Bionano Access 1.1 Software User Guide

Bionano Access 1.1 Software User Guide Document Number: 30142 Document Revision: B For Research Use Only. Not for use in diagnostic procedures. Copyright 2017 Bionano Genomics, Inc. All Rights Reserved.

Table of Contents Legal Notice... 4 Introduction... 5 Bionano Access Terms... 6 Login to Bionano Access... 7 Bionano Access Modules... 7 User Roles... 8 Add Users... 8 Change Password... 9 Help... 9 Message Center... 10 Projects... 11 Create a Project... 11 Project Browser Overview... 11 Options... 13 Delete a Project... 15 Give Users Access to View Projects... 15 Share Objects Between Projects... 15 Download Data... 16 Remove Objects... 16 Import Data... 16 Experiments... 18 Create an Experiment... 18 Monitor Run Progress... 20 Run Information... 21 Analysis Graphs... 21 Run Metrics Table... 22 Red Labeled Sample Experiment... 23 Red Only Workflow... 23 Dual Labeled Workflow... 24 Multiplex Workflow... 24 Set Up Digestion Tool Configuration Settings... 26 Add Enzymes... 26 Runs in Progress... 27 Completed Runs... 28 in silico Fasta Files Management... 28 Settings... 29 Add References... 29 Add Configurations... 29 Add Mask Bed Files... 30 Login Banner... 30 System Warning... 31 Bioinformatics Analysis... 32 Analysis Types... 32 Merge Molecule Objects... 32 Hybrid Scaffold... 33 Page 2 of 46 30142 Rev B, Bionano Access Software User Guide

Import Hybrid Scaffold... 34 Generate Hybrid Scaffold... 34 Alignment Object... 35 Import an Alignment Object... 35 Generate an Alignment Object... 36 View Structural Variations (SV)... 36 Perform Structural Variations (SV) Merge... 36 Perform Variant Annotation Analysis... 37 Visualization Features... 39 Viewer Settings... 39 Genome Maps Viewing Features... 40 Navigate to the Viewer... 41 Set Enzyme Colors... 42 View Label Stretch Match... 42 View Match Lines in Enzyme Color... 42 Create a Container for Two or More Assemblies... 42 Match Tab... 43 Administrator Tasks... 44 Modify User Accounts... 44 Delete Objects Permanently... 44 Restore Deleted Projects... 44 Delete Projects Permanently... 45 Technical Assistance... 46 30142 Rev B, Bionano Access Software User Guide Page 3 of 46

Legal Notice For Research Use Only. Not for use in diagnostic procedures. 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, reverse-engineered 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, IrysSolve, Saphyr, Saphyr Chip, and Bionano Access 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. Copyright 2017 Bionano Genomics, Inc. All rights reserved. Page 4 of 46 30142 Rev B, Bionano Access Software User Guide

Introduction The Bionano Access Software 1.1 is an application with an improved data visualization user experience. This software enables users to view Saphyr run results in real time and perform a variety of bioinformatics analyses. Saphyr and Irys users can perform bioinformatics analysis including, but not limited to, the following: Perform in silico digestion of fasta files Merge molecule data sets Generate de novo assemblies Align maps Build hybrid scaffolds Analyze structural variants Merge 2 enzyme based SV calls Saphyr users can also do the following: Create experiments Manage projects Monitor Saphyr Chip run progress This user guide provides instructions on using features and viewing maps with this software. 30142 Rev B, Bionano Access Software User Guide Page 5 of 46

Bionano Access Terms Saphyr cluster One Saphyr Compute server is included with each instrument. Users can also add up to two Bionano Compute servers with the Saphyr Compute server. The Saphyr and Bionano compute servers together represent a cluster. Objects Whenever Bionano Access performs a job, it may generate a variety of output data depending on the command, such as molecule data, map alignments, assemblies, hybrid scaffold results, variant annotations, and others. Each of these distinctive output data is an object (a set of output files). The type of objects include, but are not limited to: Alignment Assembly Container (Structural Variants Merge or Variant Annotation Results) Hybrid Scaffold Consensus CMAP Molecule BNX FASTA File BED file Page 6 of 46 30142 Rev B, Bionano Access Software User Guide

Login to Bionano Access After the installation of the Bionano Access software on the server, a URL will be provided to log on. 1. Navigate to the Bionano Access web page. 2. Enter user name and password. 3. Click Login. Bionano Access Modules The Bionano Access Home page lists the following modules: Note: Depending on the types of User Roles (see below) and the types of Access install instances (see details in 30170 Bionano Access Installation Guide), not all the modules are available at Home page. Module Description Availability Projects This module lets users create, view, edit, and manage projects. Additionally, users can import and export data. All user roles; all install instances of Access Experiments This module lets users set up and manage Saphyr experiments, view templates, and track chip run progress. All user roles; only Access install with proper configuration setup to Saphyr instrument owners In Silico Digestion This module lets users perform in silico digestion, update digestion settings, and view results. All user roles; all install instances of Access Settings This module lets users create users, manage user accounts, add references, configure software, and upload masked BED files. Only available to Administrator and Project Lead ; all install instances of Access 30142 Rev B, Bionano Access Software User Guide Page 7 of 46

User Roles There are four unique user roles in Bionano Access. The personnel with administrator privileges can assign roles to users. Roles Administrator Description The administrator can assign user roles, manage user accounts, manage refrences and delete projects permanently or restore deleted projects. Project Lead The project lead can give users access to view projects, create and manage projects and manage references. All project leads can view and edit all projects in the Projects list. User Read Only The user can view the projects that they have access to and create and edit experiments. This role can view the projects that they have access to. Add Users Users must have administrator privileges to perform this task. All users should have their own user account with a valid email address. Bionano Access notifies users via email when their job is complete. 1. From the Bionano Access main menu, select Settings. 2. Select User Accounts. 3. Click New Users. 4. Enter the user information. 5. At the Role field, choose one of the following: User Project Lead Administrator Read Only 6. At the User Status field, chose one of the following: Active: The user account is active; the user can log on to Bionano Access. Disable: The user account is disabled; the user cannot log on to Bionano Access. 7. Click Submit. The new user account appears on the User Accounts screen. Page 8 of 46 30142 Rev B, Bionano Access Software User Guide

Change Password 1. In Bionano Access, click the Menu icon. 2. Select User Profile. 3. At the Current Password field, type the password. 4. At the New Password field, type the new password. 5. At the Repeat Password field, type the new password again. 6. Click Submit. Help 1. In Bionano Access, click the Help icon. The Help page has the following information a. Access Version information b. Link to Bionano Support Webpage c. Tutorial Videos 30142 Rev B, Bionano Access Software User Guide Page 9 of 46

Message Center When there are new messages about job status in Access ready to be read, a red circle with a number will show at the message center icon. By default, the system will attempt to notify users via email and the message center. Users can disable email notifications on the user profile screen. Email notifications can also be disabled systemwide by adding disableemails : true to the access.txt configuration file and restarting the web server. If emails have been disabled in a systemwide manner, the email notification option on the user profile screen will not be visible. In Bionano Access, click the Message Center icon to read and delete individual messages. Users can also mark all messages as read and delete all read messages. Page 10 of 46 30142 Rev B, Bionano Access Software User Guide

Projects In the Projects module, users can add new projects, manage projects, edit, import, and download objects. Users can also filter the projects by project name and export all the objects in the project by clicking the select-all option. Additionally, users can perform bioinformatics analysis such as alignment, molecules merge, structural variations merge, hybrid scaffold, and other commands. Objects can be shared across all projects. Create a Project Users in the Project Lead or the Administrator role can create and delete projects and give others access to view projects. 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Click Create Project. The Project dialog box appears. 3. At the Project Name field, type the name of the project. 4. [Optional] At the Key field, type a key (e.g. a project code) to associate with the project. 5. [Optional] At the Description field, type a brief description. 6. Click Submit. The project appears in the Projects list. Project Browser Overview When users select a project, the Project Browser page opens. The page contains menu options, the list of object(s), the selected-object details, and the options for the selected object. Menu Menu Import Remove Copy Edit Jobs Samples Description To import output files from Saphyr clusters or Bionano files from another system. To remove an object from the project. To copy data from the object and paste it in another project. To edit object name, sample, reference, tags, and description. To view a list of jobs for a project. To view a list of samples associated with the objects. 30142 Rev B, Bionano Access Software User Guide Page 11 of 46

Objects List All the objects that are listed belong to the selected project. Info Name Sample Tag Description The user-defined name for the object. The sample name. The tag feature allows users to create and edit keywords that are tagged to this object. Users can filter objects with the same keywords to easily manage objects in a project. 1. To create or edit tags for the object, select the object, and then click Edit. 2. At the Tags field, type the keyword, and then press Tab. Repeat to add more keywords. 3. Click the delete x icon to delete the keyword. Type Created The object type (e.g. assembly, molecules, map, scaffold, FASTA file). The date when the object was created. Click the icon to sort and filter the information in the columns. Page 12 of 46 30142 Rev B, Bionano Access Software User Guide

[Object Type] Details Info Name Sample Reference Description Created Operation Status User Job Description The object name should be unique in a project. The same object can have different names in different projects. The sample name should be unique in a project. [Optional] The reference map. [Optional] The description of the object. The date the object was created. The type of job performed. The current status of the job. The user who created the job. The job number of the object. For additional job details, click the job number. Icon of Job Error Reporting shows the error message that was returned. Options The Options pane contains a list of operations (options) that can be performed with the selected object. The operations are dependent on the type and current status of the object. Bionano Access can perform one operation at a time. The links in the Options pane may be disabled if an operation is still in progress. Alignment Options Download Alignment Object View Maps Alignment View Molecules Alignment Assembly Options Assembly Report Molecules to Maps Maps to Reference with SV Maps to Reference Download Assembly Generate Hybrid Scaffold Generate 2-Enzyme Hybrid Scaffold Variant Annotation Pipeline (Beta) SV Merge Align Maps 30142 Rev B, Bionano Access Software User Guide Page 13 of 46

Bed Option Download BED file Container Option View [name of the merge] FASTA Option Download FASTA file Map Options Download Map Generate Hybrid Scaffold Generate 2-Enzyme Hybrid Scaffold Align Maps Molecules Options Download Molecules File Molecule Quality Report (MQR) Merge Molecule Objects Generate de novo Assembly Scaffold Options Hybrid Scaffold Report Maps to Next Generation Sequencing (NGS) with Conflicts Maps and Next Generation Sequencing (NGS) to Hybrid Scaffold Download Hybrid Scaffold Export NCBI Package Variant Annotation Options View Variant Annotation Results Download SV Annotation File View Pie Chart Merge SV View variant annotation results with original molecules It is also possible to run SVMerge on Variant Annotation containers. This works similar to merging assemblies. Select one of the Variant Annotation containers and then select the merge operation. In the merge dialog select the second annotation you want to merge. The merge output will include the extra SV annotation columns in addition to the traditional smap data. Variant annotation containers can only be merged with other variant annotation containers. They cannot be merged with assemblies. The download links have been added for Variant Annotation objects and SV merge. The import of Variant Annotation objects and SV Merge objects is not yet supported. Page 14 of 46 30142 Rev B, Bionano Access Software User Guide

Delete a Project Users in the Project Lead or the Administrator role can create and delete projects and give others access to view projects. Deleting a project is a two-step process. A project lead can delete the project from the project list. However, only the administrator can delete the project permanently from Bionano Access. For more details, see the Administrator Tasks section. 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Click the Delete icon of the project to delete. The Confirm dialog box appears. 3. Click Yes. The project is deleted from the list. Give Users Access to View Projects Users in the Project Lead or the Administrator role can create and delete projects and give others access to view projects. 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Click the Access icon of the project. The Project Access Control screen appears. 3. In the All Users pane, select the users to give access, and then click Grant Access. Users in the Project Lead or the Administrator role are not listed in the pane. The users appear in the Users with Access pane. Share Objects Between Projects Users can copy objects from one project and add it to another project. 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Select the project to view from the list. 3. In the Objects list, select an object (i.e. assembly, molecules, map) to share. 4. Click Copy. The Copy Object to Project dialog box appears. 5. At the Select the target project field, click the drop-down list to another project. 6. Click Submit. Users can view the data from the project that was selected to share. 30142 Rev B, Bionano Access Software User Guide Page 15 of 46

Download Data 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Select the project to view from the list. 3. In the Objects list, select the object to download. 4. In the Options pane, click the download [object type] option. The software downloads the data onto the workstation running the browser. Remove Objects Users can remove objects from a project. Removing an object from a project does not affect that same data that are shared with other projects. To remove an object that exists in several projects, users would need to remove them from each project. 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Select the project to view from the list. 3. In the Objects list, select the object to remove. 4. Click Remove. The Remove Object From Project dialog box appears. 5. Click Yes. 6. To permanately remove an object or restore a previously deleted object in a project (and Access database). Select the project and click on the recycle bin button corner of Access webpage to manage deleted objects. Import Data Users can import data from the following: at top-right Data output files that are generated from the Saphyr cluster, which are based on userdefined commands. Data from another system that is in the Bionano file format (e.g. Irys data generated by AutoDetect 2.1.4 or IrysSolve 2.1.0) 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Select the project to view from the list. 3. Click Import. The Import dialog box appears. 4. Select the file type to import, and then click Next. 5. Type a name for this object. 6. At the Sample field, click the drop-down list to select the sample associated with file. 7. [Optional] At the Reference field, click the drop-down list to select the reference that was used. Page 16 of 46 30142 Rev B, Bionano Access Software User Guide

8. [Optional] At the Tags field, type the keywords to associate with the file. 9. [Optional] At the Description field, type a brief description. 10. Click Choose File, and then browse and select the file. 11. Click Open. 12. Click Next. The spinning arrows indicates that the software is uploading data to the server. Important: Do not exit the screen until the spinning arrows disappear. If users exit the screen while the arrows are spinning, the uploading process may be interrupted. 13. When the spinning arrows disappear, click Close. The screen switches back to the Project Browser page. Bionano Access sends an email notification when the data are imported. The imported data can be accessible from the Objects list. In Bionano Access v1.1, the system is modified to make de novo assembly independent of MQR operation. This allows users to begin de novo assembly with the BNX file while the MQR is still running if desired. In addition, Bionano Access v1.1 supports uploading compressed bnx files in *.gz format. Also, sub-sampling for MQR has been increased from 1000 molecules to 10000 to provide more accurate metrics. 30142 Rev B, Bionano Access Software User Guide Page 17 of 46

Experiments In the Experiments module, users can add new experiments, manage experiments, track run performance, and view real-time metrics. Users can create multiple experiments for one project. Additionally, users can view and edit templates. In Bionano Access v1.1, the Experiment Design module no longer allows the submission of chips with empty flowcells. Both flowcells must be populated before you can proceed. All flowcells of a cartridges must be loaded with a sample when running to avoid crosscontamination between flowcells. In addition, the experiment list has been modified. The list now shows one row for each chip and users can expand the chip to see what is in each flowcell. Create an Experiment Each chip can only be associated to one experiment. Before inserting the Saphyr Chip into the instrument, create an experiment to associate with the chip. The run results are posted for the experiment that is associated with the chip. An experiment created on Bionano Access is in pending status until it is associated with a Saphyr chip. Users can modify or delete pending experiments. When an experiment is associated with a scan on the Saphyr, users cannot modify or delete the experiment. Only users in the Administrator or Project Lead role can create experiments. 1. From the Bionano Access main menu, select Experiments. The Experiment Design window appears. 2. Select Add Experiments. A dialog box appears. 3. At the Project field, click the drop-down list to select the project that is associated with the experiment. 4. [Optional] If users do not have a project for this experiment, click New to create a project. 5. At the Experiment Name field, type in the name of the experiment. 6. [Optional] At the Experiment Description field, type in a brief description. 7. [Optional] At the Experiment Template field, click the drop-down list to select a template. 8. Click Next. Page 18 of 46 30142 Rev B, Bionano Access Software User Guide

9. At the Workflow field, click the drop-down list to select Single Sample, Sample Multiplex or Dual Labeled Sample. Please see section Red Labeled Experiment (page 21) for details to setup Sample Multiplex and Dual Labeled Sample experiments. The instruction below describes setup for Single Sample. 10. At the Sample field, click the drop-down list to select a sample or click New. 11. At the Label filed, click the drop-down list to select Green 01 (green labeled) or Red 01 (red labeled) for Single Sample. 12. At the Recognition Enzyme field, click the drop-down list to select an enzyme or click New to add a new enzyme. 13. [Optional] At the Reference field, click the drop-down list to select a genome reference for the experiment. 14. [Optional] At the Comment field, type in a brief comment. 15. Click Add to Flowcell 1. To edit information to Flowcell 1, click Remove in the Chip pane, and then make changes. 16. Repeat step 9 through step 12 for Flowcell 2. Important: Users must populate information for both flowcells. 17. Click Add to Flowcell 2. To edit information to Flowcell 2, click Remove in the Chip pane, and then make changes. 18. Click Next. A summary of the experiment appears. 30142 Rev B, Bionano Access Software User Guide Page 19 of 46

19. By default, the Auto Assemble Human check box is selected if the reference is a human genome. Users can deselect the check box. When this option is selected, if there is at least 70X effective coverage, the de novo assembly will start using the human haplotype assembly parameters immediately after the molecules file is generated. 20. Click Done. The Experiment List screen appears. Monitor Run Progress After the instrument scans the chip for 15 to 20 minutes and raw images are being detected, users can monitor the progress of the run and view real-time metrics from the Bionano Access web site. 1. From the Bionano Access main menu, click Experiments. 2. Click Chip Runs The latest chip run appears at the top of the list. 3. [Optional] Click the Filter icon to filter data in the columns. 4. Select the experiment to monitor, and then click View Dashboard. The Dashboard screen appears showing: Run information Analysis graphs Run metrics table Page 20 of 46 30142 Rev B, Bionano Access Software User Guide

Figure 1: Dashboard Run Information Info Chip Run Project Experiment Instrument Min Length Min Labels Start Time End Time Description The Saphyr Chip bar code. The system-generated run identifier. The project name. The experiment name. The system-generated serial number. The minimum length of molecules that are used for analysis. The setting for this parameter is system generated. The minimum labels per molecule that are used for analysis. The setting for this parameter is system generated. The run start time. The run end time. Analysis Graphs Map DNA per Scan Description This graph shows the amount of DNA per flowcell that is detected per scan. Map Rate This graph shows the percentage of molecules that map to the reference genome. The map rate cannot be calculated unless there is a minimum number (1000) of molecules and labels. If the minimum threshold is not acquired, the map rate returns to zero. If no reference genome is provided, the map rate is zero. 30142 Rev B, Bionano Access Software User Guide Page 21 of 46

Run Metrics Table Metric Flowcell Sample Enzyme Total DNA Avg. N50 (>=150kbp) Avg. N50 (>=20kbp) Description The scanned flowcell. The sample name. The enzyme name. The total amount of DNA that is detected per flowcell during the run. The molecule length N50 for all molecules that are 150 kbp in length. The molecule length N50 for all molecules that are 20 kbp in length. Molecules that are less than 20 kbp are considered noise by the image detection algorithm. Avg. Label Density Avg. Map Rate The number of labels that are detected by the image detection algorithm per 100 kbp of DNA length for molecules 150 kbp. The percentage of molecules that map to the reference for molecules 150 kbp. If no reference genome is provided, the metric is blank. Estimated Effective Coverage The coverage number is calculated as follows: Average Map Rate * Total DNA / length of the reference For human structural variation detection, we recommend at least 70X effective coverage. Avg. False Positive Avg. False Negative Scans The percentage of labels that are present in the aligned molecules but not in the reference. This is an estimated false positive rate, true sample differences will also be present. The percentage of labels that are present in the reference but not in the aligned molecules. This is an estimated false positive rate, true sample differences will also be present. The scans Recd number reflects the volume of scans submitted by Saphyr Control Software to Bionano Access. The scans Done number reflects the volume of scans that have been processed by Bionano Access. The Scans Done number reflects the number of scans that have been converted to molecule data and had metrics data generated. Page 22 of 46 30142 Rev B, Bionano Access Software User Guide

Red Labeled Sample Experiment The Access system now supports experiments that use the red laser in the Saphyr instrument. These workflows are disabled by default. To enable this operation, users need to contact Bionano Customer Solutions (support@bionanogenomics.com). Red Only Workflow If capability of running red labeled sample has been enabled, when users open the Single Sample workflow they will have the choice of selecting green or red. If red has not been enabled, the system will only generate experiments using green. 30142 Rev B, Bionano Access Software User Guide Page 23 of 46

Dual Labeled Workflow If red is enabled, the Dual Labeled workflow in the experiment design module will be available. The Dual Labeled workflow is designed to process a single sample with motifs in the DNA labeled using either green or red colors. The system will generate label-related metrics on the dashboard for each color. Users can provide separate references for each color, but only one reference will be associated with the final merged BNX file. The user is prompted to select which label (the primary) would retain its reference. The Dual Labeled workflow will generate a single BNX file that contains both green and red labels. When users import a dual-labeled BNX file, the reference will automatically be associated with channel 1. Users can select the Edit option to designate either channel for the reference. When users run an assembly using a dual labeled BNX file, they will be prompted to select which channel they want aligned and assembled. After the assembly is done and users choose to show molecules in the viewer, the viewer system will show labels for both labeled motifs. Multiplex Workflow If red has been enabled, the Sample Multiplex workflow will be enabled in the Experiment Design module. This workflow is designed to process one sample using green and a second sample using red in the same flowcell. This workflow will generate separate dashboard metrics for each color. This workflow will also generate a separate BNX file for each color. It is possible to use the same sample for both red and green, but the molecules loaded should be labeled with only red or green, but not both. Page 24 of 46 30142 Rev B, Bionano Access Software User Guide

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In Silico Digestion In Silico digestion is used to create a genome reference consensus map for any sequence file. This tool uses a FASTA file and the recognition sequence of a nicking enzyme to create a CMAP file that can be used as a reference. The in silico digestion results are accessible by all users. Bionano Access saves the FASTA files that are uploaded; users can reuse the files to generate different CMAP files with different enzymes or enzyme-to-channel combinations. The in silico Digestion page contains an accordion menu that contains three panes: Create New Run, Runs in Progress, Completed Runs. Click the arrow on the pane to expand or collapse the view. Set Up Digestion Tool Configuration Settings Before performing in silico digestion, define the following settings: 1. From the Bionano Access main menu, select In Silico Digestion. 2. Click the Configuration Settings icon. The icon is at the top right-corner of the screen. The Configuration dialog box appears. 3. At the Minimum Labels field, set a value between 0 20 (default as 0). 4. At the Minimum Length field, set a value between 0 250 kb (default as 0). 5. At the Number of Channels field, set the number of channels to digest. The channel refers to the laser color used. 6. At the Number of Enzymes Per Channel field, set the number of enzymes per channel to digest. 7. Click Save. The software displays the settings in the Create New Run pane. Add Enzymes Users can add and edit nicking enzymes and recognition sequences; however, the default enzymes listed in Bionano Access are not editable. Users must have administrator privileges to perform this task. 1. From the Bionano Access main menu, select In Silico Digestion. 2. Click the Enzyme Management icon. The icon is at the top right-corner of the screen. The Manage Enzymes dialog box appears. 3. Click Add. 4. At the Enzyme Name field, type the enzyme name. 5. At the Enzyme Sequence field, type the recognition sequence of the nicking enzyme (ACGT). 6. Click Save. The software adds the enzyme to the list. Page 26 of 46 30142 Rev B, Bionano Access Software User Guide

Perform in Silico Digestion Run Depending on the digestion tool settings, users may have one or more label channels parameters to select. Bionano Access saves the FASTA files that have been digested. Bionano Access only checks if there are duplicate file names. If a user uploads a FASTA file that Bionano Access already obtains, the software will point to the existing file. For best practices, give FASTA files unique names to easily track them. The following task is an example of an in silico digestion for one label channel. 1. From the Bionano Access main menu, select In Silico Digestion. 2. In the Create New Run pane, at the Select file field, browse to the FASTA file to digest, and then click Open. In Bionano Access v1.1, files larger than 1GB will upload properly. 3. At the Label Channel 1, select the enzyme to nick from the drop-down list. 4. Click Launch. A message appears to indicate that the file is uploading if it is new, and then the software initializes the run. The digestion progress appears in the Runs in Progress pane. Once the run is complete, the digested data appears in the Completed Runs pane. 5. If this FASTA file was previously uploaded to the server, this message appears: This FASTA has been uploaded to the server. To use it, click OK. To use a new version of this FASTA, click Cancel, rename your FASTA, upload, and try again. Runs in Progress Expand the Runs in Progress pane to view the following statistics: Statistic Run Date FASTA Min Labels Min Size Enzyme(s) Progress Cancel Description System-generated number for the run. The date the file is uploaded. The FASTA file name. The user-defined setting for the minimum map label. The user-defined setting for the minimum map length. The enzyme(s) that are used. The progress of the run. Click x to cancel the file upload. 30142 Rev B, Bionano Access Software User Guide Page 27 of 46

Completed Runs Expand the Completed Runs pane to view the following statistics: Statistic Run ID Date Minimun Labels Minimum Size Files FASTA Files Cmap Files Key Files Gap Files Summary Description System-generated number for the run. The date the file is uploaded. The user-defined setting for the minimum map label. The user-defined setting for the minimum map length. The FASTA file name. The CMAP file that can be downloaded. A file to track CMAP IDs that are associated with the FASTA sequence map names. The GAP file can be downloaded. The GAP file track N based gaps in sequence of 1,000 kb in length. A complete summary of the results that can be downloaded. Enzyme(s) Channel 1 Enzyme(s) that are used to create the CMAP in channel 1 Enzyme(s) Channel 2 Enzyme(s) that are used to create the CMAP in channel 2. Enzyme(s) Channel 3 Enzyme(s) that are used to create the CMAP in channel 3. #Maps N % Nicks / 100kbp Global Number of maps in the digested sample. Percent of genome that has N bases. The number of nicks per 100 kbp Globally. Nicks / 100kbp Channel 1 The number of nicks per 100 kbp for channel 1. Nicks / 100kbp Channel 2 The number of nicks per 100 kbp for channel 2. Nicks / 100kbp Channel 3 The number of nicks per 100 kbp for channel 3. Action Add to References Clicking on this will automatically copy the newly digested cmap file to the reference pool. Users do not need to manually download the cmap file and import to Reference pool. in silico Fasta Files Management Users can use in silico Fasta Files Management to manage FASTA files that have been uploaded into the in silico digestion tool. To access the FASTA file management screen, click the file icon in the header. From the FASTA Management screen users can delete runs associated with that file and also delete the FASTA file itself. Page 28 of 46 30142 Rev B, Bionano Access Software User Guide

Settings In the Settings module, users can do the following: Add references Add and edit configurations Add Mask Bed files Perform administrator tasks to manage User Accounts Add login banner Create system warning Add References Users must have administrator or project lead privileges to perform this task. Users can add references (CMAP files) to use for map alignments. 1. From the Bionano Access main menu, select Settings. 2. Select References. The Reference List page appears. 3. Click Add Reference. A dialog box appears. 4. At the Reference Name field, type the name for the reference. 5. At the Enzyme Motif field, type the sequence. The sequence format is (ACGT). Use a comma to separate the enzyme sequences. 6. At the Genome field, type the name of the genome. 7. If the reference is human, select the Human check box. 8. At the Reference File field, click Choose File, and then browse to the CMAP file to use. 9. Click Open. 10. Click OK. Add Configurations Users must have administrator or project lead privileges to perform this task. When generating a de novo assembly or hybrid scaffold, users are required to select a configuration that contains a set of pre-defined parameters. Users can select a configuration from: A list of default configurations in Bionano Access A list of customized configurations based on the existing configurations in Bionano Access Configuration files (*.xml) that users upload to Bionano Access Bionano Access does not validate the formatting or parameter settings of the upload XML file. If the uploaded file does not contain proper parameters or formatting, the file may cause the system to crash. In Bionano Access v1.1, users can also download the configuration files in the system. For details on customizing configurations based on existing and validated configurations in Bionano Access, see the following tasks: 30142 Rev B, Bionano Access Software User Guide Page 29 of 46

Generate de novo Assembly Generate Hybrid Scaffold 1. From the Bionano Access main menu, select Settings. 2. Select Configurations. 3. Click Add Configuration. A dialog box appears. 4. At the Configuration Name field, type the name for the configuration. 5. At the Configuration Type field, select the operation type to use this configuration file from the drop-down list. 6. [Optional] At the Description field, type a brief description. 7. At the Configuration File field, click Choose File, and then browse to the configuration file (*.xml) to use. 8. Click Open. 9. Click OK. Add Mask Bed Files Users must have administrator or project lead privileges to perform this task. 1. From the Bionano Access main menu, select Settings. 2. Select Mask Beds. 3. Click Add Mask Bed. A dialog box appears. 4. At the Mask Bed Name field, type the name for the mask bed. 5. [Optional] At the Description field, type a brief description. 6. At the Mask Bed File field, click Choose File, and then browse to the mask bed file (*.bed) to use. 7. Click Open. 8. Click OK. Login Banner Users must have administrator privileges to perform this task. 1. From the Bionano Access main menu, select Settings. 2. Select Login Banner. 3. At the Content for login page field, type and edit the information. 4. Click Submit. Page 30 of 46 30142 Rev B, Bionano Access Software User Guide

System Warning Users must have administrator privileges to perform this task. 1. From the Bionano Access main menu, select Settings. 2. Select System warning. 3. At the System warning field, type the information. 4. Click Submit. 30142 Rev B, Bionano Access Software User Guide Page 31 of 46

Bioinformatics Analysis Analysis Types Users can perform the following analyses in Bionano Access: Analysis Molecules Merge de novo Assembly Hybrid Scaffold 2-Enzyme Hybrid Scaffolds Alignments Structural Variation (SV) Merge Variant Annotation Pipeline Description Merge two or more molecules objects into a single molecules object. Assemble single molecules into consensus maps for SV Detection and Hybrid Scaffold applications. Merge Bionano maps with sequence assemblies to produce long hybrid scaffolds that represent the chromosome structure for analysis. Combine two sets of Bionano maps and a sequence assembly to generate two-enzyme hybrid scaffolds, which increases specificity and resolution. Compare two different maps by aligning them to each other. Merge SV calls obtained by using two different enzyme assemblies (e.g. Nt.BspQI and Nb.BssSI) of the same sample. This analysis increases specificity of SVs size, location, and accuracy. For applications such as identification of rare and potential de novo SVs for trio (mother, father, and proband) or for cancer research. Merge Molecule Objects Starting with Bionano Access v1.1, the program will firstly select Molecule objects with the same sample name and recognition enzymes for users to merge. The user can still choose other molecule objects that do not have the same sample name, but they must have the same recognition enzymes. Dual labeled BNX files can only be merged with dual labeled BNX files with matching recognition enzymes. Best Practices: Merge molecule objects that have the best quality data. Use BNX files that have the same sample and reference. Do not merge BNX files that have different levels of quality data. 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Select the project to view from the list. 3. In the Objects list, select a molecules object to merge. 4. In the Options pane, select Merge Molecule Objects. A dialog box appears. 5. At the Merge Molecule Name field, type the new name for the molecules object. There is also a select-all option on the table that lists molecule files that can merged together. Page 32 of 46 30142 Rev B, Bionano Access Software User Guide

6. At the Sample field, select the sample from the drop-down list. 7. [Optional] At the Reference field, select the genome reference from the drop-down list. 8. [Optional] At the Tags field, type the keywords to associate with the assembly. 9. [Optional] At the Description field, type a brief description. 10. Select the check boxes of the molecule objects to merge with the molecules object that was selected in step 3. By default, Bionano Access automatically selects the molecule objects in the project that are a match with the molecule object selected to merge with. For example, it selects the molecule objects with the same reference and sample information. 11. Click Submit. Users will receive an email when the merge is complete. Generate de novo Assembly Users can generate de novo assembly with molecules data. 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Select the project to view from the list. 3. In the Objects list, select the molecules object to perform de novo assembly. 4. In the Options pane, select Generate de novo Assembly. 5. At the Assembly Name field, type the name of the assembly. 6. [Optional] At the Tags field, type the keywords to associate with the assembly. 7. [Optional] At the Description field, type a brief description. 8. [Optional] At the Mask Bed File field, select a bed file from the drop-down list. The mask bed file filters SVs from regions of the genome that are included in the mask. 9. At the Configuration field, select the configuration file from the default list or select a customized configuration. a) To customize a configuration, click the Edit icon in one of the default configurations. The configuration dialog box appears. b) Define the settings for de novo assembly, and then click Save As. c) Type the name of the configuration, and then click OK. The customized configuration appears in the list of configurations. 10. Click Submit. Users will receive an email when the assembly is complete. Depending on the volume of data and coverage, the de novo assembly process may take a while to complete. Hybrid Scaffold Users can merge Bionano maps with sequence assemblies to produce long hybrid scaffolds that represent the chromosome structure for analysis. There are two ways to generate a hybrid scaffold object: Import ZIP or TAR.GZ files to Bionano Access. Generate alignment objects using maps and assemblies that are already in Bionano Access. 30142 Rev B, Bionano Access Software User Guide Page 33 of 46

Import Hybrid Scaffold Users must have the hybrid scaffold data in a *tar.gz or *.zip file to import to Bionano Access. 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Select the project to view from the list. 3. In the Objects list, select an assembly. 4. Click Import. The Import dialog box appears. 5. Click the Hybrid Scaffold radio button, and then click Next. 6. At the Hybrid Scaffold Name field, type a name for the scaffold. 7. At the Sample field, select the sample from the drop-down list. 8. [Optional] At the Tags field, type the keywords to associate with the alignment object. 9. [Optional] At the Description field, type a brief description. 10. At the Hybrid Scaffold File field, browse to the *.gz or *.zip file and select it. 11. Click Open. 12. Click Next. Generate Hybrid Scaffold Users can use map or assembly objects to generate hybrid scaffold data. We recommend that users do not use haplotype assemblies to generate hybrid scaffold. 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Select the project to view from the list. 3. In the Objects list, select the map or assembly to use as a reference. 4. In the Options pane, select Generate Hybrid Scaffold. The Scaffold screen appears. 5. At the Scaffold Name field, type a name for the scaffold. 6. At the Selected Map field, by default the software shows the map to use for scaffolding. 7. At the Enzyme Selection field, select the enzyme from the drop-down list. 8. At the Select Fasta field, select the file from the drop-down list. 9. At the Conflict Resolution field, for best practices, select Resolve Conflicts. 10. [Optional] At the Molecules field, select the molecules object and the scripts from the previously completed assembly. If users select a molecules object, they need to also select the assembly optargument file for it in the Configuration-Molecules Alignment field. This option enables molecule alignment to the hybrid scaffolds. The output files of the alignment reside in alignmol_bionano/ and alignmol_hybrid directories. See more in document 30073 Bionano Solve Theory of Operation Hybrid Scaffold. 11. At the Configuration field, select the configuration file from the default list or select a customized configuration. a) To customize a configuration, click the Edit icon in one of the default configurations. The configuration dialog box appears. Page 34 of 46 30142 Rev B, Bionano Access Software User Guide

b) Define the settings for hybrid scaffold, and then click Save As. c) Type the name of the configuration, and then click OK. The customized configuration appears in the list of configurations. 12. [Optional] At the Tags field, type the keywords to associate with the alignment object. 13. [Optional] At the Description field, type a brief description. 14. Click Submit. Users will receive an email when the alignment is complete. Users can also generate a hybrid scaffold for two enzymes. In the Options pane, select Generate 2-Enzymes Hybrid Scaffold, and then follow the steps in this task. Users must select a second CMAP file for the software to generate hybrid scaffold for both enzymes. Alignment Object Generating an alignment object lets users compare two different maps by aligning them. There are two ways to generate an alignment object: Import map files to Bionano Access. Generate alignment objects using maps and assemblies that are already in Bionano Access. Import an Alignment Object Users must have all three of these files to import an alignment to Bionano Access:.xmap, _r.cmap,_q.cmap 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Select the project to view from the list. 3. In the Objects list, select an assembly. 4. Click Import. The Import dialog box appears. 5. Click the Alignment radio button, and then click Next. 6. At the Alignment Name field, type the name of the alignment. 7. At the Sample field, select the sample from the drop-down list. 8. [Optional] At the Reference field, select a reference from the drop-down list. 9. [Optional] At the Tags field, type the keywords to associate with the alignment object. 10. [Optional] At the Description field, type a brief description. 11. Choose one of the following options for Bionano to display the files in: Anchor to Genome Maps Display a consensus map as contigs. Anchor to Molecules Display a consensus map as molecules. 12. Browse and select the file for the following: Alignment Map (.xmap) Anchor Map (_r.cmap) Query Map (_q.cmap) 13. Click Open. 14. Click Next. 30142 Rev B, Bionano Access Software User Guide Page 35 of 46

Generate an Alignment Object Users can use map or assembly objects to generate alignment data. 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Select the project to view from the list. 3. In the Objects list, select the map or assembly to use as a reference. 4. In the Options pane, select Align Maps. The Alignment screen appears. 5. At the Alignment Name field, type the name of the alignment. 6. [Optional] At the Tags field, type the keywords to associate with the alignment object. 7. [Optional] At the Description field, type a brief description. 8. At Select Query Map, select the map or assembly to generate an alignment. 9. Click Submit. Users will receive an email when the alignment is complete. View Structural Variations (SV) 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Select the project to view from the list. 3. In the Objects list, select an assembly. 4. In the Options pane, select Maps to Reference with SV. The Viewer screen appears. 5. In the SV table, users can do the following: Find, sort, or filter data in the columns. Select an SV from the list to highlight in the viewer. Perform Structural Variations (SV) Merge When a sample is analyzed and assembled with two enzymes, the structural variants can be merged together into a combined output. The benefits include improved sensitivity and breakpoint accuracy. For more details, view the SVMerge Tutorial in the Help page. Users cannot use more than two enzymes to perform the merging of SVs. 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Select the project to view from the list. 3. In the Objects list, select the assembly object to merge. 4. In the Options pane, select Merge SV. A dialog box appears. 5. At the Container Name field, type a name for container. Page 36 of 46 30142 Rev B, Bionano Access Software User Guide

6. [Optional] At the Tags field, type the keywords to associate with the container. 7. [Optional] At the Description field, type a brief description. 8. At the Select second assembly field, select the second assembly object to perform the SV merge from the drop-down list. 9. Click OK. 10. When the merge is complete, return to the Project Browser page. The container with the SV merge appears in the Objects list. 11. Select the container name, and then in the Options pane, select the view container option. The Viewer screen shows the SV data of the two assemblies. In the SV Merge table, Query 1 column shows the first assembly data and Query 2 shows the second assembly data. Starting Bionano Access v1.1, the SV Merge operation now merges translocations and inversions as well as insertions and deletions. The SV Merge operation is now also completed in the background and on the compute server instead of the local web server, freeing the system to perform other operations, making operation faster, and providing more consistency with other bioinformatics operations. The system will message users when the SV Merge job has completed. Perform Variant Annotation Analysis This analysis lets users identify rare and potential de novo SVs for trio (mother, father, and proband) or dual (parent and proband) research. This analysis is also used for cancer research. The following task is an example of the trio variant annotation pipeline (proband, father, mother). 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Select the project to view from the list. 3. In the Objects list, select the map or assembly to use as a reference. 4. In the Options pane, select Variant Annotation Pipeline. 5. At the Variant Annotation Result Name field, type the name for the variant annotation. 6. [Optional] At the Tags field, type the keywords to associate with the alignment object. 7. [Optional] At the Description field, type a brief description. 8. At the Variant Annotation Result Type field, select Trio from the drop-down list. 9. Click Next. 10. At the Proband de novo assembly field, select the assembly from the drop-down list. 11. At the Father de novo assembly field, select the assembly from the drop-down list. 12. At the Mother de novo assembly field, select the assembly from the drop-down list. 13. Use the default values or enter the values to use for the next 8 parameter fields. For more details, see the Bionano Solve Theory of Operation: Structural Variant Calling (document #30110) for guidance on setting these parameters. 30142 Rev B, Bionano Access Software User Guide Page 37 of 46

14. Click Submit. The spinning arrows indicates that the software is uploading data to the server. Important: Do not exit the screen until the spinning arrows disappear. If users exit the screen while the arrows are spinning, the uploading process may be interrupted. 15. When the spinning arrows disappear, click Close. The screen switches back to the Project Browser page. Bionano Access sends an email to notify the user when the variant annotation is complete. The variant annotation (container file type) can be accessible from the Objects list. Starting Bionano Access v1.1, It is also possible to merge Variant Annotation containers. This work is similar to merging SV. Select one of the Variant Annotation containers and then select the merge operation. In the merge dialog select the second annotation you want to merge. The merge output will include the extra SV annotation columns in addition to the traditional smap data. Variant annotation containers can only be merged with other variant annotation containers. They cannot be merged with other types of objects. Page 38 of 46 30142 Rev B, Bionano Access Software User Guide

Visualization Features Viewer Settings Function File Anchor Range Molecules Confidence Find Query Description Allows user to select the file alignments from other files, merge SVs, and save the file. Allows user to change the view of other contigs and chromosomes in the samples. Allows user to set the range of the samples shown in the viewer for mega base pairs. Allows user to change the molecule position in the genome maps. Allows user to view molecules in the genome maps depending on their levels of confidence. Users can also enter a specific value manually. Allows user to highlight the genome map for the input. Icon Description Shows all the data in the viewer. For example, if users hide the alignment lines and maps, click this icon to view them again. Refreshes the viewer. Allows user to download the current molecules view in scalable vector graphics (SVG) file format. Allows user to customize the view settings to show or hide unmatched labels, copy numbers, NGS + BNG to Hybrid, NGS to Hybrid, molecule coverage, molecule match lines, and others. Allows user to customize the options settings, such as setting enzyme colors, SV colors, minimum and maximum map height value, molecule and copy number height value, and others. Returns to the Project Browser page. Returns to the Bionano Access Home page. Quick help menu that details all the shortcut keys and actions. 30142 Rev B, Bionano Access Software User Guide Page 39 of 46

Genome Maps Viewing Features Figure 2: Genome Maps View Insersion (left) and Deletion (right) Figure 3: Genome Maps View - Translocation Page 40 of 46 30142 Rev B, Bionano Access Software User Guide

Feature Customize the view of the samples Move maps horizontally Move maps vertically Map options Description In the first ruler, press down on the mouse, and then drag left or right to highlight the samples to view. A red box indicates the selected samples that are displayed on the viewer. Left-click on mouse; move left or right. Press Shift; move up or down. Right-click on map. Hide Hide the selected map from the viewer. Hide the others Hide the other genome maps from the viewer except for the selected map and reference map. Collapse Collapse the genome maps to a single row next to the reference map. Show Ruler Attach the ruler to the selected genome map. Show Molecule Show all the aligned molecules that are related to the selected genome map. Show Molecule for Show all the aligned molecules that are related to Proband, Mother and Father for variant annotation results. Hide Matchgroups Remove the gray line connecting to the selected genome map from the reference map. Invert Invert the orientation of the selected map. Zoom in or out Use the mouse to scroll zoom in or zoom out. Navigate to the Viewer Users can navigate to the Viewer screen from the project page. 1. From the Bionano Access main menu, select Projects. The Projects window appears. 2. Select the project to view from the list. 3. In the Objects list, select an assembly, alignment, container, or scaffold object. Users can view these objects on the Viewer screen. 4. In the Options pane, depending on the object selected, these are possible options to get to Viewer screen: Molecules to Maps Maps to Reference with SV Maps to Reference View [name of container] Maps to NGS with Conflicts Maps to NGS with Hybrid Scaffold 30142 Rev B, Bionano Access Software User Guide Page 41 of 46

Set Enzyme Colors Users can set the color view for each enzyme. 1. In Bionano Access, navigate to the Viewer screen of the project to analyze. 2. Click the Settings icon. The dialog box appears. 3. Scroll down to the enzymes to change the color. 4. Next to the enzyme, there are two columns. Click the color to change it. The first column shows the matched labels and the second column shows the unmatched labels. 5. Select a new color, and then click OK. 6. Click Save. The new settings are saved. 7. [Optional] To revert back to the original settings, click Reset Options. View Label Stretch Match Users can choose stretched alignment for molecule labels when viewing assemblies. 1. In Bionano Access, navigate to the Viewer screen of the project to analyze. 2. Click the View icon. The dialog box appears. 3. At the Show molecule labels option, click the drop-down list and select one from UnStretch, Stretch, Stretch Matchgroup. UnStretch is the system default. View Match Lines in Enzyme Color 1. In Bionano Access, navigate to the Viewer screen of the project to analyze. 2. Click the View Options icon. The View Options dialog box appears. 3. At the Show match lines in option, click the drop-down list and select Enzyme Color. 4. Click Close. The Viewer screen shows the match lines in the user-specified enzyme color. Create a Container for Two or More Assemblies Users can view more than one assembly at the same time in the Viewer screen. Additionally, users can create a container for two or more assemblies so that they can be viewed at a later time. The following task is an example of creating a container for two assemblies. 1. In Bionano Access, navigate to the Viewer screen of the project to analyze. 2. Click the File menu, and then click Select Alignments. 3. In Alignments, select the check box of the second alignment assembly to view. 4. Click OK. 5. Click the File menu, and then click Save. Page 42 of 46 30142 Rev B, Bionano Access Software User Guide

6. At the Container Name field, type the container name. 7. [Optional] At the Tags field, type the keywords to associate with this container. 8. [Optional] At the Description field, type a brief description. 9. Click Save. 10. Return to the Project page. The container that contains the two assemblies appears in the Objects list. 11. Select the container name, and then in the Options pane, select the view container option. The Viewer screen shows the two assemblies together. Match Tab The viewer now includes a Match tab that lists the contigs for an assembly. The table on the Match tab can be sorted and filtered. The table includes columns for the xmap ID, query ID, query start, query end, orientation, and confidence. The content for the Match tab is generated when the assembly is imported. Only new assemblies generated or imported after version v1.1 has been installed will show the Match tab. 30142 Rev B, Bionano Access Software User Guide Page 43 of 46