains-only computer if power cuts are an issue
  • Having an Uninterrupted power supply (UPS) would also help to mitigate unstable power supplies

Internet access

ONT platforms generally need to access the ONT server online at the start of a run. The exception to this is the Mk1C, which can sequence and basecall entirely without internet connectivity

  • To basecall your sequence in real time, you will need internet access for the duration of your run, as MinKNOW uses a cloud-based platform to basecall
  • For large sequence runs, it can take much longer to basecall than the time it takes to sequence, so a stable internet connection is important
  • ‘real time’ basecalling can really only be achieved using graphics processing unit (GPU) rather than the more standard central processing unit (CPU) (especially on big runs)
  • However, if time or the internet is an issue, then base-calling can be done separately, using the Guppy line command interface (CLI) (the same programme as MinKNOW uses)
  • You will also require internet access to run sequences through cloud-based databases such as TBprofiler, as well as the ONT programme EPI2ME
  • Offline configurations can be made available for field use and expeditions

 

References

Below are some references on the set up and use of sequncing platforms in resource poor settings:

Nature paper - Implementation of whole genome sequencing for tuberculosis diagnostics in a low-middle income, high MDR-TB burden country (using Illumina platform)

Genetics - Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Malaria Journal - Applying next-generation sequencing to track Falciparum malaria in sub-Saharan Africa