For decompression of FASTQ.ORA derived from specific species/models, the reference that has been used at compression needs to be downloaded from the ORA Support Site. The whole reference database can also be downloaded.

Linux or mac

Extract a specific reference

1. Move the downloaded archive to the oradata directory

mv yourdownloadpath/<genus_specificname>.tar.gz yourpath/oradata

2. Navigate to the oradata directory

cd yourpath/oradata

3. Extract the archive file using the following command:

tar -xzvf <genus_specificname>.tar.gz (Linux)
tar -xzvf <genus_specificname>.tar.gz (Mac)

When the extraction of the specific reference is completed on Linux OS, the orad.2.7.0.linux folder should be structured as follows with example on gallus_gallus reference:

orad.2.7.0.linux

   |___orad

   |___oradata

          |___refbin

          |___Gallus_gallus

               |___refbin

               |___readme_gallus_gallus

Extract the full database

1. Move the downloaded archive to the orad.2.7.0.linux directory

mv yourdownloadpath/oradata.tar.gz yourpath/orad.2.7.0.linux

2. Navigate to the orad.2.7.0.linux directory

cd yourpath/ orad.2.7.0.linux

3. Delete existing oradata folder

rm -r oradata

4. Extract the archive file using the following command:

tar -xzvf oradata.tar.gz (Linux)
tar -xzvf oradata.tar.gz (Mac)

When the extraction of the full database is completed the orad.2.7.0.linux folder should be structured as follows:

orad.2.7.0.linux

   |___orad

   |___oradata

     |___Homo_sapiens

          |___refbin

          |___readme_homo_sapiens

     |___Homo_sapiens_bisulfite

          |___refbin

          |___readme_homo_sapiens_bisulfite

     |___<Genus_specificname>

          |___refbin

          |___ readme_<Genus_specificname>

Windows

Extract the downloaded archive with a software that can handle gziped tarballs, such as 7-Zip. Right-click on the archive and select Extract with.

When a specific reference is downloaded, a folder with name <Genus_specifcname> is extracted. This folder contains the corresponding refbin and readme files. Move this folder in the location where orad and the default human refbin file has been extracted during the installation of the DRAGEN ORA Decompression Software procedure.

When the full database is downloaded, a folder with name oradata is extracted. This folder contains subfolders for each specific species which contains the corresponding refbin and readme files. Move this oradata folder in the location where orad and the default human refbin has been extracted during the installation of the DRAGEN ORA Decompression Software procedure.

Required commands

Use the following commands to decompress the files:

orad FILE [args]or orad [args] FILE

A folder that contains FASTQ.ORA files can also be provided for batch decompression of FASTQ.ORA files at top level directory:

orad ./pathtofolder/ [args]

On Windows, replace orad with orad.exe.

orad.exe FILE [args].

To decompress FASTQ.ORA compressed with a reference other than the default human reference, ensure the specific reference is available locally.

No change applies in the command line. The decompression software automatically detects which species/model is used.

Command Line Options

Command Examples

Using Windows, replace orad with orad.exe. Example is: orad.exe myfile.fastq.ora --check

For a fully transparent usage of fastq.ora files (no changes in the command, no overhead, no additional footprint) with third-party bioinformatics software, DRAGEN ORA Helper Suite Software is recommended and available for download on the ORA Support Site. This software is only supported on Linux.

For a semi-transparent usage of fastq.ora files with third-party bioinformatics software, use DRAGEN ORA Decompression with the pipe function or process substitution. This method improves system performance by reducing reads and writing to the disk versus a full decompression step.

• If the analysis software can read from the standard input, such as BWA, use the following command:

orad file.fastq.ora -c --raw | bwa mem humanref.fasta - > resu.sam

The -c option decompresses to standard output. The result is sent | to BWA, which uses the dash option - to read from standard input. This also works for paired reads, which uses the -p option of BWA to specify that the input contains interleaved paired reads.

• If the analysis software cannot read from the standard input, you can use process substitution:

bwa mem humanref.fasta <(orad file.fastq.ora -c --raw) > resu.sam

For the file name, use the <( ) syntax containing the command that generates the file to standard output. In this case, orad with the -c option as in the command above. This method does not work when the third-party software checks the input file name or when the third-party software does not read the file sequentially.

DRAGEN ORA Compression is a lossless compression.

If you wish to verify that no data was lost during the compression of the fastq.ora file, compare the MD5 checksum of the decompressed fastq.ora file and the MD5 checksum of the decompressed fastq.gz file.

1. Compute the md5 checksum of the uncompressed FASTQ.ORA content as follows

md5sum <(orad myfile.fastq.ora --raw -c )

2. Compute the md5 checksum of the uncompressed FASTQ.GZ content as follows

md5sum <(gzip -d -c myfile.fastq.gz)

DRAGEN ORA Decompression Software decompresses fastq.ora files into fastq.gz files. Fastq.ora files are generated using lossless compression technology as part of DRAGEN. Orad is the executable file that runs the DRAGEN ORA Decompression Software, which is a standalone piece of software.

With DRAGEN v4.3, compression of FASTQ files derived from supported non-human species and from human bisulfite data (methylated DNA application) is available. DRAGEN ORA decompression v2.7 allows for the decompression of FASTQ.ORA compressed with all supported references. Refer to the References Installation and Supported References sections.

The DRAGEN ORA Decompression Software is available for the following operating systems in three separated executable:

• Linux

• Mac

• Windows

Decompression of FASTQ.ORA stored on local storage is supported on Linux, Mac, and Windows. Decompression of FASTQ.ORA stored on AWS S3 is only supported on Linux.

Decompression of FASTQ.ORA stored on Azure Blob storage is only supported on Linux.

When FASTQ.ORA is located on AWS S3 or Azure Blob storage, the decompression occurs on a streaming mode: the FASTQ.ORA file does not need to be fully transferred before decompression can start.

DRAGEN v3.8 has introduced ORA Compression, a fully lossless compression algorithm to compress FASTQ.GZ files at a ratio up to x5 vs FASTQ.GZ. FASTQ.ORA files can be used as-is within the DRAGEN ecosystem. For users who are not using the DRAGEN secondary analysis software, FASTQ.ORA files can either be:

• used as-is, in a seamless manner, with most common downstream software, using DRAGEN ORA Helper Suite standalone software; or

• decompressed, using DRAGEN ORA Decompression standalone software.

These two standalone software can be downloaded for free, from the

Please refer to the relevant user guide for using them.

Installation Requirements

The following are the minimum requirements for the DRAGEN ORA Decompression Software:

Installing the DRAGEN ORA Decompression Software

Use the following steps to install the DRAGEN ORA Decompression Software once DRAGEN ORA Decompression has been downloaded from the .

Linux or Mac

1. Extract the archive files using the following command:

2. Navigate to the Orad directory as follows:

3. Move the executable to your preferred location as follows:

4. Add Orad to your path as follows:

5. Move the oradata folder content into the home repository as follows:

To store the folder in a different location, use the following command:

When oradata has been moved in another location, you can:

• either point to the reference by using the ORA_REF_PATH environment variable as follows:

• or use the following command at decompression

Windows

1. Extract the downloaded archive with a software that can handle gziped tarballs, such as 7-Zip. Right-click on the archive and select Extract with. The following two files are extracted:

• orad.exe

• refbin

The following steps use C:\Users\user1 as an example location. Change C:\Users\user1 to the location where you extracted the archive.

2. Open the Command Prompt application.

3. Set the environment variables to use the orad.exe and the refbin file with the set command or the setx command. The set command configures the variables temporarily (for the current console window) while the setx command configures the variables permanently.

4. Set the path to the orad.exe to the PATH environment variable as follows:

or

5. Set the path to the refbin file to an ORA_REF_PATH environment variable as follows:

or

Local vs. remote environment

In a local environment, FASTQ.ORA files are accessible on the compute node via the file system. Cloud instances with local data, or data available via a network file system, are also considered local environments. Local environment examples include the following:

• The compute node is a laptop and files are stored on the local hard disk.

• The compute node is a cloud instance and files are copied to its local storage.

In a remote environment, FASTQ.ORA files are stored in the cloud on either AWS S3 or Azure blob.

Remote using AWS S3

The DRAGEN ORA Helper Suite Software reuses the AWS Command Line Interface (AWS CLI) configuration. The location of a file is passed by specifying the file name as follows:

s3://bucket/<file name>

To make sure that the AWS CLI is configured to access your file, use the following command:

$ aws s3 ls s3://bucket/<remote file name>.fastq.ora

Remote Using Azure Blob Storage

The DRAGEN ORA Helper Suite Software reuses the URI set up for your Azure Blob Storage. The location of a file is passed by specifying the file name as follows:

https://StorageAccountName.blob.core.windows.net/<my container>/<my blob>.fastq.ora

To make sure that the Azure CLI is configured to access your blob file, use the following command:

export AZURE_STORAGE_CONNECTION_STRING=<yourURI> az storage blob list – container-name <myContainer>

If the input FASTQ.ORA file is an interleaved paired read file, it can be used as-is with downstream bioinformatics software which provide options to handle interleaved files. Make sure you are using the downstream bioinformatics software with proper interleaved options on the interleaved virtual FASTQ.

In the following example, after the oraFuse software has been mounted, the bwa command is used with the –p option to specify that the input contains interleaved paired reads.

bwa mem reference.fasta -p <interleaved file>.fastq -o result.sam

To decompress an interleaved FASTQ.ORA file into two separate files, use the orad software.

orad <interleaved file>.fastq.ora

To decompress into a single file with interleaved paired reads, use the --interleave option.

In the following example the orad software is used with the bwa command, and the -–interleave option is used to decompress into a single stream containing interleaved data.

bwa mem reference.fasta -p <( orad -c --raw --interleave <interleaved file>.fastq.ora ) > result.sam

This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license under its patent, trademark, copyright, or common- law rights nor similar rights of any third parties by this document.

The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read and understood prior to using such product(s).

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oraHelper Interactive User Guide

The oraHelper interactive user guide helps you to determine which of the DRAGEN ORA Helper Suite software to use with your intended downstream bioinformatics software. The oraHelper interactive user guide also displays the correct syntax to use.

Use the oraHelper interactive user guide as follows:

1. Open a terminal window.

2. Add the oraHelperSuite directory to your PATH as follows:

PATH=<oraHelperSuite Directory>:$PATH

3. Enter oraHelper followed by the intended downstream software command and the input *.fastq.ora file name.

$ oraHelper <command> <file name>.fastq.ora

Examples

The following example shows oraHelper used with the bwa command.

$ oraHelper bwa -i <filename>.fastq.ora

The output will print the proper syntax for each of the DRAGEN ORA Helper Suite software.

The following example shows the output of oraHelper used with the head command.

$ oraHelper head <file name>.fastq.ora

The following example shows the output of oraHelper used with the head command.

Command line Options

oraFuse Software

The oraFuse software creates a virtual FASTQ file for each FASTQ.ORA file in the directory. The virtual file created has a *.fastq file extension instead of a *fastq.gz file extension.

Local Environment

1. The oraFuse software runs with the following dependencies: fuse, fuse- libs, libcurl and openssl. If the dependencies were not installed during the DRAGEN ORA Helper Suite Software installation, run the following command.

$ sudo yum install fuse fuse-libs libcurl openssl

2. Add the oraHelperSuite directory to your PATH as follows.

PATH=<oraHelperSuite DIR>:$PATH

3. Enter the following command to mount the oraFuse software to the directory where the FASTQ.ORA files are located.

$ oraFuse

4. Run the desired command on the virtual *.fastq file.

5. When finished, enter the following command to unmount the oraFuse software from the directory.

$ oraFuse --unmount

Examples

The following examples show the results of the oraFuse software with the ls command.

• Before oraFuse has been mounted to the current directory:

r10K_1.fastq.ora

• After oraFuse has been mounted to the directory:

https: r10K_1.fastq r10K_1.fastq.ora s3:

• After oraFuse has been mounted to the current directory with ls -l:

The following example shows the head command on a virtual FASTQ file after oraFuse has been mounted.

$ head <file name>.fastq

The following example shows the bwa command on a virtual FASTQ file after oraFuse has been mounted.

$ bwa mem -t 8 -M <FASTA> -o <SAM> <file name>.fastq

Remote Environment

The steps to use the oraFuse software in the remote environment are the same as those used in the local environment.

The following command shows the remote and virtual files on S3.

$ ls s3 -l s3://bucket/<path>

Examples

The following example runs BWA on S3 files.

$ bwa mem -t 8 -M <FASTA> -o <SAM> s3://bucket/path/<file name>.fastq

When using DRAGEN v3.7 as a downstream software, you can use oraFuse in the remote environment if the location of a file gets passed by specifying the file name as follows:

.virtual/ora/s3:/bucket/<file name>.fastq

The following is an example of using DRAGEN v3.7 on a single FASTQ.ORA located on AWS S3:

Comman Line Options

Error Messages

If you receive an error message while using the oraFuse software, use the following command to get more information.

$ cat .virtual/ora/.error

ora LD-Preload Software

The ora LD-Preload software creates a virtual FASTQ file for each FASTQ.ORA file in the directory. The virtual file created has a *.fastq file extension instead of a *fastq.gz file extension.

Local Environment

1. Add the oraHelperSuite directory to your PATH as follows.

PATH=<oraHelperSuite Directory>:$PATH

2. Run the command with the ora LD- Preload shared library on the *.fastq file as follows.

$ LD_PRELOAD=<oraHelperSuite direcory>/ora-ldpreload.so <command><input file name>.fastq

Examples

The following is an example of the ora LD-Preload software with the 'bwa' command.

$ LD_PRELOAD=<oraHelperSuite DIR>/ora-ldpreload.so bwa mem -t 8 -M <FASTA> -o <SAM> <file name>.fastq

The following is an example of the ora LD-Preload software with the ls command.

$ LD_PRELOAD=<oraHelperSuite DIR>/ora-ldpreload.so ls

The following shows the output of the ora LD-Preload software with the ls command.

<file name>.fastq.ora <file name>.fastq (virtual file)

Remote Environment

The steps to use the ora LD-Preload software in the remote environment are the same as those used in the local environment. The features are the same, with the following exceptions:

• In the remote environment, the ora LD-Preload software does not work on statically compiled bioinformatics software.

• In the remote environment, the Linux shell auto-completion feature does not work on virtual files.

orad Software

The steps are the same for local and remote environments.

Local Environment

1. Add the oraHelperSuite directory to your PATH as follows.

PATH=<oraHelperSuite DIR>:$PATH

2. Enter the command with orad and the pipe or process substitution on the *.fastq.ora files. See the following examples.

Examples

Example with pipe:

When the downstream command or bioinformatics software can read from the standard input, the pipe process is used. The following is an example with the head command. The - c option decompresses to standard output.

$ orad <file name>.fastq.ora -c --raw | head

Example with process substitution:

When the downstream bioinformatics software cannot read from the standard input, for example md5sum, process substitution is used. The following example shows the command with process substitution.

$ md5sum <( orad -c --raw "<file name>.fastq.ora" )

Remote environment

The following information provides the requirements and steps to install the DRAGEN ORA Helper Suite Software.

Installation Requirements

The following table lists the minimum requirements for the DRAGEN ORA Helper Suite Software.

Install the DRAGEN ORA Helper Suite Software

Download the installer from the , and follow the installation steps below to install the DRAGEN ORA Helper Suite Software. Installation Steps:

1. Open a terminal window.

2. If the installation is for an online system, then move the installer to the directory where the software should be installed. If the installation is for an offline system, then the installer does not need to be moved.

3. Navigate to the installer location.

4. Run the following command:

sh <file name>.sh

5. To install oraFuse, install the dependencies when prompted. The following dependencies will automatically be installed on an online system:

The installer creates a folder named oraHelperSuite. If the installation is for an offline system, then move the oraHelperSuite folder to the offline system. The oraHelperSuite folder contains the following:

• orad executable

• oraFuse executable

• ora-ldpreload.so library

• oraHelper wrapper executable

Install the DRAGEN ORA Decompression References

Extract a specific reference

1. Move the downloaded archive to the oraHelperSuite directory

mv yourdownloadpath/<genus_specificname>.tar.gz yourpath/oraHelperSuite

2. Navigate to the oraHelperSuite directory

cd yourpath/oraHelperSuite

3. Extract the archive file using the following command:

When the extraction of the specific reference is completed the oraHelperSuite folder on a linux environment should be structured as follows with example on gallus_gallus reference:

oraHelperSuite   |__orad   |__orafuse   |__ora-ldpreload.so   |__oraHelper   |__refbin   |__Gallus_gallus        |__refbin        |__readme_gallus_gallus

Extract the full database

1. Move the downloaded archive to the oraHelperSuite directory

mv yourdownloadpath/oradata.tar.gz yourpath/oraHelperSuite

2. Navigate to the the oraHelperSuite directory

cd yourpath/oraHelperSuite

3. Extract the archive file using the following command:

When the extraction of the full database is completed the the oraHelperSuite folder on a linux environment should be structured as follows:

oraHelperSuite   |__orad   |__orafuse   |__ora-ldpreload.so   |__oraHelper   |__refbin   |__oradata     |__Homo_sapiens       |__refbin       |__readme_homo_sapiens     |__Homo_sapiens_bisulfite       |__refbin       |__readme_homo_sapiens_bisulfite     |__Genus_specificname       |__refbin        |__readme_Genus_specificname

The DRAGEN ORA Helper Suite support pages on the provide additional resources. These resources include training, compatible products, and other considerations. Always check support pages for the latest versions.

With DRAGEN v4.3, compression of FASTQ files derived from supported non-human species and from human bisulfite data (methylated DNA application) is available. DRAGEN ORA Helper Suite allows for transparent handling of such files, providing the correct reference has been downloaded and is available locally. Refer to the sections and .

The command orahelper <file.fastq.ora> provides information on:

• which reference has been used to compress

• the steps to follow to install the correct reference. Note: the default human reference refbin is installed by default with the Software and does not need to be downloaded again.

The DRAGEN ORA Helper Suite Software is a suite of software for Linux distributions, designed to integrate in a transparent manner compressed FASTQ.ORA files into secondary analysis in a local or remote environment.

In Illumina® DRAGEN™ Software v3.8 and later, the handling of FASTQ.ORA files is integrated within the system. DRAGEN ORA Helper Suite is intended for processing FASTQ.ORA files in environments where a secondary analysis is not performed on the DRAGEN Software or in environments that do not have access to DRAGEN v3.8 or later. If you are running secondary analysis pipelines on a DRAGEN v3.7 platform, only the oraFuse software is supported. Refer to oraFuse Software.

With the DRAGEN ORA Helper Suite, FASTQ.ORA files can be used:

• without full decompression to the disk

• with no overhead

• with no change in the current linux command (*.fastq is used instead of *.fastq.ora at input)

Software quick overview

The DRAGEN ORA Helper Suite Software package contains four software. Three of them process FASTQ.ORA files, the fourth one is an interactive user guide. You can use the FASTQ.ORA processing software in downstream analysis. The DRAGEN ORA Helper Suite Software v2.0 include:

• oraFuse v2.0

• ora-ldpreload v2.0

• orad v2.7.0

• oraHelper

oraFuse Software

The oraFuse software uses the FUSE filesystem to create a virtual ora subdirectory in the current directory, with ora symbolic links pointing to a virtual uncompressed FASTQ.ORA file. The virtual FASTQ file does not impact the storage footprint.

ora LD-Preload Software

The ora LD-Preload software uses the LD-Preload variable of the dynamic linker to intercept stystem calls. The ora LD-Preload software creates a virtual FASTQ file for each FASTQ.ORA file. The virtual FASTQ files do not impact the storage footprint.

orad Software

orad is the DRAGEN ORA Decompression software. Although it does not operate in a fully transparent manner, it works in most situations. The Orad software can be used in combination with the pipe process or pipe substitution process to reduce reads and writes to the disk.

oraHelper Interactive User Guide

oraHelper is an interective user guide that provides the following guidance:

• Which of the ORA Helper Suite software is the best fit.

• The proper command line to use with each of the ORA Helper Suite software.

• Information about the input file. For example, if a FASTQ.ORA file is a paired read interleaved file or which reference has been used to compress a FASTQ.ORA file.

The supported references are listed below. This list may not be exhaustive, the most up-to-date list of supported references can be found on the ORA Support Site.

You can use the oraHelper interactive user guide for assistance with common Linux commands, and to help troubleshoot errors encountered when using the different software included in the DRAGEN ORA Helper Suite. For details, refer to oraHelper Interactive User Guide section.

Common Linux Commands

The following table shows common Linux commands that you can use with the DRAGEN ORA Helper Suite software. This list is not exhaustive.

Bioinformatics Software

The DRAGEN ORA Helper Suite software work with some commonly used bioinformatics software. If the bioinformatics software is supported, you can use the oraHelper interactive user guide to help troubleshoot errors with your command. Below is the list of supported bioinformatics software with DRAGEN ORA Helper Suite. It does not limit the use of other thrid-party software.

Revision history of the DRAGEN ORA standalone Software family product documentation.