NanoTYPE™ 24/11

Site preparation guide

For research use only
Revision: 2
Published on 09/09/2022

© Omixon Biocomputing Ltd.

All rights reserved. Do not distribute.
Website: https://www.omixon.com
Technical contact: support@omixon.com
Sales contact: sales@omixon.com
Site preparation guide

1 Document history................................................................................................................ 3
2 Introduction ........................................................................................................................ 4
3 Safety information .............................................................................................................. 4
4 Laboratory space requirements......................................................................................... 4
5 Materials and recommendations ....................................................................................... 5
5.1 Technical equipment .................................................................................................................................. 5
5.2 Supplied reagents ....................................................................................................................................... 6
5.3 User-supplied associated reagents............................................................................................................ 6
5.4 User-supplied consumables....................................................................................................................... 7
5.5 PC requirements for Omixon NanoTYPER™ and MinKNOW...................................................................... 7
5.6 Recommended Genomic DNA isolation..................................................................................................... 9
6 Thermocycler programs ..................................................................................................... 9
6.1 HLA amplification........................................................................................................................................ 9
6.2 ExoSAP-IT purification (not for Single sample protocol) ........................................................................ 10
6.3 Barcoding .................................................................................................................................................. 10
7 Onsite and remote training .............................................................................................. 10
7.1 Site preparation ........................................................................................................................................ 10
7.2 Proposed two-day training agenda ......................................................................................................... 11
8 Technical assistance ......................................................................................................... 11
9 Legal notice ....................................................................................................................... 11
10 References ......................................................................................................................... 12
11 Checklist ............................................................................................................................ 13

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Site preparation guide
Document history

1 Document history
Revision Date Description

2 9-Sep-2022 Initial RUO version

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Site preparation guide
Introduction

2 Introduction
This guide provides specifications and guidelines for preparing your site for NGS typing by NanoTYPE™ 24/11 v2
RUO kit in combination with the MinION sequencing device from Oxford Nanopore Technologies.
This includes:
• Safety information (see page 4),
• Laboratory space requirements (see page 4),
• Recommendations regarding DNA extraction, technical equipment, consumables and PCR requirements (see
page 5).

3 Safety information
Before you start, read the Safety Information and/or Important Notes sections of each reagent or kit. For more
information, please consult the appropriate Safety Data Sheets (SDSs) available from the specified product
supplier (links in the References section (see page 12)).
When working with chemicals always wear:
• a suitable lab coat,
• disposable gloves,
• protective goggles.

After use, dispose of the product components as general medical waste.

Review the MinION Mk1B Technical Specification document for safety information on the MinION sequencer
available on the website of the manufacturer.

4 Laboratory space requirements

You must perform all pre-amplification steps in the dedicated pre-PCR area free of contamination that could
negatively affect the PCR reaction.
You must perform all post-amplification steps in the dedicated post-PCR area.
The MinION device was designed to sequence between temperatures of 18-24°C.

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Site preparation guide
Materials and recommendations

Figure 1. MinION device dimensions

You must place the MinION device in the dedicated post-PCR area. Read the MinION Mk1B Technical Specification
document for more details.

5 Materials and recommendations

5.1 Technical equipment

Item name Catalog Supplier
number

1 ONT MinION Mk1b sequencer MIN-101B ONT

2 Thermocycler with 96-well block format Not applicable Any brand

We recommend the ABI Veriti 96-well thermocycler.

3 Qubit fluorometer Q33216 Thermo Fisher

Scientific

4 Magnetic stand for 1.5-2.0ml tubes Not applicable Any brand

5 Magnetic stand for 96-well PCR plates (optional, for protocol with bead- Not applicable Any brand
based amplicon purification)

6 96-well cooler rack or ice bucket with ice Not applicable Any brand

7 1.5ml tube cooler rack or ice bucket with ice Not applicable Any brand

8 Microplate centrifuge Not applicable Any brand

9 Adjustable volume pipettes (1.0-1000 μl capacity) Not applicable Any brand

10 8-channel adjustable volume pipettes (1.0-100μl capacity) Not applicable Any brand

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Site preparation guide
Materials and recommendations

Item name Catalog Supplier

number

11 PC for the installation of (check the hardware requirements below): Not applicable Any brand
• MinKNOW software (MinION controlling software)
• NanoTYPER™ (HLA genotyping software)

5.2 Supplied reagents

Item name Catalogue number Rxns Supplier

1 NanoTYPE 24/11 v2 RUO NT2411v2 24 Omixon

5.3 User-supplied associated reagents

Item name Catalog number Supplier

ONT MinION flow cell type R9.4 FLO-MIN106D ONT

ONT Rapid Barcoding Kit 96 SQK-RBK110.96 ONT

ONT Flow Cell Wash kit EXP-WSH004 ONT

ExoSAP-IT Express (required for a multiple sample 75001-200 (200μl vial for 50 Thermo Fisher
protocol) reactions) Scientific
Each sample requires 2.1 μL of ExoSAP-IT Express 75001-1-ML (1ml vial for 250
enzyme. reactions)

75001-4X-1ML (4ml vial for 1000

reactions)

75001-10ML (10ml vial for 2500

reactions)

Agencourt AMPure XP Beads (alternative for amplicon A63880 (5 ml vial) Thermo Fisher
purification) Scientific
A63881 (60 ml vial)

A63882 (450 ml vial)

Qubit dsDNA BR Assay Kit Q32850 (100 assays) Thermo Fisher

Scientific
Q32853 (500 assays)

Molecular grade ethanol (Anhydrous Alcohol) Not applicable Any brand

Molecular grade water (DNase and RNase free) Not applicable Any brand

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Site preparation guide
Materials and recommendations

5.4 User-supplied consumables

Item name Catalog number Supplier

1 1.5ml microcentrifuge tubes Not applicable Any brand

2 1.5ml low-bind microcentrifuge tubes 0030108051 Eppendorf

3 0.5ml thin wall tubes for Qubit instrument Q32856 Thermo Fisher Scientific

4 96-well plates compatible with the thermocycler Not applicable Any brand

5 Plate seals compatible with the thermal cyclers Not applicable Any brand

6 Pipette tips. Check the compatibility with the flow cell priming Not applicable Any brand
port.

7 15ml conical tubes Not applicable Any brand

5.5 PC requirements for Omixon NanoTYPER™ and MinKNOW

5.5.1 General
The table that follows specifies the minimum hardware requirements for installing both MinKNOW (software for
controlling MinION) and Omixon NanoTYPER™ (HLA genotyping software). Contact us through Omixon Support1 to
get the necessary installers and instructions for installation.

Item name Recommendation

Operating system Linux Ubuntu 20.04 (64 bit) or Windows 10

Memory (RAM) 32 GB RAM

CPU Intel i7 with at least 8 cores or i9/Xeon* with at least 4 cores (8

threads)
AMD Ryzen 5, 7, or better, with at least 4 cores (8 threads)

GPU NVIDIA GPU RTX 2060 or better, with at least 8 GB of GPU

memory
Any of the following: RTX 2060, RTX 2070, RTX 3060, RTX 3070
OpenGL 2.0 compatible

NVIDIA version 455 or newer

CUDA version 11.1 or newer

1 mailto:support@omixon.com

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Site preparation guide
Materials and recommendations

Item name Recommendation

Storage for MinION data 1 TB internal SSD + 2 TB HDD

Size storage according to your sample throughput.

Storage for NanoTYPER™ result files ~40 MB per sample

Ports USB3

*You must verify that your i9 processor is at least quad-core.

5.5.2 Storage
The table that follows lists data storage options and estimates:

Samples/year Estimated storage (TB)

1 sample 0.008 – 0.01

500 samples or less 4.5

500 -1000 samples 4.5-9

1000 - 2000 samples 9-18

2000 - 3000 samples 18-27

5.5.3 Optimization
The table that follows lists configuration recommendations for the correct operation of MinION:

Component Configuration

User account privilege level You must have Local Administrator privileges so you can give
MinKNOW device the correct CPU priority for data acquisition.

Internet connection An internet connection is always necessary for software updates and
telemetry.
It is possible to create offline configurations for field use and
expeditions. Contact us through Omixon Support2 if you need help.

Antivirus settings Antivirus software scanning the entire hard drive uses significant
amounts of resources and can cause performance issues. For this
reason, start scans manually or configure automatic scans to run
outside work hours when the MinION is not in use.

2 mailto:support@omixon.com

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Site preparation guide
Thermocycler programs

Component Configuration

OS update settings Do operating system updates manually or configure automatic

updates to install outside work hours when the MinION is not in use. If
an OS update triggers a restart of the computer, that can cause a fatal
error in a sequencing run.

5.6 Recommended Genomic DNA isolation

Isolate human genomic DNA (gDNA) from peripheral blood, white blood cells, buccal swabs or saliva. Do not collect
blood in heparinized tubes. Do not use, lipemic or hemolyzed samples or blood samples from patients undergoing
heparin therapy.
High molecular weight genomic DNA samples of sufficient quantity are important for a reliable performance of the
protocol. To ensure the consistency of preparation, we recommend using a well-tested, commercially available
DNA isolation kit. UV absorbance is a common method used for assessing the purity of a DNA sample. The protocol
requires 200 ng of gDNA per sample, and its quality should meet with 260/280 absorbance ratio values of 1.8-2.0
and 260/230 absorbance ratio values of 2.0-2.2. Values outside this range indicate impurities or the presence of
contaminants (alcohol, salts, detergents, formaldehyde, heparin). It is critical to accurately determine the input
DNA concentration. We recommend using a fluorometric method to accurately quantify DNA. The integrity of a DNA
sample is of similar importance as the initial amplification step requires a suitable amount of template material
with more than 6.5 kilobases in length.
EDTA in gDNA elution buffer can inhibit PCR reaction so we recommend using elution buffer with only low EDTA
content. We recommend storing the prepared gDNA for extended periods at temperatures of -20°C or less and
avoid repetitive freeze/thaw of the gDNA as well to preserve its integrity and stability.

6 Thermocycler programs

6.1 HLA amplification

 Note
Omixon validated this protocol on the ABI Veriti® Thermal Cycler. When you program the thermocycler, set
the ramp rate to resemble the ABI 9600 emulation mode, which is equal to a heating speed of +0.8°C/s and
a cooling speed of -1.6°C/s.

Number of Cycles Temperature Time

Set lid temperature to 105°C

1 94°C 1 minute

98°C 10 seconds
30
68°C 4 minutes 30 seconds

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Site preparation guide
Onsite and remote training

Number of Cycles Temperature Time

1 4°C ∞

6.2 ExoSAP-IT purification (not for Single sample protocol)

Temperature Time

37°C 4 minutes

80°C 1 minute

4°C ∞

6.3 Barcoding
Temperature Time

Set lid temperature to 105°C

30°C ∞

30°C 1 minute

80°C 1 minute

7 Onsite and remote training

Omixon provides a flexible training plan to the users of NanoTYPE and Omixon NanoTYPER. All training options
involve a NanoTYPE and a NanoTYPER training seminar. We provide the seminars either on-site or remotely using a
web conference. Additionally, Omixon provides a two-day onsite training to guide users through the protocol.
However, before training can begin it is important that you follow our Site preparation guide to optimize our time
together with you. The training schedule and site preparation guide are guidelines, and we advise all customers to
work with your dedicated FAS in order to tailor training to your laboratory.

7.1 Site preparation

In addition to having the lab supplies listed in the previous sections of the Site preparation guide, your lab should
also do the following:
• Make sure that the necessary instruments and technical equipment (see page 5) are available and are in good
working condition,
• Set and save the programs (see page 9) in your thermocycler.
• Install the MinKNOW and Guppy software.
• Install the Omixon NanoTYPER™ software.
• Check if the internet connection of MinION is feasible.

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Site preparation guide
Technical assistance

• Perform a successful device control run on the MinION sequencer. Check the MinION Mk1B installation and
configuration document for instructions.

7.2 Proposed two-day training agenda

 Tip
For the sake of efficient training perform the long-range PCR before the arrival of our field application
scientist.

Depending on the situation the training can be led on-site by our FAS or remotely.

Time Day 1 Day 2

09:00 gDNA dilution and LR-PCR Amplification. gDNA dilution and LR-PCR Amplification.

10:00 Pre-training presentation. Data analysis and result presentation.

11:00 Equipment and reagent check.

Flow cell priming training.

12:00 Lunch break. Lunch break.

13:00 Amplicon quantitation and normalization. Amplicon quantitation and normalization.

14:00 Library preparation. Library preparation.

15:00 Flow Cell preparation, priming, loading, and Flow Cell preparation, priming, loading, and
the start of the sequencing run. the start of the sequencing run.

8 Technical assistance
For general assistance with this protocol contact your field application scientist.

9 Legal notice
The NanoTYPE™ 24/11 IFU and all contents are proprietary to Omixon Biocomputing Limited ("Omixon") and are
intended solely for the contractual use by its customer in regard to the product(s) described herein and for no
other purpose. This document and its contents shall not be used or distributed for any other purpose and/or
otherwise communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of
Omixon. Omixon does not convey any license under its patent, trademark, copyright, or common-law rights nor
similar rights of any third parties by this document.
Use of NanoTYPE (including software and assay) is governed by Terms and Conditions Governing Omixon Products,
which references the Omixon NanoTYPE EULA. The use of Omixon NanoTYPER™ software alone is governed by the
Omixon NanoTYPER EULA.

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Site preparation guide
References

The instructions in this document must be strictly and explicitly followed by qualified and properly trained
personnel in order to ensure the proper and safe use of the product(s) described herein. All of the contents of this
document must be fully read and understood prior to using such product(s).
FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY
RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND DAMAGE
TO OTHER PROPERTY.
OMIXON DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED
HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) OR ANY USE OF SUCH PRODUCT(S) OUTSIDE THE SCOPE OF
THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY OMIXON IN CONNECTION WITH THE CUSTOMER'S
ACQUISITION OF SUCH PRODUCT(S).
FOR RESEARCH USE ONLY
©2022 Omixon Biocomputing Ltd. All rights reserved.

10 References
Item Document

MinION Mk1B installation and configuration Guide

ExoSAP-IT Express SDS document3

Qubit dsDNA BR Assay Kit SDS document4

Rapid Barcoding Kit 96 SDS document5

MinION flow cell type R9.4 SDS document6

3 https://www.thermofisher.com/order/catalog/product/75001.200.UL
4 https://www.thermofisher.com/order/catalog/product/Q32850
5 https://store.nanoporetech.com/eu/rapid-barcoding-kit-1.html
6 https://store.nanoporetech.com/eu/flow-cell-r9-4-1.html

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Site preparation guide
Checklist

11 Checklist
Item ✔ if OK Item ✔ if OK

Thermocycler with pre-installed Flow Cell Wash kit

programs

ONT MinION Mk1b sequencer Qubit dsDNA BR Assay Kit

Qubit fluorometer ExoSAP-IT Express

Magnetic stand for 1.5-2.0ml tubes AMPure XP beads (optional)

Magnetic stand for 96-well PCR plates Molecular grade ethanol (Anhydrous
(optional) Alcohol)

1.5ml tube freezer rack or ice bucket Molecular grade water

with ice

96-well freezer rack or ice bucket with 1.5ml microcentrifuge tubes

ice

Microplate centrifuge 1.5ml microcentrifuge LoBind tubes

Adjustable volume pipettes (1.0-1000μl 0.5ml thin-wall tubes for Qubit

capacity) instrument

8-channel adjustable volume pipettes 96-well PCR plates

(1.0-100μl capacity) (optional)

Adjustable volume pipettes (1.0-1000μl PCR plate seals

capacity)

NanoTYPE 24/11 v2 RUO kit Pipette tips (check the compatibility

with flow cell priming port)

MinION flow cell type R9.4 15ml conical tubes

Rapid Barcoding Kit 96

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Site preparation guide
Checklist

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