For Research Use Only. Not for use in diagnostic procedures.

Copyright © 2017 Bionano Genomics, Inc. All Rights Reserved.

Guidelines for Running Red

Labeled Samples

Document Number: 30193

Document Revision: A

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Table of Contents

Legal Notice ............................................................................................................ 3

Introduction ............................................................................................................ 4

System Requirements .............................................................................................. 5

Overview of Workflows ............................................................................................. 5

DNA Labeling .......................................................................................................... 7

Experiment Setup for Saphyr or Irys Instrument ......................................................... 8

To setup the experiments at Irys instrument, please see 30047 Irys User Guide. ........................... 8

Sequential Imaging .................................................................................................. 8

MQR (Molecule Quality Report) in Access .................................................................... 8

Visualization of Molecule Alignments in Access .......................................................... 10

Appendix: How to run a color split command line to split a two color BNX file ................ 11

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Legal Notice

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© Copyright 2017 Bionano Genomics, Inc. All rights reserved.

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Introduction

With the release of Bionano Access (v1.1), Bionano Solve v3.1 and Saphyr ICS (v3.1), the Saphyr system is now capable of imaging and detecting either a single red color labeled sample or simultaneous red and green labeled samples. The Saphyr system now generates BNX files (v1.3) containing molecules with red and/or green labels for downstream data analysis. In Access (v1.1), users can now import and analyze red data generated by Irys or Saphyr.

Related product release and updates are listed below:

Saphyr ICS v3.1

Bionano Access v1.1

Bionano Solve v3.1

BNX File Format Update (v1.3) – Saphyr only. Bionano Access can still import Irys BNX v1.2 files o Irys BNX files should not be merged with Saphyr BNX files o There is no restriction/warning about merging BNX v1.2 and v1.3 files

CMAP File Format Update (v0.2)

BNX v1.2 is still compatible with Bionano Access v1.1 and Bionano Solve v3.1.

Note:

Access v1.1 provides o red label sample experiment setup capability (Saphyr only) o real-time cohort QC and MQR stats for red labeled samples (Saphyr only) o visualization of molecules labeled with 2 colors

To obtain a red labeling mix for red label sample preparation, please contact a Bionano Support. Bionano does not offer a NLRS kit containing a red labeling mix.

Bionano currently has not developed or validated any assays for red labeled sample prep. Users need to develop their own assays to prepare red labeled samples.

Bionano data analysis pipeline only supports alignment or assembly of label information from 1 color, but not 2 colors at the same time. For more information, see the Bionano Access training video “Two Color BNX Files”.

When running red labeled samples, throughput guarantees for either the IrysChip or the SaphyrChip are no longer guaranteed, including the minimal 100 Gbp/FlowCell for "non-supported samples" at Saphyr Chip. Bionano will still warranty the chips for physical damage.

When working with a 2 color BNX (data collected from dual labeled sample), only one "primary" reference can be associated with the BNX file at a time in Access. For more information, see the Bionano Access training video “Red Experiments Tutorial”.

Although we have no reason to believe it will not work, Bionano has no experience using a red labeling mix on non-human samples.

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System Requirements

Saphyr Instrument: o ICS version 3.1 or greater o Any commercially available SaphyrChip

Irys Instrument: o ICS version 1.6 or greater o Any commercially available IrysChip

Bionano Access: v1.1 or greater (with red label experiment setup enabled for Saphyr, please contact Support@Bionanogenomics.com for assistance)

Overview of Workflows

This release enables four new workflows, including “Red-only NLRS”, “Sample Multiplexing”, “Enzyme Multiplexing”, and “Dual-Labeled” workflows, on the Saphyr and Irys Instruments, in addition to our previously released “Green-only NLRS” workflow. The major differences among all these workflows are with the DNA labeling and data collection steps.

Green-only refers to Bionano’s traditional labeling chemistry and is the only fully validated workflow Bionano currently offers.

For “Red-only NLRS”, the workflow is essentially identical to the “green-only”, except a red labeling mix is used instead of green labeling mix in the labeling step of NLRS. When setting-up the experiment in Bionano Access, a red color is chosen for the label instead of green. The output BNX file at the end of the run will also be similar to a green-only workflow, except that in the header of the BNX file "channel 1" will be identified as red.

For multiplexing, it is possible to multiplex two different samples (sample multiplexing) or the same sample with two different enzymes (enzyme ‘motif’ multiplexing) in one flow cell.

For sample multiplexing, two different samples are prepared independently with different label colors, and then combined before loading into a flowcell. When defining an experiment in Bionano Access, the user will identify each sample’s details. During the run, Bionano Access will provide real-time dashboard stats (throughput, labels/100 kbp, map rates, etc) for each sample independently. Finally, at the end of the run, two different (single color) BNX files will be generated and two different MQR reports will be generated. (Fig 1a). For more information, please check the “MQR in Access” session in this document.

Enzyme (motif) multiplexing is similar, except that there is only one single starting sample and it goes through two independent labeling reactions, each with a different enzyme and its associated labeling color. The samples are combined before loading into 1 flowcell (Fig 1b)

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Fig 1a (left) and Fig 1b (right). Illustration of multiplexing workflow. In Fig 1a (Sample Multiplexing Workflow), two different samples (S1 and S2) are nicked and labeld separately with two different colors (C1 or C2). In Fig 1b (Enzyme Multiplexing Workflow), the same sample (S1) is nicked with two different motif-recoginzing enzymes (E1 or E2) and colors (C1 or C2). Using 2 different labeling mixes, the motifs are labeld in either green or red.

For the “Dual-Labeled workflow” (Fig 2), DNA is labeled with both red and green labels at the same time.

When setting up the experiment in Bionano Access, the user is required to define two different enzymes with two different labeling colors for the same sample. During the run, Bionano Access will provide real-time dashboard stats (labels-per-100kbp, map rates, etc) for both enzymes independently (as with multiplexing); however, as both colors are on the same molecules, throughput and N50 stats are shared.

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Fig 2. Illustration of a single sample (S) labeled sequentially with two different motif-recognizing Enzymes (E) and each enzyme is matched with a single Color (C) labeling mix.

DNA Labeling

The guidance below for preparing multiplexing samples or dual-labeled color samples is not validated by Bionano. It is only provided as a reference for users to experiment or optimize.

Red-only NLRS Sample Prep, please follow 30024 Bionano Prep Labeling NLRS Protocol.

Multiplexing Sample Prep guidance

Prepare the samples independently with different labeling colors and/or different enzymes and try to obtain similar DNA concentrations in the NLRS samples.

Combine the individual labeled and stained samples together at a 1:1 ratio by volume.

Mix gently via wide bore pipette 5 times, then store overnight to homogenize at 4°C.

Load the multiplexed sample into a flowcell as usual.

Note:

The additional mixing step may potentially affect the sample's N50.

There is no guarantee that the two individual samples will have 1:1 throughput ratios during data collection.

The maximum DNA throughput limit for Saphyr Chip counts all raw DNA (>150kbp) per FC, regardless of number of samples in the FC. The individual throughput of the two separate samples is not used to determine the maximum throughput.

Sample multiplexing for genomes larger than 1 Gbp is not recommended.

Dual-Labeled Sample Prep Guidance

Although Bionano does not offer any dual-labeling methods at this time, there is a growing list of publications that describe various dual-labeling approaches using Bionano systems. We are currently aware of:

1. CRISPR-CAS9 D10A nickase target-specific fluorescent labeling of double strand DNA for whole genome mapping and structural variation analysis

In this paper, McCaffrey et al developed a sequence-specific DNA labeling strategy, which utilizes the Cas9 D10A protein to create a gRNA-directed DNA nick. Red fluorescent nucleotides are then incorporated adjacent to the nicking site with a DNA polymerase to label the gRNA-determined target sequences (e.g.: Alu elements). Combined with information obtained from global Nt.BspQI nicking-labeling (green labeling) on the same DNA molecule, researchers mapped the DNA molecules to the reference genome and profiled the distribution of Alu elements on the whole genome scale.

2. Reduced representation optical methylation mapping (R2OM2)

In this paper, Grunwald et al presented DNA methylation patterns in the genome by using the bacterial methytransferase M.Taql in combination with a synthetic cofactor analogue, which can then fluorescently label the adenine residue (green labeling) in TCGA sites containing non-methylated CpGs. Together with a genetic pattern generated by labeling of specific sequence motifs (Nt. BspQI, red labeling), researchers were able to simultaneously assign the epigenetic labels to their specific genomic locations and study DNA methylation patterns over large genomic fragment at molecule level.

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McCaffrey, J., Sibert, J., Zhang, B., Zhang, Y., Hu, W., Riethman, H., and Xiao, M. (2016) CRISPR-CAS9 D10A nickase target-specific fluorescent labeling of double strand DNA for whole genome mapping and structural variation analysis. Nucleic Acids Res. 44(2): e11

Grunwald, A., Sharim, H., Gabrieli, T., Michaeli, Y., Torchinsky, D., Juhasz, M., Wagner, K.R., Pevsner, J., Reifenberger, J., Hastie, A.R., Cao, H., Weinhold, E., Ebenstein, Y., (2017) Reduced representation optical methylation mapping (R2OM2). BioRxiv preprint

Experiment Setup for Saphyr or Irys Instrument

Prior to setting up experiments with red-labeled sample on the Saphyr instrument, please ensure that Bionano Support has configured your version of Bionano Access to enable this capability. To setup the experiments and check run progress (dashboard) on the Saphyr instrument, please see the help tutorials in Bionano Access software (“Red Experiment Tutorial”) and 30142 Bionano Access Software User Guide.

To setup the experiments at Irys instrument, please see 30047 Irys User Guide.

Sequential Imaging

The Saphyr instrument is capable of simultaneously imaging both red and green labels. This improves the registration accuracy and increases overall system throughput. This is the recommended mode of operation for multiplexed and dual-labeled samples. However, if higher SNR values are required for a dual-labeled sample, slightly improved performance may be achieved by running the imaging in a sequential imaging mode. This can be done by selecting the checkbox below. This should only be done after consultation with a Bionano Customer Solutions representative.

MQR (Molecule Quality Report) in Access

Sample Multiplexing

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Saphyr: after data collection and image detection, molecule BNX files of each individually labeled

sample will be automatically imported separately. Below is the example screen of MQR for a

multiplex sample.

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Irys: first run the color split command line of Bionano Solve v3.1 to split the BNX into 2 BNX files (1 per color) before importing into Bionano Access. . Please see Appendix for instruction.

Dual-Labeled Samples

For dual-labeled samples, only one molecule object will be imported to Bionano Access after data collection and image detection. The MQR will give the Total DNA and N50 values for the overall set of molecules, then give mapping statistics for each color independently if references were provided.

Visualization of Molecule Alignments in Access

Currently Bionano Access does not have the capability to align molecules to a reference without going through assembly first. To enable this functionally, a command-line script outside of Access needs to be run which generates the alignments for visualization within Bionano Access. This workflow can be used for single or dual-labeled molecules. For dual-labeled molecules, the color to align with will need to be selected by the user when running the command line script. Please refer to document 30194 “How to Align BNX to Reference” for instruction.

After alignment, please import the xmap, relevant _r.cmap and _c.cmap to Bionano Access as an “Alignment” object and specify the “Anchor to Molecules” alignment to visualize the results in Bionano Access.

When visualizing dual-labeled samples, only one enzyme’s labels will align to the reference. The other enzyme’s labels will be displayed on the molecules in their correct location relative to the first enzyme.

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Appendix: How to run a color split command line to split a

two color BNX file 1.) SSH to where you have Bionano Solve pipeline installed. For example, at Bionano Computation

Solutions Servers, it is located at /home/bionano/tools/pipeline/1.0/Pipeline/1.0 directory.

2.) The script is named “ColorSplit.pl” in the directory.

3.) To get help responses for this script, please issue the following command:

perl ColorSplit.pl –h

You will get this response:

Usage: perl ColorSplit.pl [Options] <Args>

Options:

-h : This help message

-i : Input BNX file (Required)

-o : Output folder (default: input folder)

-1 : Minimum label SNR threshold for channel 1 (default: 0)

-2 : Minimum label SNR threshold for channel 2 (default: 0)

-S : Filter the output BNX using SNR threshold? (default: OFF)

-r : The minimum ratio of the number of labels in two channels

for the molecule to be classified as the one (default: 1)

-N : Minimum nicks in each channel of the molecule (default: 0)

-K : Filter the output BNX using minimum nicks? (default: OFF)

-L : Minimum length of the molecule in kb (default: 0)

-M : Filter the output BNX using minimum length? (default: OFF)

-D : Debug mode to keep all temporary files (default: OFF)

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4.) To split a multiplexed bnx file into two separate bnx files, please issue this command:

perl ColorSplit.pl –i <<bnx file>> -o <<output folder>>

For example: perl ColorSplit.pl –i /home/bionano/split/RawMolecules.bnx –o /home/bionano/split

5.) After the command completes there will be a *_p1.bnx and a *_p2.bnx file in the output directory you specified. You can then transfer and use the files as you please.

Note: If you get an ‘ERROR: Could not find RefAligner in the default path!’ message just add RefAligner to your path with the following command:

export PATH=$PATH:<<path to RefAligner>>

For example:

export PATH=$PATH:/home/bionano/tools/pipeline/1.0/RefAligner/1.0