The latest release of Basespace Sequence Hub is version 7.29.0

Look here for information about previous releases

BaseSpace Sequence Hub is a cloud environment for analysis, storage, and sharing of genomic data. Stream data directly from your sequencing run and then analyze your results with BaseSpace Sequence Hub apps. BaseSpace Sequence Hub apps perform alignment, variant calling, data classification and visualization, and interpretation of results.

BaseSpace Sequence Hub has the following features:

• Easily shares and transfers data for collaboration with colleagues and customers.

• Analyzes and stores run data streamed from your sequencing system in real time.

• Automatically converts data from standard file formats for use with any BaseSpace Core App or third-party analysis app.

• Provides 1 Terabyte of complimentary data storage. Additional storage is available with Professional or Enterprise subscription accounts.

• Records sample information and run parameters for quicker run setup.

• Monitors sequencing runs using Sequencing Analysis Viewer (SAV) charts.

• Offers an intuitive interface to prepare and launch complex analyses.

When launching an analysis, BaseSpace Sequence Hub automatically aggregates all data associated with the same biosample name. For more information, see Associating Biosample Data With Projects.

To correct aggregation of data, requeue the run with a corrected biosample name. Only the run owner can requeue a run.

1. Edit the biosample name.

   • If you are using a sample sheet, useFix Sample Sheet.

   • If you are using the Prep tab, change the sample name in the Prep tab.

2. Requeue the run.

The previous run data will be marked as failed and can be deleted to reduce storage costs.

Biosamples that were converted from samples are automatically locked if they contain data produced by two or more runs. To use the data for analyses, unlock the biosample. Before unlocking a Biosample, review the data to ensure accuracy.

Reviewing Biosample Data

1. Navigate to the Biosample summary page and select the FASTQ Datasets tab.

2. Review the data and identify any data that should be excluded.

3. To exclude data, do the following:

   1. From the Datasets tab, select one or more datasets to exclude.

   2. From the Status menu, point to Change, and then select FASTQ QC.

   3. In the Change FASTQ QC Status dialog box, select QC Failed.

   4. [Optional] Add a comment.

   5. Select Save.

Unlock a Biosample

There are several ways to unlock Biosamples:

To unlock a biosample from the Biosample details page,

1. Navigate to the Biosample summary page.

2. A locked Biosample will display a red lock icon in the Biosample Status field.

3. From the Status menu, point to Unlock, and then select Biosample.

4. Select Continue.

To unlock multiple biosamples from a biosample list,

1. Navigate to the Biosample list page or the Project Biosamples tab.

2. Use the checkboxes to select Biosamples.

3. From the Status menu, point to Unlock, and then select Biosample.

4. Select Continue.

Updated Analysis Summary View

The analysis summary tab has been updated to bring the most relevant information front and center, both while the analysis is running and after it completes.

Spotlight Section

Useful information such as the analysis status and cost, as well as the application and version number, is organized in a Spotlight section at the top of the page.

Multiple Output Projects

Analyses that write data to multiple output projects now show an expandable list with links to each project.

This, along with the project column in the files tab, allows you to easily find any project or file associated with an analysis.

Multi-Node Applications

For applications that run multiple nodes, the Spotlight section incorporates high-level node statuses.

Detailed information for each node is displayed below the Spotlight and Logs sections. In addition to the node status and analysis duration, a dropdown section shows real-time logs and (upon completion) any log files that were produced.

Logs and Log Files

As before, the Logs section displays real-time logging during the analysis. In addition, a separate Log Files section will appear for applications that output specific log files, such as FASTQ Generation.

The availability of these log files is also reflected by a link in the Spotlight section, under the Application Type.

Improvements to the Requeue Experience

Requeue Sample Sheet

The requeue sample sheet feature loads the original run sample sheet into a text editor, allowing for review and/or editing before launching the requeue. Previously this feature only supported V1 sample sheets and now supports both V1 and V2 formats. For more information, see Fix Sample Sheet.

Requeue Planned Run

The requeue planned run feature uses the Run Planning tool for reviewing and/or editing V2 sample sheets. This feature supports both starting from the original sample sheet as well as uploading a new V2 sample sheet, which can be useful when the original sample sheet is the V1 format. Previously this feature was restricted to runs from certain instrument types and storage configurations and is now available for all runs. For more information, see Requeue a Planned Run.

Supported Requeue Applications

• The V1 sample sheet format supports launching the FASTQ Generation app.

• The V2 sample sheet format supports launching BCL Convert and supported Illumina DRAGEN secondary analysis applications:

  • Illumina DRAGEN Apps version 3 or higher.

  • DRAGEN TruSight Oncology 500 Apps version 2.1 or higher.

Consolidation of New and Classic Modes

To simplify workflows in BaseSpace Sequence Hub, we are consolidating New and Classic mode interfaces. With this change, Biosamples and Samples will be visible in a single view with no need to toggle between modes. Instead, choose a preferred input type when launching applications.

For more information,

• Illumina Informatics New and Updates

• BaseSpace Data Model Documentation

Automated Run Zipping

Around the middle of 2022, we will turn on an automated process in BaseSpace Sequence Hub that will start zipping the BCL and image files associated with old runs. Any run that hasn’t been modified for more than 90 days will be eligible for zipping. Metrics, log files, samplesheets, and other files in the run folder will be left untouched, and the ability to review run metrics remains unchanged.

For more information, see Automated Run Zipping.

Archiving

BaseSpace Sequence Hub now includes archive storage. This feature allows accounts configured for New application mode to move runs and datasets to long-term storage, with significantly lower costs than standard storage.

Data transfer to and from archival storage takes several days to complete and incurs additional fees per TB transferred. Restoring data that have been archived for a short period of time can result in higher overall storage, so archiving is not recommended for data that you intend to use soon.

For more information, see Archival Storage.

Biosamples

What are biosamples?

Biosamples are the original DNA samples that needs to be prepared, sequenced, and analyzed to produce the desired results for a bioinformatician. In BaseSpace Sequence Hub, they are the central link for related physical entities and digital data such as libraries, pools, runs, lanes, analyses, and data sets.

How do I add biosamples?

Add biosamples in a biosample workflow file. You can download a template and upload completed files from the Biosamples page, available from the My Data tab. In the biosample workflow *.csv file, add information about the new biosamples, the projects you want to store data in, the library preps you want to use, the yields required to launch an app, and the analysis workflows you want to schedule. BaseSpace Sequence Hub validates the inputs and adds the biosamples to the system. For more information, see Add Biosamples.

What happened to my samples?

Samples can still be accessed through a Run or a Project, using the Samples tab.

How do I add libraries and pools?

Libraries and pools are not uploaded manually but are generated automatically using the information in a sample sheet with an instrument run upload. From the sample sheet, Sample ID is used for the biosample name, and Sample Name is used for the library name.

When a biosample name is recognized due to a previous biosample workflow upload or instrument run containing the biosample, BaseSpace Sequence Hub checks the sample sheet for a match to a library. If we find an exact match, we add sequencing data to the existing biosample and library. If we don't find exact matches, we create a new biosample, a new library, or both.

When more than one library is given for a single lane number in the sample sheet, we interpret this as a pooled sample merged together using the libraries. We automatically assign a name to the pool and link it to the biosample and libraries. If the same combination of libraries exists within the same instrument run, the generated data are linked to the same pool. Library combinations cannot be reliably matched to runs across different instruments; in those cases, new pools are created.

The libraries and pools automatically generated from a sample sheet can be found in the Libraries tab of the biosample details page.

What are library prep kits?

Library prep kits are the names of the sample preparation kits used to turn biosamples into sample libraries. They are defined in the Prep Request column in the biosample workflow upload. BaseSpace Sequence Hub uses this information to separate data during data aggregation when there are two or more library prep kits used for the same biosample. For example, if you use a TruSeq PCR-Free library prep kit to prepare your libraries but receive poor results due to a low starting concentration of DNA, then make a second attempt with a TruSeq Nano library prep kit to amplify the DNA, you can use Sequence Hub to separate the data produced by the kit used to prepare the data.

Yield for a biosample is separated per unique library prep kit. View the list of prep kits for a biosample on the Summary tab of the biosample details page.

To launch an app using only the data from specific kits, select Select Biosample button when selecting the app inputs. This options enables a biosample chooser where you can select data by library prep kit.

What is biosample metadata?

Biosample metadata are key-value pairs used to save custom information to biosamples. The metadata can be viewed from the biosample summary page. Biosample metadata can only be entered when first creating the biosamples through the biosample workflow spreadsheet upload. When you add custom columns to the spreadsheet and define the values for the biosamples, the biosamples are imported with the metadata.

Do biosamples belong in a project?

Biosamples do not belong in a project. Instead, biosamples are related to a project by producing sequencing data in the form of data sets, which do belong in projects. Biosamples can have a default project, which is the default location data is written to when it is produced through Generate FASTQ and other BaseSpace Sequence Hub apps.

Biosamples can be related to many projects by creating data sets in each of them. For example, a biosample may be assigned a default project named Project A, where its FASTQ data sets are saved to. You can select the biosample as an input to manually launch an app and specify a different project, Project B, as the output project. The app then creates general data sets in Project B. The biosample is now linked to both projects, but does not belong to either of them.

When a project is shared or transferred, some biosample data to the project is shared with the collaborator. For more information, see Sharing Data.

What happens when I import FASTQ files from my desktop now that samples were replaced with biosamples?

When you upload FASTQ files, you create a new FASTQ data set which must be linked to a new biosample and library. Our new data model uses automatic aggregation of data to exclude any failures or low quality data among the biosamples, libraries, pools, lanes, and data sets. To allow auto-app launch to work, manual uploading of FASTQ files must conform to this model. The modified file import page will allow the creation of new biosamples and libraries to support adding FASTQ files to Sequence Hub.

Can I delete biosamples?

Biosamples themselves cannot be deleted. However, the data within biosamples can be deleted, either by deleting associated analyses or by deleting individual datasets using the FASTQ Datasets or Other Datasets tabs.

What happens when I cancel biosamples?

Canceling a biosample affects further work initiated to be performed on biosample data. Analyses that have not already been completed or stopped are canceled and their delivery status is changed to Do Not Deliver. These biosamples no longer appear in the available list of biosamples to be selected for app launch. Lab requeues can no longer be created for these biosamples and new biosamples cannot be created with the same name.

Runs

Automated Run Zipping

Please see a thorough list of FAQs here.

What is yield and how is it calculated?

Yield is a measure of how much sequencing data has been produced, in units of base pairs. Yield is the most commonly used app dependency to automatically launch an analysis for a biosample. BaseSpace Sequence Hub determines how much yield was produced from each flow cell lane the biosample was sequenced on, even if the biosample was merged into a pool with other biosamples.

Yield amounts include only high quality yield and exclude failed data or data produced by an entity that was marked as failed. For more information, see Yield.

BaseSpace Sequence Hub automatically tracks yield and updates status if yield is missing so you can request more sequencing data. For more information, see Request a Lab Requeue.

How do I update my lane QC thresholds?

Lane QC thresholds are a user setting that applies to the metrics of lanes from all runs the user owns, once the run is complete. You can set the thresholds using the API. For more information, see the developer documentation at developer.basespace.illumina.com.

What happens when a Generate FASTQ appsession aborts?

The Generate FASTQ app runs immediately after a run completes to convert .bcl files to .fastq files and demultiplex any indexing that occurred. If the app fails to finish, the status changes to Aborted, which causes the sequencing run status to change to Failed.

You can use the Fix Sample Sheet and Requeue option in the Run Details page to restart the Generate FASTQ analysis. This initiates a new Generate FASTQ analysis and resets the sequencing run status to Analyzing.

Analyses

What are Analysis Workflows?

Analysis workflows are templates that contain pre-defined settings and QC thresholds for a specific app. These workflows can be scheduled in advance to automatically launch when minimum requirements, called dependencies, are met.

Use the biosample workflow upload to schedule analyses for either new or existing biosamples. The analyses remain in Pending status until they can be launched. For more information, see Analysis Workflows.

How do I use biosamples to launch new analyses?

You can schedule apps to automatically launch or you can manually launch them.

To automatically launch an app, schedule an analysis workflow in a biosample workflow file. When enough yield or other dependencies are met for the analysis workflow, the analysis uses the biosample as an input to launch automatically.

Manually launch apps through the app details page. Apps that formerly required samples now require biosamples. Select inputs from a list of biosamples that contain FASTQ data sets.

How is data gathered before auto-launching apps?

The new data model supports data aggregation for the same biosample placed in multiple flow cell lanes in multiple runs. BaseSpace Sequence Hub now automatically locates and merges different samples for you before launching an app. When a biosample is linked with multiple libraries of a similar type, placed on different lanes, and placed on different flowcell runs, we can collect all of the FASTQ files produced exclusively for the original sample and input them into the app.

BaseSpace Sequence Hub excludes data that do not meet quality thresholds, which improves the chances of success in running apps. Immediately before an app is launched with biosamples as the input, BaseSpace Sequence Hub checks the statuses of all resources that produced the FASTQ data sets, including libraries, pools, data sets, runs, and lanes. For example, if a sequencing lane had failed due to quality, the app will not include any FASTQ data sets produced from that specific lane.

You can manually override QC statuses. For example, you can set a pool to Failed, which automatically excludes all FASTQ data sets produced by the pool.

For more information, see Automatic Data Aggregation and Manual QC.

How do I reschedule an analysis workflow to run again?

The biosample workflow upload allows you to specify an existing biosample in the analysis workflow column of the spreadsheet. As long as the biosample name given is an exact match with a biosample already owned by the uploading user, the analysis workflow is added to the existing biosample.

What is an analysis' delivery status and how do I use it?

The delivery status of an analysis is a manually updated, independent status used for tracking the progress of sending data to another user. You can use this to mark the data to be delivered and track the status of review and delivery . For more information, see Share Analyses.

Lab Requeues

What are lab requeues and how do I use them?

Lab requeues are a way to request more yield when a biosample falls short of what is required to run an app successfully. When a biosample has not produced the required yield in the specified time, it is marked as Missing Yield. You can initiate a lab requeue to request the sequencing lab to produce more data to make up for the missing amount.

When you initiate a lab requeue, you specify the checkpoint in the sample prep steps the lab should begin from. You can initiate more than one requeue at the same time, but Illumina recommends that only one lab requeue be fulfilled at a time.

When yield shows up in the form of another sequencing run, the lab status transitions to Sequencing. If enough shows up to meet the requested amount, the lab requeue status updated to Fulfilled. For more information, see Request a Lab Requeue.

Datasets

What are datasets?

Datasets are bundles of one or more files output by BaseSpace Sequence Hub apps. They can be used as input to other BaseSpace Sequence Hub apps when chaining apps together. Datasets belong in projects and are included if the project they are in is shared or transferred.

Where do I find my datasets?

Datasets can be viewed two ways,

• In a Project view, using the FASTQs or Other Datasets tabs

• In a Biosamples view, using the FASTQs or Other Datasets tabs

The following diagram illustrates the data flow and automation steps in BaseSpace Sequence Hub.

1. Biosample Workflow File Import—Add biosamples, library prep instructions, and analysis workflow instructions via a biosample workflow file. BaseSpace Sequence Hub creates new biosamples and schedules analyses.

2. After validating the manifest, BaseSpace Sequence Hub creates a new biosample and associated library prep requirements, and schedules a new analysis which includes analysis QC thresholds and a yield dependency.

3. Library Prep—BaseSpace Sequence Hub reports library prep status and QC.

4. Automatic Lane QC—BaseSpace Sequence Hub automatically performs lane QC on the new sequencing data based on the configured lane QC thresholds. Streaming Sequencing Data—The lab loads a flow cell and starts sequencing. Sequencing data begins to arrive in BaseSpace Sequence Hub.

5. Automatic FASTQ Dataset Generation—BaseSpace Sequence Hub performs demultiplexing, if needed, and generates FASTQ data sets for each library by lane and index (if indexing). QC-passed files are added to the yield calculations for the biosample.

6. Automatic App Launch—BaseSpace Sequence Hub automatically launches pending analyses that have yield dependencies met with the new data. BaseSpace Sequence Hub aggregates all FASTQ data sets for a biosample as inputs to the analysis, excluding biosamples that are QC failed or are associated with a different project. Data sets can originate from multiple flow cells or other uploads.

7. Automatic Analysis QC— BaseSpace Sequence Hub automatically performs analysis QC after analysis is complete.

8. Mark Analyses for Delivery—BaseSpace Sequence Hub users manually review analyses and change the status of acceptable analyses as Ready For Delivery.

9. Deliver Data—Deliver data using your preferred delivery mechanism, and mark the data as delivered.

Illumina supports customers who need help to analyze and interpret their data by offering comprehensive bioinformatics consulting support. Whether you are a researcher who is new to bioinformatics, or you need to improve your current analytical skills, Illumina can help. Illumina Bioinformatics Professional Services are delivered by professionals with deep scientific and product knowledge, who are committed to helping researchers effectively pursue their scientific goals. Illumina experts can support a range of skill levels, and both standard and specialized workflows.

More information is available on the Illumina website at .

For technical assistance, contact Illumina Technical Support.

Website:

Email:

Illumina Customer Support Telephone Numbers

When launching apps using biosamples as inputs, BaseSpace Sequence Hub automatically aggregates all of the valid (QC Passed) biosample FASTQ data associated with the same default biosamples. You can control which data are used in analyses by setting the QC status of a resource as QC Passed or QC Failed.

To prevent unintended aggregation, biosamples are locked if the biosample was converted from a sample and has data produced by two or more runs. The data cannot be used in analysis until the biosample has been reviewed and unlocked, however you can use samples to launch analysis without aggregation. For information about unlocking biosamples to make them available for analysis, see .

When BaseSpace Sequence Hub collects data for an app launch, it automatically excludes QC-failed lanes, libraries, pools, and any downstream data they produced. For example, if you fail a flow cell lane, all FASTQ data sets produced from that lane are excluded when aggregating data for the biosamples and libraries put on those lanes. If you fail a FASTQ dataset, only that FASTQ dataset is excluded.

If a FASTQ dataset has been copied, BaseSpace Sequence Hub uses the original FASTQ dataset, or the most recent copy if the original is not available. To use a different copy, mark the other copies as QC Failed before starting the analysis.

The following resources can be excluded from data aggregation:

• Lanes—Fail lanes using Automatic Lane QC, BaseSpace Sequence Hub API, or manually in BaseSpace Sequence Hub.

• Libraries—Fail libraries using the BaseSpace Sequence Hub API.

• Pools—Fail pools using the BaseSpace Sequence Hub API.

• FASTQ Datasets—Fail FASTQ data sets using the BaseSpace Sequence Hub API, or manually in BaseSpace Sequence Hub.

If you specify a library prep kit when selecting a biosample for analysis, the analysis launches using only FASTQ datasets from libraries of the specified prep kit. In the following example, Prep Kit B is selected as input and the FASTQ files from Prep Kit A are excluded.

Free Trial Account

BaseSpace Sequence Hub offers a limited 30-day free trial for new accounts. New free trial accounts have access to the following features.

• 1 TB free storage—Additional storage can be purchased with promotional iCredits.

• 250 iCredits—Promotional iCredits are valid for 30 days and can be used to pay for additional storage, compute, and third-party app fees.

• All BaseSpace Sequence Hub Apps—Access to all BaseSpace Sequence Hub apps during the trial period.

For information about upgrading your account, .

Basic Account

After the trial period ends, accounts that have not upgraded to Professional or Enterprise subscriptions are automatically converted to Basic accounts with limited storage and app access. To ensure uninterrupted service, contact your sales representative about upgrading to a Professional or Enterprise subscription.

• Basic accounts are limited to 1 TB storage. To increase your storage, contact your sales representative about upgrading to a Professional or Enterprise Account.

• All Basic accounts can perform instrument runs, download data, and delete data.

• Basic accounts using more than 1 TB storage cannot move data, share data, or run any apps until storage is reduced to less than 1 TB

• Basic accounts have access to a limited set of free BaseSpace Sequence Hub apps

Professional, Academic, and Enterprise Accounts

Paid subscription accounts offer expanded storage options, multi-user access, and workgroup support.

Inactive Accounts

Each sequencing run produces log files, instrument health data, run metrics, base call information (*.bcl files), and other data. BaseSpace Sequence Hub demultiplexes base call information to create the FASTQ files used in secondary analysis.

Common Terms

• Biosamples represent the source biological sample being sequenced. They are associated with data aggregated from multiple sequencing runs according the sample name provided in the samplesheet of each run.

• Samples represent a set of FASTQ files from a single sequencing run, according to the sample name provided in the samplesheet.

• Libraries are produced when a biosample is prepped with a library prep kit.

• Pools are an aliquot of one or more libraries, pooled together in order to be placed in a flowcell lane.

• Datasets are sets of files produced by a Basespace application. Some views will refer to them as "Other Datasets" to distinguish them from Datasets containing FASTQ files. These were formerly referred to as "App Results."

• FASTQ Datasets are a set of FASTQ files produced by FASTQ-Generation or BclConvert apps. Given their proment place in the Basespace data model, they're often treated as a distinct type from "Other Datasets," which aids in data management tasks like filtering & sorting.

• Projects are the containers for datasets and dataset files, which can include FASTQ, BAM, and VCF files. Projects can be associated with runs, analyses, and other entities in BaseSpace Sequence Hub. If a given project contains FASTQ files, it will also be associated with one or more Samples & Biosamples.

Analyzing FASTQ Data

• Basespace apps that analyze FASTQ files can accept either Biosamples or Samples in the input form, and the system will utilize the proper set of FASTQ files in each situation.

• Basespace users can select their preferred input at the top of the form, and Basespace will load the correct controls into the form:

More on Biosamples

• A run's Biosamples tab will list all of the Biosamples that this run contributed yield to:

• A project's Biosamples tab will list all of the Biosamples associated with FASTQ Datasets that live this this project:

What Happened to the Classic Data Model?

Basespace still has full support for classic data types like Samples (see term definitions above for more info). You can continue to use Samples and the associated set of features if that model is a good fit for your lab's needs, like launching an app with input FASTQ files from a single run.

• A run's Samples tab will continue to list the samples containing FASTQ files produced from that run's sequencing data:

• A project's Samples tab will continue to list the samples associated with FASTQ files that live in that project:

Biosamples that have not produced the requested amount of yield are marked as Missing Yield. Use a Lab Requeue to request more sequencing yield from the lab. After the requeue request is set to Acknowledged, incoming yield is applied to the request. For more information about yield calculations, see Yield.

A lab requeue requests additional biosample data. For information about requeueing a run with a fixed sample sheet, see Fix Sample Sheet.

The lab requeue returns the biosample to one of three stages in the lab preparation process.

• Biosample — The original biosample is prepared in a new library for sequencing.

• Library — An existing library is used for sequencing.

• Pool — An existing pool is used for sequencing. You can track the status or cancel the requeue from the Requeues tab of the biosample details page.

Requeue a Biosample

Request a Biosample Requeue if there were problems with the sequencing data from a biosample, or the existing library or pool does not have enough volume to produce the required yield. This request initiates a new library preparation from the original biosample.

1. Open the biosample details page for the biosample you want to requeue.

2. Select the Summary tab.

3. Select the File drop-down arrow, point to Requeue, and then select Biosample.

4. In the Requeue Biosample dialog, specify the requeue parameters.

   • Library Prep kit

   • Requested yield (Gbp/Mbp)

5. Select Save.

Requeue a Library

1. Open the biosample details page for the biosample you want to requeue.

2. Select the Libraries tab.

3. Select the checkbox for the library you want to requeue. You can only requeue one library at a time.

4. Select the File drop-down arrow, point to Lab Requeue, and then select Library.

5. In the Requeue Library dialog, specify the requeue parameters.

   • Requested yield

6. Select Save.

Requeue a Pool

1. Open the biosample details page for the biosample you want to requeue.

2. Select the Libraries tab.

3. Select the pool name for the desired library. If more than one pool exists for the library, select Pools, and then select the desired pool from the list.

4. Select the File drop-down arrow, point to Lab Requeue, and then select Pool.

5. In the Requeue Pool dialog, specify the requeue parameters.

   • Required pool yield

   • Unit of measurement

6. Select Save.

Acknowledge a Lab Requeue Request

Pending Lab Requeues are listed the Requeues tab of the Biosample Details page. When you have determined that your lab can fulfill the request, change the status to Acknowledged.

For information about acknowledging requeues using the BaseSpace Sequence Hub API, see the developer documentation at developer.basespace.illumina.com.

Cancel a Requeue

You can cancel a pending requeue. You might do this if the requeue is not needed, or there were errors in the requeue.

1. Open the biosample details page for the requeued biosample.

2. Select the Requeues tab.

3. To cancel a library, do the following:

   1. Select a library.

   2. Select the Status drop-down arrow, point to Cancel, and then select Library Requeue

4. To cancel a pool, do the following:

   1. Select a pool.

   2. Select the Status drop-down arrow, point to Cancel, and then select Pool Requeue

The following examples show possible yield scenarios:

• Example 1: Required Yield Met — The lab produces the required yield specified in the biosample workflow file.

• Example 2: Missing Yield — The lab does not produce all of the required yield. A lab requeue is initated and fulfilled.

• Example 3: Missing Yield After Lab Requeue — The lab does not produce the required yield from the initial request or the lab requeue. A second lab requeue is initiated and fulfilled.

You can configure BaseSpace Sequence Hub to automatically apply a QC status to the lanes of sequencing runs as they complete. BaseSpace Sequence Hub compares the lane metrics to your predefined thresholds and assigns a lane status as QC Passed or QC Failed.

When a lane is set to QC failed, its data are excluded from downstream data analyses and total yield calculations.

Lane QC thresholds are configured per user (or per workgroup) and apply to all lanes generated for sequencing runs owned by the user.

For information about using the BaseSpace Sequence Hub API or CLI to configure lane QC thresholds, see the developer documentation at developer.basespace.illumina.com.

You can review and manually change the lane QC status after it has been applied. For information about manually applying QC status, see Manual QC.

In this example, the original request and lab requeue do not produce the required yield before the yield timeout, requiring a second lab requeue.

3a: Yield Requested

A biosample manifest is imported with a prep request required yield of 105 Gbp.

3b: Partial Yield Received

The lab produces 80 Gbp of QC-passed FASTQ data sets. Actual yield is increased and the amount still pending is changed to 15 GBP to reflect the remaining balance. The expected yield does not change because it represents the total amount expected from the lab and includes actual yield and pending yield.

3c: Yield Timeout

Yield timeout is reached with no additional yield received.

When the yield timeout is reached, BaseSpace Sequence Hub assumes that the lab is unable to produce the requested quantity. The amount pending is classified as missing, and the expected yield is reduced to the actual yield received, indicating that no additional data are expected from the lab.

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3d: Lab Requeue for Additional Yield

A lab requeue for more yield is initiated. In this example, the user requested more than the missing amount.

• Expected yield updates. The updated amount reflects the quantity received from the initial request and the additional amount requested from the lab requeue. The required yield does not change because that is the amount of data required for analysis.

• Missing yield is reset to zero.

• Requested yield updates on the Requeues tab.

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3e: Partial Requeue Data Received

The lab produces 20 Gbp of QC-passed FASTQ data sets. Actual yield is increased and the amount still pending is changed to 10 GBP to reflect the remaining balance. The expected yield does not change because it represents the total amount expected from the lab and includes actual yield and pending yield.

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3f: Lab Requeue Expires

The lab requeue expires after the timeout period. As with the yield timeout, the amount pending is classified as missing and the expected yield is reduced to the actual yield received, indicating that no additional data are expected from the lab.

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3g: New Lab Requeue

Another lab requeue is initiated for 10 Mbp. The new request is listed on the biosample Requeues tab.

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3h: Lab Requeue Data Received

The lab produces 5 Mbp of QC-passed FASTQ data. The lab requeue is not completely fulfilled but the required yield dependency is met and analyses can automatically launch if they are not waiting for other dependencies.

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Yield is the amount of sequencing data produced for a particular biosample. When you add a new prep request with a biosample workflow, you specify the amount of yield you require before launching the analysis. BaseSpace Sequence Hub tracks the QC-passed data sets it receives and adds them to the cumulative yield total.

You can use yield information to easily identify the biosamples that do not have enough data and require a lab requeue.

Yield values appear in the Summary tab of the biosample details page.

Yield Types

The Demo Data page provides a list of run and project public data sets that you can add to your account.

1. Select the Demo Data tab.

2. Select a data set name to view details.

3. Select Import to import the run or project.

4. Select Accept to confirm the import.

1a: Yield Requested

A biosample manifest is uploaded with a prep request required yield of 105 Gbp.

1b: Partial Yield Received

80 Gbp of QC-passed FASTQ data sets are received. The actual yield is increased and the amount still pending is changed to 25 GBP to reflect the remaining balance. The expected yield does not change because it represents the total amount expected from the lab and includes actual yield and pending yield.

1c: Balance of Yield Received

The remaining 25 Gbp of FASTQ data sets are received. Actual yield is updated with the received amount. The full amount expected from the lab has been received, so there is no more pending yield.

BaseSpace Sequence Hub stores data for sequencing runs, samples, projects, and analyses. You can manage the data in your account and save space by deleting files that you no longer need.

The process to delete data requires two steps: move the data to trash, and then empty the trash. You can restore files that have been moved to trash.

Moving items to the trash, especially items with large amounts of data (eg, projects), can take time. The 'Trash Status' column indicates the percentage of data that has been moved into the trash. Once the status is 'Complete', the item can then be deleted by emptying the trash, or restored back to your account.

Delete Runs

1. Select the Runs tab, and then one or more runs to delete.

2. Select Move to Trash.

3. Select the run file types to delete.

   • Data files only—Deletes the data folder but keeps SAV data.

   • Data files and the run record—Deletes the data folder and any links to projects but keeps SAV data.

   • All run related files—Deletes all files, data, and project links associated with the run.

4. Verify the deletion, and then select Confirm.

Delete a Project

1. Do 1 of the following on the Projects tab:

   • Select a project to delete.

   • Open a project to delete.

2. Select Move to Trash.

3. Verify the deletion, and then select Confirm.

Delete a Sample

1. Select the Projects tab.

2. Select the project containing the sample to delete.

3. Select the checkboxes for the samples to delete.

4. Select Move to Trash.

5. Verify the deletion, and then select Confirm.

Delete an Analysis

1. Select the Projects tab.

2. Select the project containing the analysis to delete.

3. Select Analyses, and then select the checkbox of the samples to delete.

4. Select Move to Trash.

5. Verify the deletion, and then select Confirm.

Delete Datasets

1. Select the Projects or Biosamples tab.

2. Select the project or biosample containing the datasets to delete.

3. Select either the FASTQs or Other Datasets tab.

4. Select the checkboxes for the datasets to delete.

5. Select Move to Trash.

6. Select the dataset file types to delete.

   • Data Files Only deletes the files but keeps the dataset record.

   • All Files deletes the files and removes the record of the dataset.

7. Verify the deletion, and then select confirm.

Restore Deleted Data

1. Select the Trash icon.

2. In the Trash window, select the files you want to restore.

3. Select Restore.

Permanently Remove Deleted Data

1. Select the Trash icon.

2. Select Empty Trash.

BaseSpace Sequence Hub tracks the status of biosample data from library prep through sequencing and delivery of analysis files.

For information about changing statuses, see and .

For information about sequencing yield status, see .

Analysis Delivery Status

The delivery status of the analysis. Manually update statuses to track the progress of analysis deliverables. Possible values are:

• On Hold—The analysis is not ready for delivery.

• Do Not Deliver—The analysis files should not be delivered. You can use this status to confirm that a QC Failed analysis has been reviewed and should not be delivered, or to mark R&D and test analyses that are for internal use only.

• Deliver—The analysis files can be delivered.

• Ready For Delivery—The analysis files have been reviewed and are ready for delivery.

• Delivery in Progress—The analysis files are currently being transferred.

• Delivery Failed—Delivery of the analysis was attempted and failed.

• Delivered—The analysis files have been delivered.

Analysis Status

The status of the analysis through execution and QC. Possible values are:

• Pending—The analysis is waiting for dependencies (eg, yield or completion of another analysis) to be met.

• Queued for Analysis—The analysis workflow has met its dependencies (eg, yield or completion of another analysis) and is queued for analysis. Analyses with this status cannot be deleted.

• Awaiting Authorization—The analysis has been created but has not been accepted.

• Initializing—The app has launched and is awaiting execution.

• Running—The analysis is running. Analyses with this status cannot be deleted.

• Timed Out—The analysis did not complete within 48 hours and was terminated.

• Needs Attention—There is a problem with the run sample sheet.

• Canceled—A user manually canceled the analysis before its dependencies were met.

• Aborting—The analysis is in the process of being stopped. Analyses with this status cannot be deleted.

• Aborted—The analysis has been stopped.

• Complete—The analysis has finished and is ready for use.

• QC Passed—All analysis metrics met established thresholds or were reviewed and manually passed.

• QC Failed—One or more analysis metrics did not meet established thresholds or were reviewed and manually failed.

• Discarded—The QC Failed analysis has been reviewed and can be ignored.

Biosample Status

Higher-level status of the biosample, from library preparation through sequencing. If more than one library has been prepared from a biosample, the status refers to the most recent library being processed. Possible values are:

• New—The default state for a biosample created by LIMS systems.

• Active—The biosample is being worked on in the lab. The default state for a biosample created by non-LIMS systems.

• Done—The analysis files have been delivered. This status is set manually

• Canceled—The biosample is canceled and will no longer be used or processed.

• QC Failed—The biosample has failed Automatic QC in a LIMS system or was manually failed based on metrics.

• On Hold—The biosample needs attention or further review. This status is set manually.

Biosample Lab Status

The status of lab tasks for the biosample, from library preparation through sequencing. If more than one library has been prepared from a biosample, the status refers to the most recent library being processed. Statuses are automatically updated by BaseSpace Sequence Hub. Possible values are:

• New—The biosample has been added to BaseSpace Sequence Hub. This is the default status.

• Visual QC Failed—The container of biosamples failed visual inspection quality control check and cannot continue.

• Visual QC Passed—The container of biosamples passed visual inspection quality control check.

• Quant QC Failed—The biosample did not meet concentration threshold at the quantification step.

• Quant QC Passed—The biosample met concentration threshold at the quantification step.

• Sequencing—The biosample is being sequenced by an instrument.

• Requeued—A requeue for the biosample or library has been initiated and acknowledged by the lab.

• Missing Yield—BaseSpace Sequence Hub has not received the required yield before the process has timed out.

• Complete—The required yield for the biosample has been met.

FASTQ Status

The status of the generated or uploaded FASTQ dataset.

• Default status for FASTQ data. Datasets in the default state are included in data aggregation for analysis.

• QC Passed—The FASTQ data passed QC.

• QC Failed—The FASTQ data failed QC.

Lab Requeue Status

The status of the requeue. Possible values are:

• Pending—The requeue request has been initiated and is awaiting acknowledgment by a lab or LIMS.

• Acknowledged—The requeue request has been acknowledged manually by a lab or automatically by a LIMS.

• Canceled—The requeue has been stopped manually by a user.

• Expired—The requeue has been acknowledged but has not met the minimum yield required within the configured amount of time.

• Fulfilled—The requeue has met the requested yield.

• Failed—The requeue request has been rejected because there is not enough volume to perform the requeue.

Lane Status

The status of the individual lane. Lane status can be changed by Automatic Lane QC, manually, or via API. Possible values are:

• QC Passed—The lane data passed QC.

• QC Failed—The lane data failed QC.

Lane and QC Status

The overall status of the lanes on a run. Possible values are:

• Initial—The default status for a lane until QC has been performed or is manually overwritten.

• Lane Pending QC—Automatic lane QC is being processed against the run thresholds (set via an API).

• Lane QC Failed—The lane has failed the automatic QC thresholds set by the user (via an API) or has been manually overwritten.

• QC Passed—All lanes passed QC.

• Rehybed—The run has been stopped because of a problem with the flow cell. After the flow cell is rehybed, it is used in a new run.

Library Status

The status of the library. Library status is updated via the BaseSpace Sequence HubAPI. Possible values are:

• Active—The default status of a library.

• Canceled—The library has been marked as canceled and will no longer be worked on.

• Failed—The library has failed QC metrics.

• Consumed—The library has exhausted its volume and can no longer be requeued.

Pool Status

The status of the pool. Pool status is updated via the BaseSpace Sequence HubAPI. Possible values are:

• QC Passed—The data from the pool has passed QC. This is the default status.

• QC Failed—The data from the pool do not pass QC.

• Failed—The pool failed due to contamination or other physical reason.

• Consumed—The pool contents have been used and there is not enough volume for a requeue.

• Requeued—A requeue has been initiated for the pool.

Prep Request Status

• Active—A library kit and yield are specified in an imported biosample workflow file.

• Missing Yield—BaseSpace Sequence Hub has not received the required yield before the yield timeout.

• Canceled—The request to sequence the library has been canceled.

Run Status

The status of the sequencing run: from Prep Tab planning, sequencing, file-upload, and FASTQ analysis. Statuses are automatically updated from the instrument.

Prep Tab statuses

• Planning—The run is being planned in the Prep Tab.

• Ready—The run prepared on the Prep Tab is ready to be sequenced.

• Unknown—The status of the run is unknown.

Sequencing statuses

• Running—The instrument is sequencing the run.

• Paused—A user paused the run on the instrument control software.

• Stopped—The instrument has been put into a Stopped state through the instrument control software.

File Upload and Analysis statuses

• Uploading—The instrument completed sequencing and is uploading files.

• Failed Upload—The instrument failed to upload files to the BaseSpace Sequence Hub.

• Pending Analysis—The file has uploaded completely and is waiting for the Generate FASTQ analysis to begin.

• Analyzing—The Generate FASTQ analysis is running.

• Complete—The sequencing run and subsequent Generate FASTQ analysis completed successfully.

• Failed—Either the sequencing run was failed using the instrument control software or Generate FASTQ failed to complete.

• Rehybing—A flow cell has been sent back to a cBot for rehybing. When the new run is complete, the rehybing status is changed to Failed.

• Needs Attention—There is an issue with the sample sheet associated with the run. The run can be requeued after the sample sheet is fixed.

• Timed Out—There is an issue with the sample sheet associated with the run. The run can be requeued after the sample sheet is fixed.

In some cases, you might need to override a QC status that was automatically set by BaseSpace Sequence Hub. When a resource is set to QC failed, its data and any data produced from it are excluded from downstream data analyses and total yield calculations. For more information about excluding data from analyses, see .

You can fail Lanes, Libraries, Pools, and FASTQ Datasets using the API. For more information, see the developer documentation at .

Change Lane QC Status

For information about automatic lane QC, see .

1. Open the Run & Lane Metrics page for the desired run.

   1. Select the Runs tab.

   2. From the Runs list, select a run.

   3. From the Run Summary page, select the Run & Lane Metrics icon

2. Select one or more lanes that have the same QC status.

3. Select the Status drop-down arrow, point to Change, and then select Run Lane QC.

4. In the Change Lane Status dialog, review the selected lanes and do the following:

   1. Select the Change Status to drop-down arrow and then select a new status, QC Passed or QC Failed.

   2. [Optional] Add a comment.

Change FASTQ QC Status

You can change the status of a generated or uploaded FASTQ dataset.

1. Open the Output Files tab for the desired biosample.

   1. Select Biosamples.

   2. From the Biosamples master list, select a biosample.

   3. From the Biosample Details page, select the Output Files tab.

2. Select one or more FASTQ data sets that have the same QC status.

3. Select the Status drop-down arrow, point to Change, and then select FASTQ QC.

4. In the Change FASTQ QC Status dialog, review the selected FASTQ data sets and do the following:

   1. Select the Change Status to drop-down arrow and then select a new status, QC Passed or QC Failed.

   2. [Optional] Add a comment.

Change Analysis QC Status

You can change the status of analyses that have completed. Changing the analysis QC does not affect the QC of the FASTQ data sets.

1. Select Analyses to open the Analyses master list.

2. Select one or more analysis workflows that have the same QC status.

3. Select the Status drop-down arrow, point to Change, and then select Analysis QC.

4. In the Change Analysis Status dialog, review the selected analyses and do the following:

   1. Select the Change Status to drop-down arrow and then select a new status.

   2. QC Passed—All analysis data sets passed QC.

   3. QC Failed—One or more analysis data sets failed QC.

   4. Discarded—The QC-failed analysis has been reviewed and can be ignored.

   5. [Optional] Add a comment.

Import Files into a Project

The file uploader imports the following file types to any project you have write access to: FASTQ (.fastq.gz), analysis (VCF and gVCF), manifest (.txt), or other file types. Use the file uploader when you want to analyze files generated outside of BaseSpace Sequence Hub, or to attach other information related to the project.

1. Open the project.

2. From the project, select File, Upload, and then select Files.

3. Select type of files to upload.

4. If you are uploading a FASTQ file, do as follows.

   1. To upload FASTQs to a sample,

      1. Set the "Save Upload To" toggle to "Sample".

      2. Select Finish Upload.

   2. To upload FASTQs to a biosample,

      1. Select Select Biosample, then select an existing or create a new biosample to associate the FASTQ dataset with.

      2. Enter a library name.

      3. Select a prep kit.

      4. Select Finish Upload.

5. If you are uploading a VCF file, do as follows.

   1. [Optional] Select Select Biosample, then select or create a biosample that the VCF will be associated with.

   2. Select Finish Upload.

6. If you are uploading a manifest file, do as follows.

   1. [Optional] Select Select Biosample, then select or create a biosample that the manifest will be associated with.

   2. Select Finish Upload.

7. If you are uploading other file types, do as follows.

   1. [Optional] Select Select Biosample, then select or create a biosample that the files will be associated with.

   2. Select Finish Upload.

If you do not need all of the files in a data set, you can download files individually.

1. Navigate to the analysis file you want to download.

2. Select the file.

3. Select Download.

Download data from a run as a package of FASTQ files or SAV files. Use the following steps to download a package.

1. Open the desired run.

2. Select File, point to Download, and then select Run.

3. Select the file type you want to download.

• FASTQ—FASTQ files.

• Sequencing Analysis Viewer (SAV)—InterOp and other files required to run SAV.

Data is downloaded in a compressed (*.zip) file format.

Use the BaseSpace Sequence Hub Downloader to download FASTQ or general datasets. Datasets are linked to biosamples and are listed on the Datasets tab of the biosample details page. To download a package of datasets from a run, see Download Run Data Files.

1. From the Biosamples or Projects list, select the biosample or project.

2. Select the FASTQs or Other Datasets tab.

3. [Optional] Select a dataset name to view a list of file details.

4. Select the checkboxes for the desired datasets.

5. Select File, Download, then select Dataset.

6. Select Download. The BaseSpace Sequence Hub Downloader guides you through the download process, and starts the download of the files to the desired location.

BaseSpace Sequence Hub allows you to download data as a package, individually, or as a group of FASTQ files.

The BaseSpace Sequence Hub Downloader supports downloading files through a proxy server and automatically inherits appropriate settings from the host system. The proxy server must be configured to support the SOCKS4/5 protocol for TCP connections. Contact your network administrator for help with establishing these settings.

The FASTQ file is a text format file used to represent sequences. Each record has four lines of data: an identifier (read descriptor), the sequence, +, and the quality scores. For a detailed description of the FASTQ format, see FASTQ Files.

Make sure the FASTQ file adheres to the following upload requirements:

• FASTQ files are generated on Illumina instruments and saved in gzip format

• The name of the FASTQ files conforms to the following convention: SampleName_SampleNumber_Lane_Read_FlowCellIndex.fastq.gz

  • Examples: SampleName_S1_L001_R1_001.fastq.gz SampleName_S1_L001_R2_001.fastq.gz

• The read descriptor in the FASTQ files conforms to the following convention: @Instrument:RunID:FlowCellID:Lane:Tile:X:Y ReadNum:FilterFlag:0:SampleNumber:

  • Examples: Read 1 descriptor: @M00900:62:000000000-A2CYG:1:1101:18016:2491 1:N:0:13 Corresponding Read 2 descriptor has ReadNum field: @M00900:62:000000000-A2CYG:1:1101:18016:2491 2:N:0:13

Quality considerations:

• The number of base calls for each read equals the number of quality scores.

• The number of entries for Read 1 equals the number of entries for Read 2.

• The uploader determines whether files are paired-end based on matching file names in which the only difference is the ReadNum.

• For paired-end reads, read 1 and read 2 files need to be uploaded together or can be combined later.

• Each read has passed filter.

Use the BaseSpace Sequence Hub Downloader to download a package of all files in a project.

1. Select Projects.

2. Select a project.

3. Select Download.

The BaseSpace Sequence Hub Downloader guides you through the download process, and starts the download of the files to the desired location.

Archival storage moves data into long-term archives with lower data storage rates. It is intended for data that must be retained and do not require immediate access.

Data transfer to and from archival storage incurs additional fees per Terabyte transferred in the move. Restoring data that have been archived for a short period of time can result in higher storage costs.

For more information about storage costs, see the illumina.com iCredits information page.

The following data can be archived:

• Runs—Includes all files within the Data folder.

• FASTQ and data set files—Includes all files within the data set.

Use the BaseSpace Sequence Hub Downloader to download whole samples or individual files from a specific sample.

Download Samples

1. From the Projects list, select the project.

2. Select the Samples tab.

3. Select the checkboxes for the desired sample or samples.

4. Select File, Download, then select Sample.

5. Select Download. The BaseSpace Sequence Hub Downloader guides you through the download process, and starts the download of the files to the desired location.

Download Sample Files

1. From the Runs or Projects list, select the run or project.

2. Select the Samples tab.

3. Click the name of the desired sample to access the sample details page.

4. In the Files section, select the checkboxes of the file or files to download.

5. Select "Download Selected". The BaseSpace Sequence Hub Downloader guides you through the download process, and starts the download of the files to the desired location.

Copy data sets from one project to another to do any of the following:

• Analyze a dataset in the context of two different projects.

• Transfer ownership of a dataset, but keep a copy.

• Correct a dataset assigned to the wrong project.

After copying, both the original and the copy appear in the data sets tab on the biosample details page. When aggregating data for analyses, BaseSpace Sequence Hub uses the original dataset or the most recent copy if the original is not available. To use a different copy, mark the other copies as QC failed before starting the analysis.

1. From the Biosamples page, select the biosample.

2. Select the Datasets tab

3. Select the checkbox of each dataset you want to copy.

4. Select File, point to Copy, and then select FASTQ Dataset.

5. [Optional] Select New to create a new project.

6. Select a project to copy the data sets to. You can only copy data sets to projects you own.

7. Select Confirm.

Use the BaseSpace Sequence Hub Downloader to download a package of analysis files.

1. Select Analyses.

2. Select the checkboxes for the analyses you want to download.

3. Select File, point to Download, then select Analysis.

4. Select the file type you want to download.

• All files including VCF, BAM, & FASTQ

• VCF files only

• BAM files only

1. Select Download. The BaseSpace Sequence Hub Downloader guides you through the download process, and starts the download of the files to the desired location.

Configure the settings for the analysis type selected for your run.

1. Select the Runs tab, and then select the New Run drop-down.

2. Select Run Planning.

3. Run Settings wizard will be loaded.

4. In the Run Name field, enter a unique name of your preference to identify the current run. The run name can contain a maximum of 255 alphanumeric characters, spaces, dashes, and underscores.

5. [Optional] In the Run Description field, enter a description of the current run. The run description can contain a maximum of 255 alphanumeric characters.

6. Select the Instrument Platform.

7. Select the analysis location. Depending on the selected instrument type, not all options may be available.

   • BaseSpace - Analyze sequencing data in the cloud.

   • Local - Analyze sequencing data on-instrument or generate a Sample Sheet v2 for Local or Hybrid mode.

8. [Optional] In the Library Tube ID field, optionally enter the library tube ID of the current run. The library tube id can contain a maximum of 255 alphanumeric characters.

9. Select Next

10. Configuration wizard will be loaded

11. Select an analysis type and version. For more information about secondary analyses, see DRAGEN Secondary Analysis Output Files on the system guide for your instrument or the BaseSpace Sequence Hub app documentation. If you selected DRAGEN Single Cell RNA analysis, see the NextSeq 1000/2000 Products Files page for information on third-party single cell RNA library prep kit compatibility.

    For on-instrument analysis, the version selected must match the version of DRAGEN installed on the instrument. To confirm the version of DRAGEN installed on the instrument, see DRAGEN Workflow and License Updates on the system guide for your instrument.

12. [Optional] Set up custom index kits as follows. If you are using more than one library, the libraries must have the same index read lengths.

    1. Select Add Custom Index Adapter Kit under the Index Adapter Kit dropdown.

    2. Select a template type and enter the kit name, adapter sequences, index strategies, and index sequences. Make sure the second index (i5) adapter sequences are in forward orientation.

    3. Select Create New Kit.

13. [Optional] Set up custom library prep kit as follows.

    1. Select Add Custom Library Prep Kit under the Library Prep Kit dropdown.

    2. Enter the name, read types, default read cycles, and compatible index adapter kits for your custom library prep kit.

    3. Select Create New Kit.

14. Select the following instrument settings. Depending on the library prep kit, recommended options are automatically selected. Some library prep kits have hard-coded number of indexes reads and read types, which cannot be changed.

    • Library prep kit

    • Index adapter kit

    • Number of index reads

    • Read type

    • Number of sequencing cycles per read

    If Not Specified is selected for library prep kit, enter the number of Index sequencing cycles to be used in the run.

15. Enter sample information into the Sample Data spreadsheet using one of the following options. To group samples for data aggregation during downstream analysis, assign a name for the group in the Project column.

    • Select Import Samples and the type of the import source file in the dropdown, either CSV or Sample Sheet, and then select your source file. If CSV file is selected, make sure that your file follows the template that can be downloaded by selecting Download Template button.

      If Sample Sheet is selected, make sure that your sample sheet meets the formatting requirements. If CSV is selected, make sure to use the correct template as the template can be different depending on the selected index adapter kit and index strategy. For more details, see Import Samples.

    • Paste sample IDs and either index plate well positions or i7 and i5 indexes directly from an external file. Before pasting, enter the number of sample rows in the Rows field, and then select +. Sample IDs can contain up to 100 alphanumeric characters, hyphens, and underscores.

      Fixed-layout index plates require entries for well position. Indexes that do not have a fixed layout require entries for i7 and i5 indexes. i5 indexes must be entered in the forward orientation.

    • Manually enter sample IDs and corresponding well positions or indexes. If Not Specified is selected for the library prep kit, enter Index 2 (i5) sequences in the forward orientation.

You can correct errors in your index and regenerate FASTQ files using the Prep tab up to five times. This feature is available only for runs set up in BaseSpace Sequence Hub.

1. Go to the Summary page for the run you want to fix.

2. Select the Settings tab.

3. Select the Pool ID link at the bottom of the screen.

4. Select the Plate ID link in the table.

5. Select Edit.

6. Select the correct indexes from the index drop-down lists.

7. Select Save.

8. Return to the Summary page for the run.

9. Select Status, point to Requeue, and then select FASTQ Generation.

BaseSpace Sequence Hub regenerates the FASTQ files with the corrected indexes. The new FASTQ files are added to the biosample list and the original files are marked as QC Failed.

The sample sheet is a comma-delimited file (SampleSheet.csv) that stores the information needed to set up and analyze a sequencing experiment. The file includes a list of samples, their index sequences, and the sequencing workflow.

Every run in BaseSpace Sequence Hub requires an associated sample sheet to define projects and samples, assign indexes, and run workflow apps.

Use Illumina Experiment Manager software to set up a sample sheet for your library prep protocol.

NovaSeq X Series systems allow you to perform multiple DRAGEN analyzes in a single sequencing run. Before setting up secondary analysis, make sure you have installed the appropriate DRAGEN application on your instrument. For more information on installing DRAGEN applications, refer to the NovaSeq X Series Product Documentation.

If storing data in the cloud, you can create up to seven analysis application and reference genome combinations with an additional BCL Convert-only application. If storing data locally, you can create up to three analysis application and reference genome combinations with an additional BCL Convert-only application. For each combination, you can use up to eight configurations using a different library prep kit, index adapter kit, or configuration settings for an analysis application and reference genome combination already used.

The following combinations are included in the seven or three configuration limit:

• The same analysis application and application version with a different reference genome

• The same reference genome with a different application or application version

• A different application or application version with a different reference genome

To perform a secondary analysis, an Application may require certain types of files, such as:

• AuxCnvPanelOfNormalsFile - for Enrichment workflow

• AuxNoiseBaselineFile - for Enrichment workflow

• BedFile - for Enrichment workflow

• RnaGeneAnnotationFile - for RNA workflow

Reference files for NovaSeq X analysis are managed from Resources page.

• Select Resources from the user menu on the top right of Sequence Hub page

• Select Reference Files tab to see the list of reference files available for use in the run planning.

• Both standard and custom files are included in the list.

Import a Custom Reference File

1. Select Import Custom Reference File to upload a custom file.

2. After file upload is completed,

   • Select the correct File Type. Run Planning tool will associate the new custom file with the Application based on the selected File Type.

   • Select one or more reference genome(s) that should be associated with the file.

   • [Optional] Enter a description for the custom reference file.

3. Select Save.

Edit a Custom Reference File

To edit a custom reference file's metadata, go to the listing page and select the file. Update the information on the Edit page and save it upon completion.

To update the file content, select Import Custom Reference File from the listing page and upload the new file.

Delete a Custom Reference File

To delete a custom reference file, go to the listing page, and select the delete icon beside the file.

If using somatic mode, you can generate a custom noise baseline file. The noise baseline file is built using normal samples that do not match to the subject the samples are from. The recommended number of normal samples is 50.

To generate a custom noise baseline file, use one of the following methods:

• Use the DRAGEN Bio-IT Platform server. See the DRAGEN Bio-IT Platform Online Help for instructions.

• Use DRAGEN Baseline Builder App on BaseSpace Sequence Hub. Use the BCL Convert pipeline in BaseSpace Sequence Hub Run Planning to generate FASTQ files. After the sequencing run is complete and 50 samples are available, input the FASTQ files into the DRAGEN Baseline Builder App.

Import Samples from CSV file is an alternative way to populate Sample table and sample-level analysis settings on the Configuration page.

CSV Template

The CSV template only includes editable columns in the Sample Table and the sample-level analysis settings if applicable (e.g. for RNA Seq application with Differential Expression). Import will auto-populate the derived columns from the input data, e.g. Index names and sequences are derived from the respective Well Position.

• For custom or standard kit with fixed plates:

  • Sample ID*, Well Position*, Project, and sample-level analysis settings (if applicable)

• For custom or standard kit with fixed indexes:

  • If single index strategy is selected: Sample ID*, I7 Index* , Project, and sample-level analysis settings (if applicable)

  • If dual indexes strategy is selected: Sample ID*, I7 Index* , I5 Index*, Project, and sample-level analysis settings (if applicable)

• For Not Specified kit:

  • If single index strategy is selected: Sample ID*, Index 1*, Project, and sample-level analysis settings (if applicable)

  • If dual indexes strategy is selected: Sample ID*, Index 1*, Index 2*, Project, and sample-level analysis settings (if applicable)

Columns marked with asterisks are mandatory.

Import behaviors

• Import will fail if an incorrect CSV template is used (columns do not match the Sample table).

• All rows in the input CSV file will be imported.

• Invalid inputs will result in blank cells in the Sample table.

  • Invalid well position or Index name when custom or standard kit is used

  • Incorrect data type, e.g. string input when a number is expected

  • Out-of-range value

  • Invalid option for a select or dropdown list field

• If there are multiple rows defining the same Sample ID, the sample-level analysis settings must be consistent.

  • If different values are specified, the resulting sample-level analysis settings will be blank.

Lane numbers

For instruments that support loading separate lanes, the CSV template also includes Lanes column.

• Enter one or more lane numbers in this column. When multiple lane numbers are specified, separate the values by commas.

• If a Lanes cell contains an invalid lane number, the resulting cell will be blank.

Use the following instructions to import libraries to the Libraries page from a CSV file.

This method is appropriate for the following:

• Prepared libraries

• Samples with assigned indexes If your samples do not have indexes assigned and are not in libraries, use Import Biological Samples.

1. On the Libraries page, select Import.

2. [Optional] Create a CSV file as follows.

   1. Select the spreadsheet image to download a template.

   2. For each sample, enter the following information.

   3. Sample ID—The unique sample ID.

   4. Name—A descriptive name for the biological sample.

   5. [Optional] Species—The appropriate species.

   6. [Optional] Project—The name of the project to save samples to. Although optional at this step, a project is required later to store the data.

   7. NucleicAcid—The nucleid acid, either RNA or DNA.

   8. Well—The plate well.

   9. Index1Name—The Index 1 name.

   10. Index1Sequence—The Index 1 sequence.

   11. Index2Name—The Index 2 name.

   12. Index2Sequence—The Index 2 sequence.

   13. Save the file.

3. Select Choose .csv File.

4. Browse to and select the appropriate CSV file, and then select Open. The information from the file populates the Import Sample Libraries page.

5. [Optional] Select additional libraries as follows.

   1. Select Save & Continue Later.

   2. Select the checkbox of each library you want to use.

6. Select Pool Libraries.

1. On the Pools page, drag a library from the plate to a pool. For Nextera Rapid Capture libraries, assign samples from the same enrichment to 1 pool.

2. In the Pool ID field, enter a unique name for the pool.

3. [OptionalSelect Add Pool to add a pool.

4. [Optional] Use 1 of the following methods to pool libraries from multiple plates:

   • Select the Plate ID drop-down arrow to switch between plates.

   • Select Save & Continue Later, select the checkbox of each pool to merge, and then select Merge Pools.

5. Select Plan Run.

1. On the Plan Run page, click the Select Instrument drop-down arrow, and select a sequencing system, either MiniSeq or NextSeq.

2. In the Name field, enter a name for the sequencing run.

3. [Optional] In the Reagent Barcode field, enter the barcode ID of the reagent kit used for the run. Entering the barcode ID links the reagent kit to the run.

4. [Optional] Select Use Custom Primer options:

   • R1 — Use custom primer for Read 1.

   • R2 — Use custom primer for Read 2.

5. Select a read type, either Single Read or Paired End.

6. Enter the number of cycles for each read in the sequencing run:

   • Read 1 Cycles — Enter a value up to 151 cycles.

   • Read 2 Cycles — Enter a value up to 151 cycles. This value is typically the same number of cycles as Read 1.

7. Review the indexing scheme for the run. To make changes, override the defaults as follows.

   1. Select the Override default indexing scheme checkbox.

   2. Select an indexing scheme:

      • Single Index — Performs a run with 1 index read.

      • Dual Index — Performs a run with 2 index reads.

      • No Index — Performs a non-indexed run.

   3. Enter the number of cycles for each index read*:

      • Index 1 Cycles — Enter the number of cycles required for the Index 1 (i7) primer.

      • Index 2 Cycles — Enter the number of cycles required for the Index 2 (i5) primer.

8. Make sure that a pool is present.

9. Select 1 of the following buttons to continue:

   • Sequence — The run appears on the Planned Runs list with a status of Ready. The run becomes available from the control software of your sequencing system.

   • Save and Continue Later — The run appears on the Planned Runs list with a status of Planning. When you are ready to sequence, select the checkbox for the run and select Sequence. The run then becomes available from the control software of your sequencing system.

If your library prep kit is not available on the Libraries page, use the following instructions to create it.

1. When prepping a library, select + Custom Library Prep Kit in the Library Prep Kit dropdown menu. The Custom Library Prep Kit Definition page opens.

2. Fill out the name of the custom prep. It has the following requirements:

   • Unique for your account.

   • Characters: only alphanumeric, hyphen, underscores, and spaces accepted.

   • Less than or equal to 50 characters.

3. Select at least 1 of the supported read types.

4. Select at least 1 of the indexing strategies. Only selecting None is not allowed.

5. Fill out the default number of cycles.

6. Select template to download the index definition file template.

7. Fill out the Settings section the following way:

   • For single read only: no adapter (blank), or 1 adapter sequence for Read 1.

   • For paired-end: no adapter (blank), or 2 adapter sequences, 1 for Read 1 and 1 for Read 2.

   • Each adapter sequence meets the following criteria:

     • Sequence of A, T, C, or G character.

     • Length from 1 to 20 characters.

8. Fill out the Index1Sequences and Index2Sequences sections the following way:

   • For Single Index, with or without None: 1 to 100 Index 1 names

   • For each Index 1 name an associated Index 1 sequence

   • For Dual Index, with or without None and Single Index: 1 to 100 Index 1 names

   • For each Index 1 name an associated Index 1 sequence: 1 to 100 Index 2 names

   • For each Index 2 name an associated Index 2 sequence

   • Each index name meets the following criteria:

     • Unique within the file

     • Length from 1 to 8 characters alphanumeric, hyphen, or underscore characters.

     • Each index sequence meets the following criteria

     • Sequence of A, T, C, or G characters

     • Length from 1 to 20 characters

     • All index sequence lengths (Read 1 and Read 2) are equal

     • Index 1 sequences are unique within the file set of Index 1 sequences

     • Index 2 sequences are unique within the file set of Index 2 sequences

9. If the supported indexing strategy specifies Single Index, you can set up Default Layout By Well the following way:

   • Each well unique from A01 to H12

   • For each well, an associated index name exists in the specified Index1Sequences section

10. If the supported indexing strategy specifies Single Index or Dual Index, you can set up Default Layout By Column the following way:

    • Each column number unique from 1 to 12

    • For each column, an associated index name exists in the specified Index1Sequences section

11. If the supported indexing strategy specifies Dual Index, you can set up Default Layout By Row the following way:

    • Each row letter unique from A to H

    • For each row, an associated index name exists in the specified Index2Sequences section

12. Select the Choose.csv File button to select and upload your custom index file.

13. Select Create New Kit to complete the process.

Your custom library prep has been added to the library kit drop-down!

1. From the Runs page, select New Run, and then select Prep Tab.

2. From the Prep page, select NeoPrep.

3. From the NeoPrep Runs page, select Create New Run to open the Configure Run screen.

4. Select the Protocol drop-down arrow, and select a protocol, either TruSeq Nano DNA or TruSeq Stranded mRNA.

5. Select the Version drop-down arrow, and select a version of NeoPrep control software.

6. [Optional] In the Notes field, enter any notes.

7. In the Run Name field, enter a name for the run.

8. Select the Default Project field, select the project to configure for the run, and then select Confirm.

9. Select the checkbox for each process you want to run:

   • Prep Library—Requires preparation of libraries.

   • Quantify—Quantifies libraries after library prep is complete.

   • Normalize—Normalizes libraries after quantification is complete.

10. Complete the following fields as applicable. Read-only or unavailable fields are default selections for the run.

    • Select the Sample Count drop-down arrow, and select the number of samples to include in the run.

    • Select the Index Type drop-down arrow, and select the indexing scheme for the samples.

    • Select the Insert Size drop-down arrow, and select the insert size of the libraries. Mixed insert sizes are specified on the next page.

    • Select the PCR Cycles drop-down arrow, and select the number of PCR cycles for the run.

11. Select Next.

The following prerequisites are needed to get started with DRAGEN Array Cloud:

• Illumina Connected Analytics subscription: An ICA Basic, Professional or Enterprise subscription can be used which include access to BaseSpace Sequence Hub. Follow the Illumina Software Registration Guide to register the software.

• Workgroup setup: Workgroups must be created before login. Using a workgroup allows all members of the workgroup to share access to resources, analyses, and data. Learn more about managing a Workgroup.

  • Designating a workgroup as ‘Collaborative’ allows projects to be shared with collaborators or Illumina Tech Support to assist with troubleshooting. To create a collaborative workgroup, select the Enable collaborators outside of this domain checkbox during workgroup creation.

• Software consumables: iCredits can be purchased for storage on the cloud platform and analysis pipelines with a compute charge. Per sample analysis can be purchased for relevant pipelines as listed in DRAGEN Array Applications and Polygenic Risk Score Software. Follow the Illumina Software Registration Guide (found under Example 3: Configuring the Software Consumables) to register the software consumables.

• [Optional] iScan integration: The iScan System is integrated with Illumina Connected Platform and can send IDATs for further analysis. The iScan System must be running iScan Control Software version 4.2.1 or later.

  • Instructions to Use Illumina Connect Analytics (ICA) with the iScan System

  • Troubleshooting iScan integration

• EULA acceptance: Accept all necessary End User License Agreements in BaseSpace Sequence Hub before scanning begins.

• Internet connection: For uploading product files or IDATs, a network connection 1 GbE or faster is recommended.

Note: Accessioning BeadChips before scanning and starting analysis is no longer a required step and has been automated within the system.

BaseSpace Sequence Hub offers a wide variety of powerful NGS data analysis apps, including:

• Illumina Core Apps: Developed or optimized and fully supported by Illumina

• BaseSpace Lab Apps: Developed using an accelerated process to make them available to BaseSpace Sequence Hub users faster than conventional Illumina Apps, and provided as-is

• Third-party apps: Developed and supported by a thriving ecosystem of third-party app providers in BaseSpace Sequence Hub

In addition, BaseSpace Sequence Hub enables users to develop custom bioinformatics apps in BaseSpace Sequence Hub. App developers are able to keep apps private, share with collaborators, or submit for publication.

Together, these apps cover the common data analysis methods used with Illumina sequencing data. These methods include RNA-Seq, exome /enrichment, amplicon, whole-genome sequencing (WGS), amplicon, de novo assembly, 16S metagenomics, and more.

For more information about BaseSpace Sequence Hub Apps, refer to the BaseSpace Sequence Hub Developer Portal

For information about manually launching apps, see Launching Apps.

For information about scheduling an analysis to automatically launch, see Analysis Workflows.

Analysis workflows are packaged templates of BaseSpace Sequence Hub Apps with predefined settings and QC thresholds to support automation of running analyses. Use an analysis workflow to automatically launch the same app configurations on different biosamples in the project.

When you assign an analysis workflow using a biosample workflow file, the workflow status is listed as Pending. After data are received and conditional dependencies are met, the analysis is launched and the status is listed as Running.

Create an Analysis Workflow

For information about creating an Analysis Workflow for an existing app, using your own settings or QC thresholds with an existing BaseSpace Sequence Hub app, see the developer documentation at developer.basespace.illumina.com.

Modify an Analysis Workflow

Analysis Workflows can be modified by their owners or members of the workgroup in which the workflow was created. If you need to modify a workflow, Illumina recommends creating a new workflow and adding a version number to the workflow name.

Schedule Analysis Workflow to Automatically Launch

1. Upload a Biosample Manifest with analysis workflows set for one or more biosamples. See Biosample Workflow.

2. Sequence data for the biosample.

3. BaseSpace Sequence Hub checks that dependencies are met, collects data sets associated with the biosample, and launches the analysis.

Cancel an Analysis

1. Select one or more analyses that are Running or Pending.

2. Select the Status drop-down arrow, point to Cancel, and then select Analysis.

When an analysis fails or doesn't meet quality standards, you can schedule a new analysis using the original input or different inputs.

• In cases where the analysis failed to successfully complete in BaseSpace Sequence Hub, you can relaunch the analysis using the original input.

• In cases where the analysis did not meet quality standards, you can exclude or include data and relaunch the analysis using only data that passed QC.

Requirements:

• If you are scheduling an analysis workflow that uses more than one sample as input (eg, Tumor Normal), all existing biosamples must have been imported from same original biosample workflow file.

• The biosample workflow must be imported using the account that owns the biosamples. If the biosample workflow is uploaded to an account that does not own the biosample, the biosamples are added to the account as new biosamples.

1. [Optional] Exclude or QC fail any of the following resources. Excluding a resource also excludes any downstream data produced by the resource. For information about changing QC, see Manual QC.

   • Biosample

   • Library

   • Pool

   • Lane

   • Run

   • FASTQ Dataset

2. Add the following information to a new biosample workflow file.

   • Biosample Name

   • Analysis Workflow

   • [Optional] Analysis Group

   • [Optional] Sample Label

   • [Optional] Delivery Mode

You can launch apps that perform additional analysis, visualization, or annotation of data. Running apps can incur a charge.

1. To start an app, do one of the following:

   • Navigate to the project or sample that you want to run the app on, select the Launch Apps button, and select the desired app from the drop-down list.

   • From the Apps page, select the desired app from the list and select Launch.

2. Read the End-User License Agreement and permissions, and then select Accept.

The app guides you through the start-up process. BaseSpace Sequence Hub has limited storage capacity and checks the free space available before starting an app. If there is not enough available space, BaseSpace Sequence Hub displays an error message. See Delete and Restore Data to learn how to save space in your account.

Data owners can share data with you via a unique URL or email invitation. You can accept the share into your personal account or one of your workgroups and then view the data when you are signed into that account.

1. Do one of the following:

   • Select the link to the shared data.

   • Select Accept in the dashboard or email notification.

2. In the Share dialog, select the context (personal or workgroup) to accept the invite.

3. Select Accept.

BaseSpace Sequence Hub includes two types of apps:

• Native applications — Run within the BaseSpace Sequence Hub infrastructure in the Amazon cloud. Native apps consist of an input form, analysis engine, and an in-browser report. Native apps can be automated using the command-line interface and BaseMount.

• Web applications — Run outside of the BaseSpace Sequence Hub infrastructure, web apps connect to BaseSpace Sequence Hub using the API or SDK and can run on secure cloud infrastructure, user desktops, or mobile devices. With web apps, analysis can be configured and started from the BaseSpace Sequence Hub input form or from within the app.

BaseSpace Sequence Hub offers four categories of apps:

• Illumina Core applications — Apps that are developed by Illumina bioinformaticians and software engineers and rigorously tested and documented. Illumina Technical Support fully supports core apps.

• BaseSpace Labs applications — Apps that are developed by Illumina bioinformaticians and software engineers and released as beta apps. Illumina Technical Support does not fully support BaseSpace Labs apps.

• Third-party applications — Apps that are developed by third-party groups outside of Illumina. Illumina reviews these native and web apps for quality and robustness before being published. The apps are supported by the third-party developers.

• Private applications — Any apps that are developed but not published to the BaseSpace App store. These apps can be shared directly with collaborators to maintain privacy and access to the app.

When you share data in BaseSpace Sequence Hub, you give collaborators access to it while you retain ownership and write access. To transfer ownership of data, see Transfer Ownership.

You can share data with collaborators in the following ways:

• Add collaborators to a workgroup—Workgroup collaborators automatically gain read and write access to all data in the workgroup. For information about sharing data between Enterprise accounts (eg, illumina.com) and Personal or Professional accounts, see Create Workgroups and Assign Administrators.

• Share a project—You can grant access to a project and any associated biosamples, libraries, analyses, and data sets.

• Share a run - You can grant read-only access to a specific run. Associated biosamples, libraries, analyses, and data sets are not shared. However, some biosample names appear on run data pages.

If you want to share FASTQ data sets without sharing project data, download the data set and share it separately. Shared data cannot be used in biosample workflow files because biosamples are not transferred.

Workgroups

Share a Project or Run With Collaborators

Share Analyses

Use analysis delivery status to track the delivery of analysis reports or to make sure that analyses for test runs or R&D projects are not shared with customers.

Use the Biosample Workflow to specify an initial delivery status.

1. Select Analyses.

2. Select one or more analyses from the list.

3. Select Status, point to Change, and then select Analysis Delivery Status.

4. In the Change Delivery Status dialog, select the Change status to drop-down arrow, and then select a delivery status.

5. [Optional] Add a comment.

6. Select Save.

Use the Send Invitation option to manage collaborator permissions. You can review the list of collaborators, change the access level for a project, or revoke access to a run or project.

1. Select the shared project or run.

2. Select Share, and then select Send Invitation.

3. In the Collaborator Settings section of the Share dialog, change or revoke collaborator access.

4. Select Save Settings.

The following tables list the data read and write permissions after shares and transfers associated with projects and runs.

For Shared Resources:

For Transferred Resources: