BioNano Genomics Irys User Manual

Irys® User Guide

Document Number: 30047 Document Revision: A 07/28/2015

Irys® User Guide

Contents

Legal Notices ................................................................................................................................................................ 4

Revision History ........................................................................................................................................................... 5

Safety Warnings ............................................................................................................................................................ 6

Chapter 1 Overview ...................................................................................................................................................... 7

1.1 Introduction ....................................................................................................................................................... 7

What's New! ................................................................................................................................................................ 7

Audience and Intended Use ........................................................................................................................................ 7

1.2 Irys Overview .................................................................................................................................................... 7

Irys Description ............................................................................................................................................................ 7

Irys Exterior ................................................................................................................................................................. 9

Front ............................................................................................................................................................................ 9

Right Side .................................................................................................................................................................... 9

Rear Panel ................................................................................................................................................................... 9

Irys Inside .................................................................................................................................................................. 10

1.3 Irys Workstation Configuration ...................................................................................................................... 11

Data Network Connections and Power ..................................................................................................................... 11

Computer System Workflow ...................................................................................................................................... 12

1.4 First Use .......................................................................................................................................................... 12

1.5 User Supplied Consumables ......................................................................................................................... 13

IrysChip ..................................................................................................................................................................... 13

IrysPrep ..................................................................................................................................................................... 13

1.6 Run Type Recipes (M2, M3, M3x and Optimized) .......................................................................................... 13

Chapter 2 Irys Instrument Control Software ............................................................................................................ 14

2.1 Best Practices ................................................................................................................................................. 14

2.2 Irys ICS Main Screen Overview ..................................................................................................................... 14

Select User Screen ................................................................................................................................................... 14

Chapter 3 Starting a Project ...................................................................................................................................... 15

3.1 Irys System Project Workflow Overview ....................................................................................................... 15

3.2 Sample Preparation Summary ...................................................................................................................... 15

IrysPrep Plug Lysis Long DNA Isolation (BNG PN 30026) ....................................................................................... 15

IrysPrep Labeling-NLRS (BNG PN 30024) ............................................................................................................... 16

IrysPrep Human Blood Protocol (BNG PN 30033).................................................................................................... 16

3.3 IrysChip Preparation Summary ..................................................................................................................... 16

Preparing IrysChip..................................................................................................................................................... 16

3.4 Instrument Preparation Summary ................................................................................................................. 16

System Startup .......................................................................................................................................................... 16

3.5 Logging In ....................................................................................................................................................... 18

Select Existing User or <Add New User> ................................................................................................................. 19

Configuring the User Settings and Remote Data Storage Location ......................................................................... 19

Chapter 4 Performing a Run ...................................................................................................................................... 20

4.1 Loading the Sample ........................................................................................................................................ 20

Load Inlets ................................................................................................................................................................. 21

Load Outlets .............................................................................................................................................................. 22

Cleaning and Oiling the Objective ............................................................................................................................. 22

Forward ..................................................................................................................................................................... 23

Sample Detect ........................................................................................................................................................... 23

4.2 Barcode............................................................................................................................................................ 24

Forward ..................................................................................................................................................................... 24

4.3 Focus & Register ............................................................................................................................................ 25

4.4 Select Recipe .................................................................................................................................................. 26

Irys® User Guide

07/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

4.5 Scanning Flowcell........................................................................................................................................... 27

4.6 Results ............................................................................................................................................................. 28

Forward ..................................................................................................................................................................... 29

4.7 Clean ................................................................................................................................................................ 29

4.8 After a Run ...................................................................................................................................................... 30

Load Another Sample ............................................................................................................................................... 30

Shutting down the Instrument ................................................................................................................................... 30

Chapter 5 Optimized Recipes .................................................................................................................................... 31

5.1 Creating an Optimized Recipe ....................................................................................................................... 31

Getting Started: .......................................................................................................................................................... 31

Workflow .................................................................................................................................................................... 31

Optimization Setup .................................................................................................................................................... 32

Labels A through G ................................................................................................................................................... 33

Step 1: Selecting a Base Recipe ............................................................................................................................... 33

Step 2: Focusing ....................................................................................................................................................... 34

Step 3: Initial Loading ................................................................................................................................................ 34

Loading Step: Concentration ..................................................................................................................................... 35

Loading Step: Spike .................................................................................................................................................. 35

Loading Step: Bump .................................................................................................................................................. 36

Concentration Time Optimization .............................................................................................................................. 37

Loading Step: Bump Time Optimization .................................................................................................................... 37

Selecting Yes Pathway ............................................................................................................................................. 39

Load Time Optimization ............................................................................................................................................. 39

Example Scan ........................................................................................................................................................... 41

Selecting No Pathway ............................................................................................................................................... 41

Save Recipe .............................................................................................................................................................. 42

Chapter 6 Data Analysis ............................................................................................................................................ 44

6.1 Data Transfer for Analysis .............................................................................................................................. 44

Appendices ................................................................................................................................................................. 45

1.1 Appendix A - Irys Maintenance ..................................................................................................................... 45

Introduction ................................................................................................................................................................ 45

Basic instrument care ................................................................................................................................................ 45

Routine Maintenance ................................................................................................................................................ 45

1.2 Appendix B - Troubleshooting ....................................................................................................................... 47

1.3 Appendix C – GUI Navigation Buttons ......................................................................................................... 48

Navigation Buttons .................................................................................................................................................... 48

1.4 Appendix D - Terms ........................................................................................................................................ 49

1.5 Appendix E - SMS Domain Names ................................................................................................................ 50

Technical Support ....................................................................................................................................................... 51

Irys® User Guide

7/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Legal Notices

For Research Use Only. Not Intended For Diagnostic Purposes.

This material is protected by United States Copyright Law and International Treaties. Unauthorized use of this material is prohibited. No part of the publication may be copied, reproduced, distributed, translated, reverseengineered or transmitted in any form or by any media, or by any means, whether now known or unknown, without the express prior permission in writing from BioNano Genomics. Copying, under the law, includes translating into another language or format. The technical data contained herein is intended for ultimate destinations permitted by U.S. law. Diversion contrary to U. S. law prohibited. This publication represents the latest information available at the time of release. Due to continuous efforts to improve the product, technical changes may occur that are not reflected in this document. BioNano Genomics reserves the right to make changes in specifications and other information contained in this publication at any time and without prior notice. Please contact BioNano Genomics Customer Support for the latest information.

BIONANO GENOMICS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL BIONANO GENOMICS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF, WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT BIONANO GENOMICS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES.

Patents

Products of BioNano Genomics may be covered by one or more U.S. or foreign patents.

Trademarks

The BioNano Genomics logo and names of BioNano Genomics products or services are registered trademarks or trademarks owned by BioNano Genomics in the United States and certain other countries.

BioNano Genomics®, Irys®, IrysView®, IrysChip®, IrysPrep® and IrysSolve® are trademarks of BioNano Genomics, Inc. All other trademarks are the sole property of their respective owners.

No license to use any trademarks of BioNano Genomics is given or implied. Users are not permitted to use these trademarks without the prior written consent of BioNano Genomics. The use of these trademarks or any other materials, except as permitted herein, is expressly prohibited and may be in violation of federal or other applicable laws.

Irys® User Guide

07/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Part Number 30047 Revision A

Date

Summary

Initial Release

07/28/2015

Released per CO 0131

• Created a reformatted, rebranded workflow-styled Irys User Guide. Replaces PN 30028.

• V1.6 New Features include:

• Generate Optimized Recipe creation

• Multicolor live molecules

• All-flowcell abort, customize recipe and cycle number

• Z Stage, autofocus, system tests

• Power supply, Micos Z Stage motor, general bug fixes

Revision History

Table 1: Current Revision History

Irys® User Guide

7/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Warning: Indicates a warning concerning operations that may lead to personal injury or potential damage to the Irys instrument if not performed correctly.

Standby: The switch does not fully disconnect the device from its power supply but places in standby state.

USB Connectivity

Ethernet Connectivity

Safety Warnings

This guide is written and organized for use by individuals skilled in the operation and function of chemical, high voltage, and industrial light sources and their subsystems, particularly excimer lasers. It is assumed that this skill and knowledge will be used when following the procedures outlined in this guide.

Following is an explanation warnings and symbols:

Safety Warnings

This product has a laser system classification of Class 1 which uses four embedded Class 3B lasers. With nominal outputs of 300 mW (532 nm), 140 mW (637 nm), 75 mW (473 nm) and 90 mW (785 nm)

optical power (nominal wavelength). A Class 1 laser is safe under all conditions of normal use. A Class 3B laser is hazardous if the eye is exposed directly, but diffuse reflections such as those from paper or other

matte surfaces are not harmful. Protective eyewear is typically required where direct viewing of a class 3B laser beam may occur. Class-3B lasers must be equipped with a key switch and a safety interlock. DO NOT OPEN the front cover of the Irys Instrument as you may come in contact with a Class 3B Laser.

1. Do not connect any item to the Irys Instrument unless specifically stated within this manual or if you are instructed to do so by BioNano Genomics Technical Support.

2. Do not use adjacent to or stacked with other equipment such that it is difficult to operate or disconnect from power.

3. Do not attach the power cord to an extension cord or to a multiple portable socket. Doing so may compromise shielding and/or grounding.

Table 2: Symbols

Irys® User Guide

07/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Chapter 1 Overview

1.1 Introduction

Complex genomes are composed of multiploid chromosomal DNA with each individual chromosome ranging from hundreds of thousands to hundreds of millions of base pairs in length. Once extracted from cells, these long biopolymer molecules form tight ball-like coils that are difficult to image and analyze. Irys uses a patented method of DNA linearization to unravel the DNA and then image it in the NanoChannel Array, allowing the structure of the genome to be imaged and then analyzed at the molecular level.

The Irys System is a groundbreaking technology for performing single molecule and whole genome mapping and analysis in a nanoscale fluidic environment designed to enable the identification of biological samples on a molecule-by-molecule basis. It is suitable for a variety of applications including whole-genome sequence scaffolding and structural variation (SV) region mapping, identification, and quantitation. The Irys System uses a straightforward and flexible process, and consists of five main steps:

1. Sample preparation — Performed using the IrysPrep sample preparation kit for your specific application.

2. Chip preparation — Performed on the Irys.

3. Molecule imaging — Performed on the Irys.

4. Data viewing and genome analysis—Performed using IrysView.

5. Data analysis — Data analysis starts with AutoDetect, the image detection software, which is performed off- instrument. Further data analysis is performed off-instrument using IrysView and IrysSolve software applications.

What's New!

• New version recipes enable 2-color.

• Version M3 and M3x are new base recipes.

• M3 and M3x Generate Optimized recipes are new user-defined configurations to improve workflow for

throughput.

• Graphical user interface enhancements include additional options for file saving in the Scan Flowcell window.

Audience and Intended Use

This guide contains information about the Irys system. It provides an overview of instrument components and software features, and full instructions for performing a run.

This guide is for laboratory personnel and other individuals responsible for:

• Operating the instrument

• Performing runs

• Performing instrument and component maintenance

• Training personnel on the instrument

Irys is intended for RESEARCH USE ONLY (RUO) and is not for use in diagnostic procedures.

1.2 Irys Overview

Irys Description

Irys is an epi-fluorescent imager capable of detecting single DNA molecules in submicron channels etched into a silicon substrate. The system is comprised of optical and mechanical components that are designed and optimized to provide imaging capabilities for single molecule analysis.

Irys® User Guide

7/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

The system is specifically designed to work with the IrysChip. The IrysChip provides a flowcell for preparing and controlling the sample for imaging. Only very small amounts of fluorescently labeled sample is needed for loading. The IrysChip is then placed on an automated stage for imaging. During the imaging process, a low-voltage electric field is used to concentrate and flow the sample through the flowcell.

Fluorescent labels bound to the sample are excited by solid-state lasers that are directed to the underside of the NanoChannel Array by mirrors, dichroic filters, lenses, and a high numerical aperture objective lens. The optical components are carefully selected and placed to optimize energy transmission that is captured via a highsensitivity, low noise EM-CCD camera. This powerful combination of lasers, optics, and camera coupled with a proprietary autofocus system and proprietary software, maintains focus while capturing images at an extremely fast frame rate. Fluorescent samples can be imaged in multiple color channels with high signal-to-noise discrimination with coordinate color data in real time.

Single Molecule Confinement and Molecular Linearization

The Irys instrument is specifically designed to detect fluorescent molecular data from within a linear NanoChannel etched into silicon. Each NanoChannel permits only a single molecule to travel through the flowcell in a straight line preventing fluorescent data overlap while reducing fluorescent noise caused by sample buffer or substrate autofluorescence.

Consistent, Disposable, High Capacity Flowcell

Taking advantage of semiconductor manufacturing, the IrysChip is designed for single use disposability. This avoids the complications of sample contamination, background fluorescence and flowcell clogging, and reduces data collection time. And with thousands of identical, parallel NanoChannels in each flowcell, tens of thousands of target molecules can be confined and imaged at the same time with nearly identical environments for each molecule.

Size Independent, Solution Phase Analysis

IrysChips are not preconfigured for any specific type of sample or pre-loaded with any specific type of buffer or polymer, avoiding any practical limit to the length of molecule that can be investigated. Samples are loaded in an aqueous buffer that maintains the samples’ original state and remains in this aqueous state throughout the analysis. This avoids surface phenomena from affecting or biasing results and allows for small molecule interactions (like protein binding events) to be investigated.

Guided Optimized Recipe Creation

The user can exit Generate Optimized Recipe by clicking the back arrow to return to the Select Recipe screen or the Abort button to return to the User Select screen. If the user was creating two Generate Optimized Recipes and already finished saving one, the saved recipe will be available in the Select Recipe screen. A dialog box warns the user that going back will cancel the Generate Optimized Recipe creation.

Multicolor Live Molecules

From single quantum dots or fluorophores to thousands of large polymeric molecules like DNA, the Irys can image any type of fluorescent molecule that can be confined within the IrysChip. Samples that contain a wide range of sizes (from less than 2 kilobase pairs to greater than 1 Megabase pair of DNA), fluorescent intensities or colors can be analyzed in the same run on the same flowcell.

Independence of Recipe and Cycle Number

With selectable multi-laser excitation and emission filters and the solid-state, semi-conductor quality of the IrysChip, the Irys ensures consistent results across a wide array of sample and feature types.

Irys® User Guide

07/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Irys Exterior

Figure 1: Irys Exterior

Front

Touch Screen Monitor: perform instrument configuration and run setup. The graphical user interface touch screen monitor has been designed with large buttons and follows an easy workflow facilitating the instrument’s operation and run execution.

Sample Loading Door and XY Stage: The sample loading door is accessible during the workflow using the touch screen monitor, and can be manually opened.

Right Side

Power button: The ON/OFF button is located on the right-hand-side of the instrument. USB: Two USB 2.0 ports are located on the right-hand-side next to the power button. Field Service Engineers

periodically use USB to perform instrument software updates.

Figure 2: Right-side view of Irys showing USB ports and power button

Rear Panel

Power Connectors: Irys requires a single 100 to 240 VAC/50 to 60 Hz (+/- 10%) rated at 1.5KVA power connection with a dedicated ground. The main power switch and the power source connection are located on the rear panel. The main power switch must be turned to the ON position before the power button on the right-hand-side of the instrument can operate.

Irys® User Guide

7/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Ethernet Connectors – The Ethernet connection is located on the rear panel. The instrument must be connected to a network server via the 1 gigabit Ethernet port before system operation can begin. This port is provided for data archival and subsequent in-depth data analysis with BioNano Genomics IrysView software suite. Data archival can be automated or done manually depending upon user preference.

Note: The user’s network may need to be pre-configured to allow instrument to successfully connect to

network. This will be coordinated before installation with the Field Service Engineer.

Figure 3: Image of rear view of Irys showing power switch and connector and Ethernet connection port

Irys Inside

Key components:

1. Ultra-low noise EM-CCD camera

2. High Speed Filter Wheel

3. Automated XY Stage

4. Lasers

5. Automated Autofocusing System

6. Image Controller

7. Electrode Bundle

1. Ultra-low noise EM-CCD camera

EM-CCD camera has single photon detection capability combined with > 90% QE (quantum efficiency). This frame transfer format delivers unequalled thermoelectric cooling, industry-lowest clock induced charge noise, and operates at 35 frames/sec (full resolution) while offering distinct speed advantages and minimizes vertical smear.

2. High Speed Filter Wheel

A high-performance servo motor with rare-earth magnets and backlash-free power transfer with filter distance travels at 23 to 30 microseconds between positions.

3. Automated XY Stage

Stages with slotted base plates and dovetail slides designed to reduce shock and vibration provide greater stability.

4. Lasers

Laser platform is made of component-based lasers at 473 nanometer wavelength and an integrated controller and power source. Beam asymmetry is <= 1:1.2, with a beam divergence of < 1.1 and a pointing stability of < 30 urads (after two hours of warm-up). Peak to peak noise is < 0.5% megahertz (after five minutes of warm-up). Laser beam safety is Class 3B with a Level 4 electrostatic discharge protection. Laser optimal temperature operating conditions are 10 to 50°C with a shock tolerance of 30 grams/6 microseconds.

Irys® User Guide

07/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Specification

Dimension

Illumination modes

Fluorescence excitation (primary), brightfield (auxiliary, for registration)

Excitation light source type

Lasers, closed-loop power controlled diode and DPSS

Number of excitation wavelengths

3 (1 molecule stain channel and 2 label channels)

Excitation wavelengths

473 nm, 532 nm, 637 nm

Brightfield source type

LED

Specification

Dimension

Imaging modes

Widefield fluorescence (primary), brightfield (auxiliary, for registration)

Emission bands 525 ± 25 nm

579 ± 20 nm 679 ± 20 nm (configurable)

Camera

TE-cooled back-illuminated EMCCD, 512x512 pixels, <1e- read noise

Objective

100 X NA = 1.4 oil-immersion

Focusing

Hardware autofocus

5. Automated Autofocusing System

Automated Autofocusing System provides continuous nanometer scale focusing control.

6. Image Control Board

Image control board synchronizes all excitation, imaging and stage movements.

Table 3: Imaging Compartment Specifications: Illumination

Table 4: Imaging Compartment Specifications: Imaging

7. Electrode Bundle

The electrode bundle mates with the IrysChip to control the motion of molecules in the NanoChannels using electric fields.

1.3 Irys Workstation Configuration

Data Network Connections and Power

Your information technology (IT) resource is responsible for:

• Preparing a stable and sustainable network connection speed of > 10 MB/sec

• Providing internet connection for BioNano Genomics Technical Support services

• Connecting an uninterrupted power supply battery backup system (UPS) or line conditioner

• Allowing local administration rights for software installation and firewall modification

• Ensuring Port 21 is open for outbound communications and remote technical support

• Ensuring TCP connections to ports 80, 443 or 8200 are open for outbound communications and real-

time monitoring

Irys ICS, AutoDetect, and IrysView share data using a networked drive and a server for data storage. The IrysSolve analysis pipeline processes data on a separate, larger Linux-based compute resource.

• Irys ICS: Irys ICS (Instrument control software) performs the runs based on the recipe selected and parameters specified in the Select Recipe screen.

• AutoDetect: AutoDetect processes image files generated by Irys ICS and creates assemble-ready digitized molecule and label files for further analysis through IrysView and IrysSolve.

• IrysView: Genomics analysis software used to analyze and visualize data generated by Irys.

Irys® User Guide

7/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Environment Specifications

Dimension

Operating temperature range

20 to 27° Celsius

Dimensions: Height Depth Width

35.6 cm (14 inches) 65 cm (25.5 inches)

86.4 cm (34 inches)

Mass

77.3 kg (170.5 pounds)

Operating altitude above sea level

0 to 2000 m (0 to 6562 feet)

Operating humidity range

15 to 65 % relative humidity

Sample environment operating temperature

Room temperature

Shipping and storage temperature range

-28 to 50° Celsius

Shipping and storage humidity range

0 to 90 % relative humidity

• IrysSolve Computational Solutions: IrysSolve is the data analysis pipeline which performs automated de novo assembly and structural variation detection in an integrated pipeline and outputs visualization-

ready assemblies in IrysView.

Computer System Workflow

Irys ICS requires one computer to process image detection and visualization of the genome maps using IrysView; and one computer in a cluster or cloud-based environment to perform data assembly using IrysSolve.

The typical Irys workflow includes Irys, a dedicated computer for image detection and analysis through IrysView, a dedicated computer for IrysSolve, and a storage server.

Figure 4: Computer System Workflow

1.4 First Use

A Field Service Engineer will complete performance testing to ensure proper installation. Calibration protocols and setting adjustments are performed during the installation and training sessions.

Note:

Moving the instrument carries a high risk of misaligning the optics of the instrument, which will result in a substantial degradation in imaging quality. If you require relocation of your instrument, please contact

support@bionanogenomics.com.

Table 5: Environment Specifications

Do not attempt to move the instrument without the assistance of a BioNano Field Service Engineer.

Irys® User Guide

07/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Electrical Specifications

Dimension

Operating supply voltage

100 to 240 VAC

Line frequency

50/60 hertz

Power

≤ 250 watts

Power connector

Region-specific power cord supplied

Fuse type

T3A

Catalogue Number

Product

FC-020-01

IrysChip v2 (1 chip)

Cat #

Product

RE-011-10

IrysPrep 1-Color Assembly Reagent Kit (10 reaction)

RE-111-10

IrysPrep DNA Stain (10 reaction)

Material

Reference or Catalog Number

AmScope Sparkle Microscope Optical Lens Cleaner

Cat #CLS or equivalent non-ammonia, non-alcohol cleaner, or high-purity methanol or isopropanol for cleaning the Objective

Microscope immersion oil

Zeiss Immersol® 518 F halogen-free

Table 6: Electrical Specifications

1.5 User Supplied Consumables

IrysChip

At the heart of the Irys technology is a disposable NanoChannel fluidic IrysChip within which DNA molecules move and are confined in an orderly, linear format. Each IrysChip (FC-020-01) has two flowcells capable of processing two DNA samples.

Table 7: IrysChip

IrysPrep

IrysPrep reagents are used for DNA extraction and labeling to prepare samples for imaging. Each IrysPrep Kit (RE-011-10) comes with enough material to perform 10 1X-nick label repair stain reactions (each requires 300 ng of raw DNA).

Table 8: IrysPrep

Other User-Supplied Consumables

The following table lists any other user-supplied materials needed to run the Irys workflow.

Table 9: Consumables

1.6 Run Type Recipes (M2, M3, M3x and Optimized)

There are three base recipes in Irys: M2 - legacy recipe; M3 - new high throughput recipe; M3x - modified high throughput recipe. There is also the ability to create Optimized Recipes (A and B). When any base or optimized recipe is selected, the user can customize laser colors and concentration time for individual flowcells.

Irys® User Guide

7/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Chapter 2 Irys Instrument Control Software

2.1 Best Practices

Turn off when not in use.

2.2 Irys ICS Main Screen Overview

Figure 5: Select User Screen

Select User Screen

The Select User screen is the opening screen to log in and proceed to set up a run, add new users, or select or modify an existing user. The screen also serves as a focal point for shutting down the application. Labels A through F are:

A. User Settings: Pressing the User Settings button accesses a list of logged users from which to log in, or

provides the menu to create a new user.

B. Status of Events: Pressing the green check mark accesses the Events screen. Here users will find a list of

events, warnings and errors that may have occurred on the instrument while the application was in use. The

green check mark changes to a red exclamation mark or an hour glass to communicate when the instruments’

status changes. For more information, see the Troubleshooting section. The Events log is available from all

views throughout Irys ICS accessing it does not interrupt any work being done in Irys ICS. C. Help: Pressing the question mark accesses the online user guide. D. Exit: Pressing this button returns user to the Select User screen. While on the Select User screen, pressing

this button safely shuts down the Irys ICS application. E. Navigation Arrows: The Forward arrow button proceeds the workflow to the next step. The Back arrow button

returns the user to the previous screen. F. Clean: The Clean button initiates the cleaning cycle. This resides here as a precautionary button in case the

previous user neglected to clean after their run (not recommended). G. Select User: Select a User name from the drop-down menu or leave <Add New User> highlighted and

proceed to the User Settings button to create a new user profile.

Irys® User Guide

07/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Chapter 3 Starting a Project

Figure 6: Workflow Overview

3.1 Irys System Project Workflow Overview

The workflow is divided into 5 discrete steps:

1. Sample Preparation: Samples must be prepared in advance according to IrysPrep instructions.

2. Chip Preparation: The IrysChip is stored at -20ºC. The IrysChip must be warmed to room temperature before

performing the run. Once warmed, any moisture on the IrysChip packaging must be dried off before removing the IrysChip.

3. Instrument Preparation: After the sample has been prepared and the IrysChip is at room temperature, the

instrument can be turned on and the run preparation begun. A user is assigned to the run data, the IrysChip is loaded into the instrument, and sample information is saved. The sample is then loaded into the IrysChip following the on-screen instructions.

4. Performing the Run: After all sample information is entered and the sample is loaded, the run recipe is

chosen and the run is started. The instrument will verify run and data quality as the run proceeds.

5. Data Analysis: After the run completes, the IrysChip is removed and the sample data are exported off Irys to a

workstation for further analysis with IrysView and IrysSolve software.

3.2 Sample Preparation Summary

Samples must be prepared in advance according to IrysPrep reagent kit instructions. Log into

www.bionanogenomics.com/support/Forums/ to access the current protocol.

IrysPrep Plug Lysis Long DNA Isolation (BNG PN 30026)

The IrysPrep Plug Lysis protocol involves immobilizing a low complexity biological material (animal cells, nuclei from plant and animal tissue, or bacteria) in an agarose matrix for subsequent Proteinase K digestion followed by RNAse treatment and washes. The plug is then melted and the agarose matrix is digested in order to recover megabase-containing genomic DNA, which is further cleaned by drop dialysis.

As you will note in the IrysPrep Plug Lysis protocol, you will need to allow 2 to 3 hours of pre-processing of tissue to harvest nuclei before embedding in agarose before the Proteinase K digestion step and before stabilizing the DNA in plugs. RNA removal and DNA recovery occur on the second day. DNA quantitation is performed on the third day following overnight equilibration at room temperature to achieve DNA homogeneity.

Irys® User Guide

7/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

IrysPrep Labeling-NLRS (BNG PN 30024)

The IrysPrep Labeling-NLRS assay provides sequence-specific labeling of megabase-containing gDNA for Irys mapping at the single molecule level. Sequence specificity is provided by the Nickase. Labeling is carried out by a limited-drive nick translation process in the presence of a fluorophore-labeled nucleotide. The labeled nicks are repaired to restore strand integrity. The labeled DNA is stained for backbone visualization (NLRS). Labeled nicks are seen as dots on a string.

IrysPrep Human Blood Protocol (BNG PN 30033)

The IrysPrep Human Blood Protocol targets recovery of white blood cells using differential red blood cell lysis. The resulting white blood cells are quantitated, immobilized in agarose plugs, and subjected to plug lysis treatment for megabase DNA recovery. White blood cell quantitation enables targeting the proper number of cells per plug to stay within the optimal range for plug lysis.

3.3 IrysChip Preparation Summary

The IrysChip is stored at -20ºC. The chip must be warmed to room temperature before performing the run. Once warmed, dry off any moisture on the chip packaging before removing the chip.

Preparing IrysChip

1. Allow the IrysChip to warm up in its pouch and allow it to equilibrate to room temperature for at least 30

minutes prior to use.

Note: Removing the IrysChip from its pouch before it has warmed to room temperature will cause

performance problems or chip failure.

2. While the IrysChip is warming, turn on the Irys instrument if it is not yet on.

3. Wipe off any external moisture from the pouch before opening.

4. Prior to loading the IrysChip onto the stage, inspect the underside of the chip for dust or other debris which can

impact optical performance. A lens paper should be used to gently wipe the underside of the chip if necessary.

Note: Do not touch the underside of the IrysChip with anything other than a lens paper. Do not use dusting

gas to clean the IrysChip.

3.4 Instrument Preparation Summary

Follow the instrument boot-up process to initialize software and hardware components.

System Startup

1. Connect the power and communication cables.

2. Open the system LCD display by lifting the front edge of the display until it is in a full up-right position. Then,

rotate the bottom of the screen until it is in a convenient viewing position.

3. Turn ON the instrument using the main power switch located on the lower right-hand side.

Irys® User Guide

07/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Figure 7: Power Up

During the power up, the touch-screen LCD should illuminate. The instrument will execute a boot-up sequence and launch to the LCD’s desktop. Select the Irys Instrument Control Software (ICS) application icon located on the desktop to launch Irys ICS.

Upon launching Irys ICS, the system will perform self-diagnostics (initialize hardware, validate firmware, validate hardware, and perform stage calibration) during which time the system start-up screen displays.

Figure 8: Irys Startup Screen

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3.5 Logging In

After initialization, the Select User screen displays for logging in. Here you can create a new user or select the user’s name to log in. If a password is required, a pop up window will ask for the password.

Figure 9: Select User Screen

Figure 10 Enter Password (optional)

Entering the incorrect password will deny access. If the user does not require a password, Irys will automatically log in as the selected user. The system is designed to allow each user to create a password in order to prevent users from accidentally logging in as the wrong user and overwriting another’s files. It is not a security feature; the password log is not a hidden file.

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Select Existing User or <Add New User>

Click on the <Add New User> drop-down menu item to create a new User Name. Once selected, the User Settings screen opens to configure user-specific settings for the new user, after which time the new User Name will appear in the Select User drop-down menu and in the workflow settings.

Figure 11: User Settings

Configuring the User Settings and Remote Data Storage Location

Data transfer to an external client server occurs automatically during and after a run as long as a server location has been specified. In order to export data from the instrument to an external workstation, the remote data storage location must be mapped to the instrument.

1. First Export Directory: This location is the primary location Irys ICS will attempt to export data to. The directory

can be a local or remote destination. If the destination is a remote location, Irys ICS will require network connectivity in order to export data to remote locations.

2. Second Export Directory: This location is the backup location Irys ICS will attempt to export data to when

unable to export to the first location.

3. Email Notification: In order to receive email notifications on the status of the instrument or events of interest, a

user may opt to enter an email address where Irys ICS will send messages to. The user can test this functionality with the “Test” button.

4. SMS Notification: In order to receive notifications on the status of the instrument or events of interest to a

cellular device, a user may opt to enter an SMS address where Irys ICS will send messages to. The user can test this functionality with the “Test” button. This address is a ten* digit number followed by the provider domain. Example for T-Mobile USA: XXXXXXXXXX@tmomail.net

Note: May be country specific. See the Appendix F for most popular USA carrier’s SMS address or a free

web link to query your carrier’s code.

5. Password: A user may provide a password for account protection. This field is optional but will be required for

this user login until turned off. This is not intended as a security feature.

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Chapter 4 Performing a Run

1. After selecting the user, press the Forward arrow button to proceed to the Load Sample screen.

Note: There will be a delay in going from the Select User screen to the Load Sample screen while the

system calibrates.

2. The XY Stage will eject and the Objective will rise.

Figure 12: Proceed to Load Sample Screen

4.1 Loading the Sample

Once the XY Stage is fully ejected, place the empty IrysChip onto the XY Stage cutout with the label side up. There is only one orientation. Secure the IrysChip in place by sliding the lock bars over the corners of the chip.

Figure 13: Load IrysChip

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Load Inlets

Note:

The Load Inlet checkbox activates a 2-minute timer; while the Load Outlet and Oil Objective

checkboxes are task completion checkboxes, which move the workflow to the next phase.

1. Using a standard pipettor with a P10 pipette tip, press the pipette plunger down and hold at the first stop. Lower

the pipette tip into NLRS (Nick-Label-Repair-Stain) DNA (IrysPrep Assay NLRS BNG 30024) sample and gently stir the sample with the tip. Slowly release the plunger to extract 8 µL of the sample.

2. Slowly dispense the 8 μL of the NLRS DNA from the loaded pipet into the Inlet wells of each flowcell on the

IrysChip:

• Align the tip over the respective Inlet well and slowly depress the plunger until the bottom of the Inlet well is coated with sample solution. Then slowly dispense the remainder of the sample in the pipettor again to fully fill the Inlet well. Repeat with Flowcell 2.

• The Inlet wells are located on the front of the IrysChip, closest to the operator. Outlet wells are located towards the back of the IrysChip, furthest from the operator.

Note: Always keep positive pressure on the plunger to avoid introducing bubbles.

Figure 14: Load Inlet Well

3. On the Load Sample screen, click the Load Inlet checkbox to start the 2-minute timer (status bar indicator is filled at 2 minutes).

Note: The timer is set for two minutes to allow proper loading and to prevent trapping air bubbles in the

NanoChannels.

4. A virtual popup keyboard opens. Type the Flowcell Sample name in the text box and press Enter.

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Figure 15: Load the Inlets, Outlets, and Oil the Objective

5. After the 2-minutes timer is complete, proceed to load the Outlet well.

Note: If only one flowcell is used, Irys ICS will prompt the user to confirm they are running only one flowcell.

Load Outlets

1. Slowly dispense 8 μL of the NLRS DNA into the Outlet well of each flowcell on the IrysChip using the same extraction and dispensing process described in the Load Inlet section.

2. Select the respective Load Outlet checkbox when complete. No 2-minute timer is necessary.

Note: Use the same NLRS reaction for both Inlet and Outlet wells of the same flowcell to prevent problems

with ionic variation between the two wells.

Cleaning and Oiling the Objective

Note: Failure to clean the Objective will result in reduced system performance.

Figure 16: Oil the Objective

1. To wipe off dust or excess oil from the Objective, use a lens tissue. Fold the tissue in half, hold on the left and right side and gently wipe the lens with the open center area of the tissue. Avoid touching the top of the Objective with anything except the tissue.

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2. Then using another clean tissue, put a few drops of Sparkle on the tissue and following the same process to wipe the Objective, clean with Sparkle to remove any excess oil. Dry the Objective with new clean tissue.

3. Using the Zeiss oil and paddle, dispense one drop of oil onto the Objective lens.

4. If a bubble forms when oil is dispensed, wipe off the Objective, clean with Sparkle, and repeat the application of the oil.

Note: Do not apply excessive oil to the Objective. Enough oil should be applied to form a rounded droplet that

does not run down the side of the lens.

5. Select the Oil Objective checkbox when finished oiling the Objective.

Figure 17: Load Sample Screen Complete

Forward

1. After all required tasks are complete, press the Forward button to proceed to the Focus & Register screen.

2. The Status icon will change from the green checkmark to the transparent hourglass timer to indicate a process is in action.

Note: There are intermediate steps described below, which alert the user if the loading is out of specification.

Sample Detect

1. When the XY Stage retracts, Irys will check the IrysChip cartridge for a present sample.

2. If a sample is not detected, Irys will notify the user and ask to fix the problem or continue with the workflow.

3. If the user selects to fix the problem, Irys will eject the XY Stage to have the sample loaded.

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Note: Irys ICS will prompt the user with a warning if it detects no sample or a low sample volume and ask the

user to load the sample flush with the well.

Figure 18: Error Message for Low Volume

4.2 Barcode

1. After the Sample Detect step, Irys reads the IrysChip barcode and moves the workflow to the Focus & Register screen.

a. If unable to read the barcode because of smeared ink or missing IrysChip, Irys will prompt the user to

enter the barcode Part Number, Lot Number and Serial Number manually in the Barcode screen.

Figure 19: Manually Enter Barcode

Forward

1. After all required tasks are complete, press the Forward button to proceed to the Focus & Register screen.

2. The Status icon will change from the green checkmark to the transparent hourglass timer to indicate a process is in action.

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4.3 Focus & Register

The Focus & Register workflow engages the camera and raises the Objective to the focal plane of the IrysChip. The Focus & Register screen allows for manual verification of system focusing and registering; and manual intervention, if necessary.

1. Focus: When the workflow progresses to the Focus & Register screen, the camera and Objective automatically engage onto the Fiducial. If the image is not in focus, follow the steps below to troubleshoot:

a. Is the focus z-position inaccurate?

i. If so, press Stop Lens and then press Search For Focus, which engages a search algorithm to find focus.

Allow the search to proceed for approximately two minutes. If still not in focus, press the Stop Lens button and manually attempt to find focus using the Up and Down Arrow buttons. If focus is achieved, press the Autofocus button. If focus is unsuccessful, reoil lens and try again. If still unsuccessful, contact Support. Once the image is in focus, proceed to step 3.

b. Is the oil on the Objective distorting the clear image?

i. If so, press Reoil Objective. Then follow the steps for cleaning and oiling the Objective. Then press the

Retract Stage button (previously the Reoil button) to initiate the automatic focusing process again. This action can be repeated until the Objective is properly oiled. Once the XY Stage retracts, return to step 2 and allow Irys to focus on the IrysChip.

2. Center and Register: Once the Objective is in position and focused, manually center on the Fiducial by tapping the Fiducial touchscreen in the direction to center the red calibration marker onto the intersection point.

3. When the Fiducial image is in focus and centered, press Set Fiducial.

Note: A red calibration marker indicates the image is in focus. The Set Position and Set Fiducial

buttons will become active once focus is achieved.

4. Then press Find Position 1. (A successful Find Position 1 will present the red calibration marker centered over the intersection of the single tie and crossarm (cross).

a. If the Find Position 1 is not centered, find the Position 1 tie and crossarm manually.

i. Tap on the Fiducial image in the quadrant you want to move the red calibration marker to until you

center the calibration marker over the Position 1 tie and crossarm.

ii. Then press Set Position 1 to register.

Figure 20: Proper Registration of Fiducial Mark

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5. Repeat step 4 for Find Position 2 centering the Position 2 tie and triple-crossarm. a. Then press Set Position 2 to register.

6. Press the Forward button to go to the Select Recipe screen.

4.4 Select Recipe

1. Select a recipe for each flowcell. There are three base recipes in Irys, along with the ability to generate optimized recipes from any of the base recipes. When selected, the user can customize label colors and concentration time for individual flowcells.

a. M2: Legacy recipe. b. M3: New high throughput recipe to be used alongside sample with increased DNA stain. c. M3x: Modified high throughput recipe. d. Generate Optimized Recipe A & B:

i. The recommended workflow consists of generating two independent Optimized Recipes, one for

each flowcell (e.g., Generate Optimized Recipe A for one flowcell and Generate Optimized Recipe B for the other flowcell. When this is the selection, two instances of optimization take place, one for each flowcell. This is the recommended workflow whether each flowcell is loaded with different or the same samples.

ii. The system does accommodate running any combination of recipes. For example, if Generate

Optimized Recipe A is selected for both flowcells, a single optimization recipe is generated and applied to both flowcells.

Note: All user-optimized recipes Note: Users may edit non-default recipes by clicking on the recipe name. A popup window opens up

will populate below the base recipes in alphabetical order.

that allows the user to edit the recipe’s concentration time, concentration voltage, bump time, and load time, as well as enabling or disabling the red and green lasers. The recipe can only be saved as a copy with a new name. The popup window also allows the user to delete the recipe. Recipes that do not have a base recipe format cannot be edited.

2. Select a cycle count for the IrysChip: The cycle count will always be the same for both flowcells. a. The default Cycle Count is set to 30 cycles. Any value selected here will apply to both flowcells. Users

may change the cycle count by pressing the up and down arrow buttons.

3. Set Automatic Pauses after appropriate cycles if necessary: Automatic Pause is an optional field not required to

proceed. The user may select up to two automatic pause cycles, and if no cycles is/are selected this field will display ‘N/A’. Users may change the cycle using the up and down arrow buttons. Pressing the up and down arrow buttons will move the count to the desired cycle count number.

a. Recommendations:

i. For M2 Base or M2 Optimized Recipes, run 20 cycles with an Automatic Pause to rehydrate/reoil at

cycle 6.

ii. For M3 and M3x Base or M3 and M3x Optimized Recipes, run 30 cycles with an Automatic Pause

to rehydrate/reoil at cycle 8.

Note: Although it is recommended that users rehydrate/reoil at specific cycle counts, for convenience users

may rehydrate/reoil anytime between cycle 6 and 10 for all recipes.

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Figure 21: Select Recipe Screen

4. Set/Configure Flowcell Settings: a. Red labels: Select Red labels (analysis of the Red Label Channel is currently not supported in IrysView). b. Green labels: Select Green labels. c. Conc. time: Input a concentration time in seconds based on measured NLRS concentration.

Note: NLRS sample concentration should be 5 ng/µl for optimal performance. See the IrysPrep NLRS

Experienced User Card (PN 30024).

5. Press the Forward button to go to either the Optimized Recipe or the Scanning Flowcell screen. For

Optimized Recipes, see the section on Generating Optimized Recipes for further instructions.

Note:

Once an Optimized Recipe is saved, Flowcell Settings cannot be edited for that recipe.

4.5 Scanning Flowcell

The Scanning Flowcell screen allows users to monitor the loading and scanning of the IrysChip. This screen also allows the user to select to Pause the Run, Rehydrate/Oil, or Abort the run.

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Figure 22: Scanning Flowcell

1. Pause Run: To Pause, press the Pause Run button. The flowcell status will change to “Pausing Run at end of

cycle” and the system will stop scanning at a stable point, which may require up to 30 minutes to reach.

Once the system is paused, the user may select to either Rehydrate/Oil or Continue Run.

a. Continue Run: Press the Continue Run button to cancel the pause and continue scanning.

Note: The Abort feature is unavailable during Pause Run. Irys will pause automatically if

a focus failure

occurs. Focus failures are logged in the Event Log and marked with a red exclamation mark.

2. Rehydrate/Oil: After the Pause, press the Rehydrate/Oil button to eject the XY Stage to the load position. a. Promptly clean the oil from the Objective and the underside of the IrysChip cartridge. b. Rehydrate the sample with deionized water until the sample is flush with the well. c. Reoil the Objective. d. Press the Forward button to retract the XY Stage and proceed to the Focus and Register screen (the

user must register the Fiducial and positions once again).

e. Press the Forward button again to resume scanning.

i. See the section “Focus and Register” for help on using the tools on the screen.

4.6 Results

The Results screen serves as a transitional screen for the user and summarizes the run. Information on the screen is organized per flowcell and contains the user who ran the scans, the name of the sample, which recipe was used, how many cycles completed and the time of completion.

1. The Results screen provides a summarized scan information by flowcell: a. User name b. Sample name. c. Recipe name d. Number of Cycles used e. Time of Completion

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Figure 23: Results Screen

Forward

Pressing Forward arrow advances to the Clean screen.

4.7 Clean

The Clean screen outlines the tasks the user must follow in order to prepare the instrument for another run or for shutdown. When entering this screen, the XY Stage will eject and the Objective will rise to the Oil position.

Figure 24: Clean Screen

1. Remove the chip from the stage: If there is an IrysChip cartridge on the XY Stage, remove it here and select

the checkbox.

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2. Clean Oil from the Objective: This task only becomes enabled after the previous task has been completed. If

there is oil on the Objective, clean it here following the proper guidelines for Objective care. Refer to Cleaning and Oiling the Objective in the Load Sample section (section 4.1).

3. Insert the Wash Cartridge: This task only becomes enabled after the previous task has been completed.

Insert the wash cartridge filled with deionized water (approximately 300ul) and check the task complete. Once checked, Irys will retract the XY Stage and begin washing the electrodes. If the incorrect wash cartridge has been inserted, Irys will extend the XY Stage and request the correct cartridge be inserted. A timer of five minutes will begin once the task is complete.

4. Dry Electrodes: This button only becomes enabled after the wash task has been completed and the five

minutes have lapsed. Click ‘Dry Electrodes’ to command Irys to eject the XY Stage and dry the electrodes. It is recommended that a user stand by for this task as solution may drip from the electrodes. A timer of five minutes will begin once the button in pressed.

5. After the 5-minute timer to dry the electrodes has completed:

a. Press the Forward button to return to the Load Sample screen with the XY Stage extended to load

another IrysChip (See Load Sample section); or

b. Press the Exit button to return to the Select User screen.

Note: If an IrysChip was accidentally left on the instrument for multiple days, or if the electrode bundle

suspected of being contaminated, contact Customer Support.

4.8 After a Run

After all data collection and transfer is complete, Irys is ready to either start a new run or to be shut down properly.

Load Another Sample

To load another sample, press the Forward button from the Clean screen. This will return to the Load Sample screen and eject the XY Stage. Follow the Run Instructions section to process another sample.

Shutting down the Instrument

To remove all power from the instrument, turn off the main power breaker on the back panel.

Note: Leaving the main power to the system on when not in use will reduce the lifetime of the lasers.

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Chapter 5 Optimized Recipes

5.1 Creating an Optimized Recipe

Optimize key parameters in the Recipe to maximize throughput per cycle. Using M2 or the new M3 and M3x recipes as the Base Recipe for the optimization process, follow Irys ICS through a series of guided steps to customize:

 Concentration Voltage  Concentration Time  Bump Time  Load Time

Getting Started:

Users may create Optimized Recipes for each flowcell by selecting Generate Optimized Recipe A or Generate Optimized Recipe B from the Select Recipe screen. By selecting Generate Optimized Recipe (A or B), the option to configure the flowcell’s settings become available, similar to selecting a base recipe. Users may select the label colors appropriate for their sample and enter a concentration time in seconds.

Note: Analysis of red label channel is not supported at this time.

Figure 25: Select Recipe Screen

It is recommended that the user generates two independent Optimized Recipes, one for each flowcell (e.g., select Generate Optimized Recipe A for one flowcell and Generate Optimized Recipe B for the other flowcell).

Workflow

When Generate Optimized Recipe is chosen for either flowcell, the user will be taken through a 20 to 45 minute step by step process to determine a customized cycle.

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Figure 26: Optimized Recipe Workflow

Optimization Setup

The Generate Optimized Recipe user-interface is designed to guide users through the optimization process with ease.

Note: The user can exit Generate Optimized Recipe by pressing the Back arrow button to return to the Select Recipe screen or the Exit button to return to the Select User screen. If the user was creating two Optimized Recipes and already finished saving the first one, the saved recipe will be available in the Select Recipe screen. A dialog box warns the user that going back will cancel the Generate Optimized Recipe step.

Figure 27: Description of User-Interface

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Labels A through G

A. Process Step: Status indicates the state of the optimization process, which includes: Focusing, Preparing

Chip, Loading Step, Imaging Example Scan, and Assembling Mosaic.

B. Detail Information: In the description, users are asked to make a series of judgments and selections based on

observations in the live image screen.

C. Proceed Step: Action Buttons are OK, Yes, and No. These action buttons lead to the next step in the

Generate Optimized Recipe screen.

D. Camera Live Screen: The live image exists in two states: Camera ON (active) and Camera OFF (inactive).

In the Camera ON state, the images will be blue to reflect the laser color used for the DNA backbone, and users will be asked to make judgments during the optimization steps. When the live image is active, users may also use the touchscreen to move vertically and horizontally similar to the Focus and Register screen. In the Camera OFF state, all touchscreen-based movement is disabled and the live image will indicate Camera

OFF showing the last image taken by the camera. There are some restrictions on vertical movements. E. Flowcell: This displays the flowcell being used to generate an Optimized Recipe. F. Revert to Live: Pressing the Home button brings live image back to the instrument calibrated home position.

Home is the starting position when the live image became active for a particular step. G. Time Remaining: The timer shows the time remaining to complete a step. H. Navigation Buttons: Press right arrow to proceed to next step; press left arrow to go to previous screen.

Step 1: Selecting a Base Recipe

M2, M3 and M3x are base recipes that can be optimized. M2 is the legacy recipe offered in previous versions of Irys; and M3 is the new high-throughput recipe. M3 is the recommended base recipe to be used with IrysPrep NLRS Experienced User Card (PN 30024); M3x modified high throughput recipe is used in certain cases. See

http://www.bionanogenomics.com/support/.

1. Use the drop-down menu to select a base recipe and press OK to proceed.

Note: The Generate Optimized Recipe will optimize the following parameters: Concentration Voltage,

Concentration Time, Bump Time, and Load Time.

Figure 28: Selecting a Base Recipe

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Step 2: Focusing

After selecting a Base Recipe, the Process Step text indicates “Focusing…” as the XY and Z stages move to the appropriate flowcell and begins the focus and optimization stage.

Figure 29: Focusing

Step 3: Initial Loading

The three parts to the Initial Loading step are:

 Concentration

 Spike

 Bump

After focus is achieved on the flowcell, the Concentration step begins. At the beginning of Concentration, a timer starts that corresponds with the concentration time from the Select Recipe screen. The Live Image will be inactive for the first minute of Concentration.

Figure 30: Initial Loading: Camera Off

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DNA Leakage

Leakage zone

Once the Live Image resumes (Camera ON), the user will see the Live feed of the IrysChip concentration zone (blue gradient at bottom border of Live Image screen) and leakage zone (blue gradient entering remaining portion of the Live Image screen). The blue gradient is the Live Image of the laser-illuminated DNA backbone; this laserillumination is at a low exposure to minimize photo-degradation.

Loading Step: Concentration

Once the user is able to visualize the DNA (horizontal blue band at the bottom of the Live feed window), Irys guides the user through Concentration optimization. Irys asks the user to press the OK button each time they see a vertical stream of DNA entering the leakage zone. Pressing OK will decrease the force, which stops the stream. There is a five-second period after each force decrease during which the OK button is disabled.

Figure 31: Concentration: Force Optimization

Figure 32: Example of DNA Leaking into Runway

Loading Step: Spike

Once the user completes the Concentration steps, Irys moves to the next loading step, Spike, where the DNA that is concentrated at the front (horizontal blue band indicates DNA is gathered at edge of the Inlet) is loaded into the Flowcell. The Live image becomes inactive, displaying Camera OFF.

Note: Because the Spike occurs quickly, there is no timer for this step.

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Figure 33: Spike

Loading Step: Bump

Following Spike, the image becomes active and user is asked to press the OK button when the DNA reaches the target zone, which will complete the bump measurement. The number of elapsed seconds for the DNA to reach the target zone is recorded as a Bump Time Measurement Trial. If the user does not press the OK button, indicating DNA has arrived at the target zone, Irys initiates an automatic 60-second timeout and returns to the Concentration step.

Note: At times, the target zone may be difficult to see. With training, these target zone become recognizable.

Figure 34: Bump Time Measurement

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Sufficient DNA

Insufficient DNA

Concentration Time Optimization

The user is then asked to determine if there was sufficient DNA to make an accurate assessment of Bump Time. Pressing the Yes button displays the Bump Trial history, while pressing the No button adjusts the DNA concentration time by adding 50 seconds. The last concentration time used before accepting the Bump Time

will be the time saved for that Optimized Recipe.

Figure 35: Concentration Time Optimization

Note:

can move horizontally along the IrysChip view to more accurately assess the Bump Time measurement.

The live image remains active (camera ON) during Concentration Time Optimization so that the user

Figure 36: Example of Not Enough DNA

Note:

perform a preparation step and then proceed to Concentration. Irys live image will deactivate during preparation steps.

If the user chooses to repeat Bump Time measurement with a longer concentration time, Irys will first

Loading Step: Bump Time Optimization

Measure Bump Time at least twice to get an accurate assessment. The user will need to press the No button to begin additional Bump Time trials.

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Each Bump Time trial alternates between one of two horizontal positions on the IrysChip: either one-third of the way across, or two-thirds across the flowcell. Before each Bump Trial, the DNA will be concentrated and spiked into the IrysChip, without the live view from the Concentration Voltage Optimization step.

Note:

For each additional Bump Time Optimization, there is an additional one minute preparation step. Up to four of the most recent Bump Time measurements are displayed under Recorded Bump Time(s); and Optimized Bump Time is displayed as an average of all recorded Bump Trials in a user-editable field. It is recommended that the user continue to measure Bump Time until two consecutive measurement are within 2 seconds of each other.

Figure 37: Bump Time Optimization

Users have the option to finish the Generate Optimized Recipe creation after Bump Time optimization; however, optimizing the Final Load Time is recommended. If Yes, continue to Yes Pathway. If No, continue to No Pathway.

Figure 38: Option to Stop Optimization After Bump Time

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Selecting Yes Pathway

By pressing the Yes button, the user has committed to performing the Intermediate Loading steps, where the DNA is moved to the load position.

Note: The amount of time it takes to perform the intermediate loading steps depends on which Base Recipe

was used and can take up to 15 minutes.

Figure 39: Intermediate Loading Steps

Load Time Optimization

Once the intermediate loading steps have completed, Irys will wait for the user to indicate that they are ready to

optimize the Load Time by pressing the OK button.

Figure 40: Begin Load Time Optimization

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Loading zone

During Load Time Optimization, the live image shows an area three fields of view (FOV) away from the outlet. The user is instructed to press the OK button to complete load measurement when the wave of DNA reaches the top of the screen. The elapsed time is the Optimized Load Time.

Note: The maximum measurable Load Time is 120 seconds.

Figure 41: Optimized Load Time Measurement

Users are then asked if the DNA loaded successfully.

Note: The live image remains active (Camera ON) during this step so that the user can move horizontally

along the IrysChip to more accurately assess the Load Time measurement.

Figure 42: Determine if Load Time Measurement was Successful

Pressing the No button means a timeout has occurred before DNA is present or the wave of DNA is too sparse. No takes the user back to Concentration, Spike, and Bump Time Optimization. Pressing the Yes, while pressing the Yes button takes an example scan to assess the overall loading.

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Example Scan

Irys takes backbone-only raster scan images of the NanoChannel Array. Each image is shown in the live view, and the images are then compiled into a mosaic showing the entire array and marking the entrance and exit.

Figure 43: Imaging Example Scan

Figure 44: Assembling Mosaic

Selecting No Pathway

By pressing the No button in Figure 39, when asked to optimize the final load time, begins a timer as Irys performs a series of preparation steps to start the run.

Note: Mosaic will not be generated for “No Pathway.”

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Figure 45: Preparing Chip to Save and Run Optimized Recipe Post Bump Time Optimization

Save Recipe

The final Optimized Recipe values are displayed in editable text boxes, including the Load Time, which retains its default value. The user may save the recipe with a custom name in the first text box.

Pressing the Yes button saves the recipe while pressing the No button returns the user to the Select Recipe screen. If the recipe is saved, it will be also be available in the Select Recipe screen for future runs.

Note: It is important to give each Optimized Recipe a unique name; otherwise any pre-existing recipes with

the same name will be overwritten.

Figure 46: Save Optimized Recipe Post Load Time Optimization

Irys will continue to either start the Run or begin Optimization for Flowcell 2, depending on the flowcell configurations in the Select Recipe screen.

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Figure 47: Custom Recipe is saved

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Chapter 6 Data Analysis

6.1 Data Transfer for Analysis

Data analysis is performed off the instrument using the IrysView analysis software package provided with the system. Please see the IrysView user guide for more information. User-developed software may also be used.

1. Irys ICS automatically exports the data (unless ICS is closed) to a pre-selected network storage location during and after the run.

2. To set or change the server export location, press the User Settings button. Type the drive and directory for the primary data export location in the First Export Directory box. Then type a drive and directory for a backup export location (in case the first location does not have sufficient free space) in the Second Export Directory.

Note:

locations be designated as writable by the instrument but read only for other users so that the raw information is not accidentally lost.

Once a transfer occurs, this data will be removed from the local drive. It is suggested that the export

Irys® User Guide

07/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Appendices

1.1 Appendix A - Irys Maintenance

Introduction

Several maintenance and troubleshooting procedures are outlined within this section to help the user maintain the optimal performance of the Irys system, to properly store the instrument for extended idle period; and to troubleshoot most problems a user may encounter while using the system. The user is encouraged to perform these routine maintenance procedures periodically, as specified, to ensure top performance.

Basic instrument care

a. Please do not lean on or use the top of the instrument, sample door, or XY stage as a work bench. b. Please keep the instrument area clean. It is important to keep the instrument in a clean, dust-free

environment. Dust can build up on optical elements, causing degradation of data quality, or disrupt electronic components.

c. Please use proper technique when oiling objectives. Only a small drop of oil (just enough to cover the

optical lens element) is needed. Be careful not to over oil, as this can cause leakage into the optical beam path or other components and lead to poor image quality or mechanical issues. Please see training video for reference.

d. Please make sure to clean the oil off the objective and XY stage after every run (within 1 hour of run

completion) or before every rehydration to limit the possibility of oil leakage into the instrument. Please see training video for reference at http://www.bionanogenomics.com/support/training.

e. Cleaning electrodes after every run is mandatory to prevent buffer component/contaminants build up

resulting in decreased loading performance, and to eliminate sample cross-contamination. Please use the wash chip provided to clean the electrode bundle using ddH2O. Please empty and clean the wash cartridges after use.

Routine Maintenance

Note: Read all safety information and warnings before operating or performing routine maintenance on the

Irys system.

Washing the Electrode Bundle

Electrodes: The electrode bundle requires cleaning after every run. Additional washing may be required depending on the sample type. Follow the cleaning instructions at the end of the run to ensure proper operation of the electrodes.

Air Filter: It is required that the air filter be cleaned every 6 months or sooner if in a dusty environment. A spare air filter is supplied in the accessory kit, however the air filter is reusable and can be cleaned by washing under running water and air dried overnight.

System Enclosure Cleaning and Care

Use a damp lint free cloth with mild detergent when cleaning the instrument enclosure. While wiping the surfaces, care must be taken to prevent liquids from dripping inside the enclosure, where they may come into contact with electrical components.

Note: Do NOT use abrasives when cleaning the enclosure as they will damage the surface.

XY-Stage Cleaning Protocol

Use a damp lint free cloth with mild detergent when cleaning the XY stage plenum, which holds the IrysChip.

Irys® User Guide

7/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

LCD Touch Screen Cleaning and Care

Use a damp lint free cloth with mild detergent when cleaning the LCD touch screen display.

Electrical Requirements

Replacing the Fuse:

1. Locate the fuse housing (circled in red) on the rear right of the instrument, next to the power cable.

Figure 48: Fuse Location

2. Turn instrument power off and unplug the power cable.

3. Pull on the tab on the left side to open the fuse housing.

Figure 49: Open Fuse Housing

4. With pliers, gently pull the fuse assembly out of the housing.

Figure 50: Replace Fuse

5. Locate the fuse and replace with new fuse. Make sure the correct voltage (e.g., 115V for US) is showing through the fuse housing window.

Irys® User Guide

07/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Indication

Symptom

Things to Check

Instrument failure

Instrument fails to turn ON

1. Make sure instrument is plugged in.

2. Check for blown fuses, or tripped

circuit breakers (including GFCI outlets), make sure instrument is plugged in.

3. Instrument power switch or main

power switch is OFF, switch power ON.

LCD display is blank

LCD fails to turn ON, but instrument is ON

Touch screen is inoperable, contact Customer Support.

LCD is ON, but accepts no input

Touch screen is inoperable

1. Reboot the system using an external

keyboard and mouse to control system.

2. Cycle power to instrument to reboot,

try touch screen again.

3. If re-booting does not correct the

problem, contact Customer Support.

LCD is ON, but dark

Instrument is in “sleep” mode

Touch the screen to exit “sleep” mode

Bar code scanner fails

Scanner failure

Verify instrument is ON and functioning, see instrument failure

Bar code is illegible

Check the label and see if something is covering or obscuring the label

Chip is too far away from reader

Make sure chip is seated correctly in XY-stage

Scanner reads bar code, but no data is recorded

Contact Customer Support No signals seen in ANY channel

Instrument is inoperable

Contact Customer Support.

A dialog box opened with a message saying validation errors and then Irys ICS shut down.

Contact Customer Support

Notifications are sent when the run pauses or aborts.

An email address and SMS texting number can now be specified for each user. Notifications are sent when the run pauses or aborts. There are buttons that send a test notification on the same screen.

1.2 Appendix B - Troubleshooting

The troubleshooting portion of this section includes a troubleshooting guide and instructions for locating and resolving problems that may occur that are deemed user resolvable. Other more complex issues/problems should be deferred to a qualified Service Technician.

Irys® User Guide

7/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

The Help button appears on every screen. It accesses the indexed Irys User Guide with topic search capabilities.

This button will shut down the system software from the Select User screen. This button will return to the Select User screen from any other screen.

This status icon indicates that the system is operating correctly.

This status icon indicates that the system is busy.

Alert indicator. If you press it, you can get a log of all the notifications.

Directional buttons.

Abort run and return to main screen (only visible during scanning).

1.3 Appendix C – GUI Navigation Buttons

Navigation Buttons

Irys® User Guide

07/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

1.4 Appendix D - Terms

1. Irys – a high resolution imaging instrument capable of single molecule detection.

2. Irys System – a complete platform for fluorescently staining, imaging, detecting and analyzing single molecules.

3. Irys Chip – a disposable cartridge that contains a silicon chip with 2 embedded NanoChannel array flowcells, each with an inlet and outlet well.

4. NanoChannel array – the location within the IrysChip that linearizes the DNA sample.

5. Well – a location on the chip where sample or buffer is introduced.

6. Run – a single analysis on a NanoChannel array.

7. E-field – the electrical conditions that determine the flow of DNA through the NanoChannel array.

8. Recipe – a set of pre-configured instrument settings that determines the outcome of a run.

Irys® User Guide

7/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

USA most popular

Where ########## is your phone number without the country code

Metro PCS

##########@mymetropcs.com

T-Mobile

##########@tmomail.net

Verizon Wireless

##########@vtext.com

AT&T

##########@txt.att.net

Sprint PCS

##########@messaging.sprintpcs.com

Nextel

##########@messaging.nextel.com

Cricket

##########@sms.mycricket.com

US Cellular

##########@email.uscc.net

Cingular (GSM)

##########@cingularme.com

Cingular (TDMA)

##########@mmode.com

1.5 Appendix E - SMS Domain Names

Table 8: SMS Domain Settings

For more SMS Domain names, contact your mobile phone carrier or find on a free open texting website such as

http://www.opentextingonline.com/emailtotext.aspx.

Irys® User Guide

07/10/2015

For Research Use Only. Not Intended For Diagnostic Purposes.

Technical Support

You can contact BioNano Genomics for technical support by telephone, e-mail or through the Internet. You can retrieve BioNano Genomics user documentation, MSDSs, certificates of analysis, frequently asked questions, and other related documents from www.bionanogenomics.com/support or by request through e-mail and telephone.

To contact Technical Support by E-Mail: support@bionanogenomics.com Toll Free Telephone Technical Support: 858.888.7600 Technical Support Telephone Hours: Monday through Friday, 9:00am to 5:00pm, PST Customer Support Website: www.BioNanoGenomics.com/support

BioNano Genomics Irys User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual