Table of Contents

guest
2022-09-24
   Panorama: Targeted Mass Spectrometry
     Panorama Partners Program
     Configure Panorama Folder
     Panorama Data Import
     Panorama Experimental Data Folder
       Panorama: Skyline Document Management
       Panorama: Skyline Replicates View
       Panorama: Protein/Molecule List Details
       Panorama: Skyline Lists
       Panorama: Skyline Annotation Data
       Panorama: Skyline Audit Log
       Panorama: Calibration Curves
       Panorama: Figures of Merit and Pharmacokinetics (PK)
       Panorama: Instruments Summary and QC Links
       Working with Small Molecule Targets
       Panorama: Heat Maps
     Panorama Multi-Attribute Method Folder
     Panorama Chromatogram Library Folder
       Using Chromatogram Libraries
       Panorama: Reproducibility Report
     Panorama QC Folders
       Panorama QC Plots
       Panorama QC Plot Types
       Panorama QC Annotations
       Panorama QC Guide Sets
       Panorama: Pareto Plots
       Panorama: iRT Metrics
       Panorama: Configure QC Metrics
       Panorama: Outlier Notifications
     Panorama and Sample Management

Panorama: Targeted Mass Spectrometry


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Panorama, implemented as a module for LabKey Server, provides web-based tools for targeted mass spectrometry experiments that integrates into a Skyline SRM/MRM/PRM/DIA workflow.

By leveraging LabKey Server's core features, Panorama offers the following solutions for targeted mass spec research:

  • Easy aggregation and curation of results
  • Guidance for new experiments based on insights from previous experiments
  • Search and review over a large collection of experiments, including multiple versions of documents
  • Secure sharing of results
  • Automated system suitability checks, tracking a variety of metrics and reports
  • Support for both peptide and small molecule analysis
Researchers have two options for using Panorama:

Topics

Experimental Data Folders

Multi-attribute Method Folders

Chromatogram Library Folders

Panorama QC Folders

Panorama Partners: Premium Resources

PanoramaWeb Documentation




Panorama Partners Program


The Panorama Partners Program is a premium offering for users of Panorama, the LabKey-based repository for targeted proteomics.

Panorama Partners Program Benefits

The Panorama Partners Program is designed to help members make the most of Panorama and provides a unique opportunity for members to guide development. Members engage directly with developers at regularly scheduled conference calls. Developers will present recent changes and improvements related to Panorama, and provide input on how to best use Panorama based on the organization's needs. Members are invited to provide input on how Panorama might be improved and suggest changes for future development.

LabKey Server Starter Edition

Membership also includes a LabKey Server Starter Edition subscription, including full support and training for installation, maintenance, configuration, and general usage of LabKey Server, plus access to Panorama targeted mass spectrometry features.


To inquire about membership in the Panorama Partners Program, please contact LabKey.

Documentation: Panorama: Targeted Mass Spectrometry




Configure Panorama Folder


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Panorama is a web server database application for targeted proteomics assays that integrates into a Skyline proteomics workflow. The LabKey Panorama module supports management of targeted mass spectrometry data and integration with Skyline workflows (SRM-MS, filtering or MS2 based projects).

Panorama Folder Types

To begin working with Panorama, create a new folder, choosing Panorama as the folder type. On the Configure Panorama Folder page, select one of the available configurations. Each type in this list links to a set of documentation.

  • Experimental data: A repository of Skyline documents, useful for collaborating, sharing and searching across multiple experiments.
  • Multi-attribute method (MAM): An experimental data folder variant with additional reporting for multi-attribute method analyses.
  • Chromatogram library: Curated precursor and product ion expression data for use in designing and validating future experiments.
    • Check Rank peptides within proteins by peak area if your data contains relative peptide expression for proteins.
  • QC: Quality control metrics of reagents and instruments.

Create New Panorama Folder

  • Create a new folder. Hover over your project name in the menu bar (On PanoramaWeb, right below the Panorama icon) and click on the New Subfolder icon circled in the image below.
  • Give the new folder a Name, then select the Panorama option under Folder Type. This is the folder type that should be selected for all workflows supported by Skyline (SRM-MS, MS1 filtering or MS2 based projects).
  • On the Users / Permissions page, select one of the available options and click Next.
    • You can also change permissions on a folder after it has been created.
  • The next page, Configure Panorama Folder, asks you to choose the type of Targeted MS folder you would like to create, detailed above.
  • Click Finish.

Topics for each Panorama Folder Type:




Panorama Data Import


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

To import targeted mass spectrometry data, first create the appropriate type of Panorama folder for the work you will be doing. This topic describes how to import data to any Panorama folder.

Import Directly from Skyline

Open the document that you want to publish to Panorama.

  • Either click the Upload to Panorama button (circled below) or select File > Upload to Panorama.

Import Data from Panorama Interface

  • Navigate to your Panorama folder.
  • Click the Data Pipeline tab.
  • Click Process and Import Data.
  • Drag the files or directories into the web part. The upload will begin.
  • If files are being zipped during upload, the upload progress window will inform the user that it is zipping files.

Zip Files on Upload

Certain file formats and directory layouts used by different instrument vendors can be zipped upon upload to reduce overhead. See the list of recognized patterns below. When you upload a directory or collection of directories that match known mass spectrometry patterns, the files will be zipped. Files or directories that do not match the known patterns will be uploaded as usual.

In either case, the progress message will inform the user of the action and status. After the successful upload, the files and/or archives will be available in the file browser.

Browser support: Chrome provides the best performance for zipping upon upload. Edge is not supported.

Recognized File Patterns

The following patterns used by these instrument vendors are supported:

  • Waters: A directory with the extension '.raw' containing at least one file matching the pattern '_FUNC*.DAT', e.g. _FUNC001.DAT
  • Agilent: A directory with the extension '.d' containing a subdirectory named 'AcqData'
  • Bruker: A directory with the extension '.d' containing at least one file of name 'analysis.baf'
  • Bruker: A directory with the extension '.d' containing at least one file of name 'analysis.rdf'

Related Topics




Panorama Experimental Data Folder


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

A Panorama folder of type Experimental data provides a repository of Skyline documents, useful for collaborating, sharing and searching across multiple experiments.

Tutorial

The Sharing Skyline Documents Tutorial on the PanoramaWeb site provides an introduction to using this folder type and covers the following areas:

  • Requesting a project on panoramaweb.org
  • Data organization and folder management in Panorama
  • Publishing Skyline documents to Panorama
  • Data display options and searching results uploaded to Panorama
  • Providing access to collaborators and other groups

Topics




Panorama: Skyline Document Management


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

As proteomics methods are developed and refined, multiple documents are often produced that need to be tracked and linked together. For example, a first runs attempt may include many proteins, precursors, and transitions, which later run attempts will progressively narrow down to the best performing ones. In order to track the method development, documents can be marked with comments and linked together as a series of different versions.

Document Details

You can view a detailed profile for each document in Panorama by clicking the document name in Targeted MS Runs web part.

The Document Summary panel shows key summary information and links to actions and views.

  • Click the icon to rename the Skyline document.
  • Click to export. Learn more below.
  • Click the # versions link to jump to the Document Versions panel which shows the document's position in the series of versions.

Download the Document

In the Document Summary, the icon gives the full document size. Click to open a download menu.

Options:

  • SkyP file: A .skyp is a small text file that makes it easy to open the full document in Skyline. Skyline will automatically download the full file from Panorama (if needed). Unlike the .sky.zip file extension for the full Skyline file, Windows associates the .skyp file extension with Skyline so you can just double-click the file.
  • Full Skyline file
  • Copy relative URL to clipboard
  • Copy full URL to clipboard

Automatically Link Skyline Documents

The server will automatically link Skyline documents together at import time, provided that the Skyline documents provide a document ID. When importing a Skyline document whose ID matches one already in the folder, the incoming document will automatically be linked to the previous document(s) as the newest version in the document chain.

The document’s import log file will indicate if it was attached as a new version in the document chain.

Manually Link Document Versions

To manually chain together a series of document versions, select them in the Targeted MS Runs web part and click Link Versions.

The Link Versions panel will appear. You can drag and drop the documents into the preferred order and click Save.

Note that an individual document can be incorporated into only one document series -- it cannot be incorporated into different document series simultaneously.

Add Comments

To add comments to a document, click in the Flag column.

The Review panel will appear. Enter the comment and click Ok.

Comments are displayed in the Document Versions panel as notes.

Related Topics




Panorama: Skyline Replicates View


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Skyline Replicates

A summary of replicates contained in a Skyline document is available by clicking the "replicates" link in the Document Summary panel.

The Replicate List provides annotation details and sample information for each replicate.

For graphical views that incorporate replicate data, see the following documentation on PanoramaWeb:

Sample File Scoped Chromatograms

Skyline can extract and store chromatogram-style data that is scoped to a raw data file. Examples include keeping track of the pressure or voltage traces recorded by the instrument during its data acquisition. This topic describes how to access the sample-file scoped chromatogram data within Panorama.

Chromatogram Data Storage

Users import raw data files as normal into Skyline. Skyline automatically inspects them and extracts whatever pressure traces or other time-series data is available, and stores it in its SKYD chromatogram file format. When these Skyline files are imported into Panorama, this information is stored in the targetedms.SampleFileChromInfo table. No additional user action is needed to include the chromatogram data.

User Interface Access

Users will be able to go to the replicate list for a given Skyline document and click to see details, including the plots of the time-series data.

  • Open the Skyline file of interest.
  • In the Document Summary, click the replicates link.
  • On the list of replicates, hover over the row of interest to reveal the (Details) link. Click it to open the details page.
  • The plots for each series of data present in the imported file will be shown below the Sample File Summary which provides about the sample and the file it came from.

API Access

The targetedms.SampleFileChromInfo table is exposed for API-based access. Developers can view the table by selecting (Admin) > Developer Links > Schema Browser.

Targeted MS Chromatogram Crawler

Administrators can obtain a listing of chromatograms stored on their site via a crawler which will check each container and list chromatograms found. This assists admins in locating chromatograms that can be deleted from the database.

  • Select (Admin) > Site > Admin Console.
  • Under Configuration, click Targeted MS Chromatogram Crawler.
  • Click Start Crawl.
  • You will see the pipeline progress.
  • When the status shows as complete, click the word COMPLETE to see the crawl log.
  • The log will begin with key statuses, show you a per-container record of the crawling completed, and conclude with a "Crawl summary by Skyline document counts."

Crawl Results: For each container crawled, the job will log the number of chromatograms that have byte offsets in the DB (which will be zero for non-Panorama folders or Panorama folders which have no imported Skyline files, or which were all imported before we started storing byte indices). It will iterate through all of the Skyline files in the container and inspect five chromatograms (the most typical problem will be that the server can’t find a SKYD file, so there’s little benefit to check out every chromatogram in each file). It will report the status for the chromatograms it inspects:

  • No data available at all: the server never had a SKYD file when it loaded data.
  • dbOnly: DB contains bytes, but not offset so impossible to retrieve directly from file.
  • diskOnly: DB does not contain bytes, but does have indices and data was retrieved successfully from file.
  • noSkydResolved: DB contains bytes and offset, but we can't resolve the SKYD path, perhaps because the S3 bucket is no longer configured.
  • skydMissing: DB contains bytes and offset, but we can't find the SKYD file that's referenced.
  • mismatch: Both DB-based and file-based bytes available, but they do not match.
  • match: Both DB-based and file-based bytes available, and they do match.
  • skipped: we hit our limit of 5 for the current file and didn’t examine the rest.

Related Topics




Panorama: Protein/Molecule List Details


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Chromatograms present in the Skyline file can be viewed from within Panorama with several display customization options. Users have the option to see multiple peptides or small molecules in the same chromatogram.

After loading your Skyline document into Panorama, click the name of the file to open the details page. Then click the name of a protein / label to open the Protein/Molecule List Details page.

Multiple Peptides Per Chromatogram

When the protein contains many peptides, it can be useful to see the chromatograms for several peptides in one plot.

On the protein details page, scroll down to the Chromatograms section where you will a legend showing the colors assigned to the individual peptides present. By default all peptides are included, unless you select a subset using the checkboxes. The chromatograms below all use this scheme to show the same series of peptides for each replicate.

Use the Row Size menu to control the display size of the SVG plots; options are 1, 2, 3, 4, 5, or 10 per row.

Select Peptides for Chromatograms

By default, all peptides will be included in the chromatograms, unless you use the checkboxes in the Peptides panel to select only a subset.

Display Chart Settings

Open the Display Chart Settings panel to adjust your chart.

  • Set the Chart Width and Height and opt to Synchronize the X- and/or Y-axis.
  • Use the Replicates dropdown to multi-select the specific replicates for which you want to see the chromatograms, making it easier to compare a smaller number of interest.

Click Update and you will see only the multi-peptide chromatograms for your selected replicates.

Related Topics




Panorama: Skyline Lists


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Within Skyline, users can create lists which have one or more columns of different data types. This topic describes how to access these lists within Panorama.

Skyline Lists

When a Skyline Document import includes lists, you can access them by clicking the "lists" link in the Document Summary panel.

Click the name of any list to see the contents. Annotations within the Skyline document that reference the given list will resolve as a lookups in LabKey to the relevant row.

These lists are functionally similar to LabKey Server lists, but stored, scoped, and surfaced in different ways. Skyline lists are saved in Skyline's XML file format, then parsed and imported to Panorama specific tables. They can be viewed, queried, and exported from LabKey in the targetedmslists schema. Skyline lists are read-only inside of LabKey, like other Skyline data, so you will need to make any changes in Skyline and upload a new version.

Skyline lists are scoped to the specific Skyline document which includes them. If the document is deleted from LabKey, the list will also be deleted.

Multiple Skyline documents could each have a list with the same name without raising conflicts since the name is stored with the unique RunId as a prefix. The superset of all lists with the same name is stored with the prefix "All_". For example, if runs 106 and 107 each have a "DocumentProperties" list, the targetedmslists schema would contain:

  • 106_DocumentProperties
  • 107_DocumentProperties
  • All_DocumentProperties

Related Topics




Panorama: Skyline Annotation Data


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

When a Skyline document is imported, Panorama will parse and expose any annotation data, which can be used to create custom grids and visualizations.

Developers can determine where annotation data (such as Condition, BioReplicate, and Run) are stored using the (Admin) > Developer Links > Schema Browser. For example, peptide and small molecule annotations are stored in tables like targetedms.precursorannotation, targetedms.generalmoleculeannotation. All such tables have lookups (foreign keys) to the tables being annotated.

In addition, there is an "Annotations" column on each entity (targetedms.replicate, for example). In this column, all annotations are shown as name/value pairs. Upon import, any annotation columns will also be exposed as discrete "virtual" columns on the entity table itself. Such fields can then be exposed in grids using the UI, such as in the following example using replicate annotation data.

Replicate Annotation Data

Replicate annotation data is exposed in the query targetedms.Replicate.

Users can access the annotation data by customizing their data grids:

  • Starting from a data grid, select (Grid Views) > Customize Grid.
  • In the Available Fields pane, open the node Sample File and then the node Replicate.
  • Scroll down the Available Fields pane to see the available fields.
  • Select fields to add them to the data grid. The screenshot below shows how to add the Time annotation field.



Panorama: Skyline Audit Log


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Panorama will import audit logs from Skyline documents, provided audit logging is enabled in Skyline.

When Panorama detects a valid audit log, the (three bars) icon is displayed in the Document Summary.

  • Click the document file name on the Runs tab.
  • In the Document Summary panel, click to see the audit log in a summary form.
  • In the Skyline Audit Log panel, select (Grid Views) > AllMessagesView for the even more detailed grid.

Related Topics




Panorama: Calibration Curves


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Calibration curves plot how a given metric changes with the concentration of an analyte and are used to calibrate an instrument.

View Calibration Curve

In the Document Summary panel, click the calibration curves link to see the available calibration curves.

Hover over any row to reveal a (Details) button. Click to see the calibration curve plot.

Calculations round to 5th decimal digit. The calibration curve plot is rendered with a regression line.

Click a point in the plot to see its calculated concentration. Values are shown in the legend.

Below the plot, a panel of Figures of Merit provides limit of detection and limit of quantitation details (when available). Learn more in this topic: Panorama: Figures of Merit and Pharmacokinetics (PK)

Summary Charts

Summary charts show peak areas and retention times for the replicates in a bar and box plot format.

Quantitation Ratios

Below the summary charts, a table of Quantitation Ratios includes columns for the sample file, Q1 Z, Sequence, RT, Intensity L, Intensity H, L/H Ratio, Exp Conc, and Measured Conc.

Related Topics




Panorama: Figures of Merit and Pharmacokinetics (PK)


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Panorama provides the following built-in reports:

Figures of Merit

The Figures of Merit report:

  • For a given Skyline document, click the ## Calibration Curves link.
  • In the Peptides Calibration Curves panel, click the peptide name (or hover for a link). Click either for details for that peptide.

Scroll below the Calibration Curve on the details page to the Figures of Merit panel. Summary information, including LOD/LOQ Values, is shown here.

Click Show Details for the more detailed report.

The Figures of Merit report includes several sections.

The report includes the following values for both Concentrations in Standards and Concentrations in Quality Control, if present:

  • n - The Number of samples used for above calculations.
  • Mean - The mean of all replicates for a given sample.
  • StdDev - Standard deviation of all replicates for a given sample.
  • CV(%) - Coefficient of Variation for all replicates for a given sample (the standard deviation divided by the mean).
  • Bias(%) - The difference between expected and observed values.
Point exclusions are imported, and any excluded raw data is shown as strike-through text. Excluded values are not included in summary calculations.

Summary: LOD/LOQ Values

Within Skyline, you can set the limit of detection (LOD) calculation, the coefficient of variation (CV) limit, and limit of quantitation (LOQ) bias. These settings are picked up automatically by LabKey and included in the Figures of Merit report in Panorama.

Key terms:

  • Limit of detection (LOD) - the lowest quantity of a substance that can be distinguished from the absence of that substance
  • Limit of quantitation (LOQ) - the lowest concentration at which the analyte can not only be reliably detected but at which some predefined goals for bias and imprecision are met. It is typically higher than the LOD. Note that LOQ can come in lower (LLOQ) and upper (ULOQ) varieties.
  • Bias - difference between expected and observed values, often represented as a percent. If no value is provided for either bias or CV, the bias cutoff will default to 30% and the CV will be ignored.
  • Coefficient of variation (CV) - ratio of the standard deviation to the mean.
To set these values in Skyline, open Settings > Peptide Settings > Quantification tab.

Enter:

  • Max LOQ bias: Maximum (percentage) allowed difference between the actual internal value and the calculated value from the calibration curve.
  • Max LOQ CV: Maximum allowed coefficent of variation of the observed peak areas of the set of internal standard replicates with a specified concentration.
  • Select how to Calculate LOD from the following options:
    • None
    • Blank plus 2 * SD: Base the limit of detection on the observed peak areas of the blank samples. Add twice the standard deviation.
    • Blank plus 3 * SD: Add three times the standard deviation to the observed peak areas of the blank samples.
    • Bilinear turning point: Impute the limit of detection using the turning point of bilinear fit.
  • Qualitative ion ratio threshold
Both limit of detection and limit of quantification results are shown in the summary Figures of Merit section below the calibration curve (as well as at the top of the details page).

Blanks

The raw data for blanks is show at the bottom of the figures of merit report. The same statistics, except bias, are displayed as in the concentrations sections.

Pharmacokinetic (PK) Calculations

To view the Pharmacokinetic values:
  • For a given Skyline document, click the Calibration Curves link.
  • In the Peptides Calibration Curves panel, click PK for a given peptide.
    • The link is only shown if your replicates have the Dose and Time annotations.

PK Calculations Panel

For successful PK calculations, the following annotations should be present on the Replicate data as configured in Skyline:

  • Dose: number (Dose that was administered)
  • DoseUnits: text (Units for what was administered, such as "mg/kg")
  • Time: number (Timepoint after administration)
  • ROA: text (Route of Administration, typically "IM" or "IV")
  • SubGroup: text (Optional) Grouping of data within a single Skyline document

The Pharmacokinetic report provides a panel of PK values, and if subgroups are provided, a panel for each subgroup.

Use the checkboxes to set values for the C0 and Terminal columns. These indicate which timepoints are used as the starting and ending points of the calculations. By default the earliest 3 timepoints for C0 and the last 3 timepoints for the Terminal values are selected.

The Time column in the spreadsheet will be a custom replicate annotation in the Skyline document. There will also be replicate annotations for Dose, DoseUnits, ROA (Route of Administration), and SubGroup. Dose and DoseUnits may be added to one or more replicates per SubGroup. When Dose is included on more than one replicate is must be the same value. The same applies for DoseUnits and ROA.

You can print the data to Excel by clicking the icon in the Statistics header.

Non-IV Route of Administration

If the ROA (Route of Administration) is any value other than “IV” the subgroup will have an input field for “non-IV C0” and a button to Recalculate.

Charts and Raw Data

Scroll down the page to see charts and raw data panels for this subgroup, followed by sets of panels for other subgroups.

Related Topics




Panorama: Instruments Summary and QC Links


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Panorama provides a summary of all the data on this server acquired from each instrument. This Instrument Summary also provides automatic links between data in Experiment folders and the QC folder(s) that contain data from the same instrument, making it quick to see how the instrument was performing at the time of acquisition.

Instruments Summary

The Instruments Summary panel lets you see which instrument(s) acquired the data in a Skyline document. It is shown on the replicate page for a single Skyline document.

The summary lists, for each experiment in the folder:

  • Instrument Name
  • Serial Number
  • Start/End Dates for the experiment
  • Number of Replicates
  • QCFolders: If any QC folders exist for the instruments and experiment dates, there will be one or more links here to go to the folder, where you will see a combined view of experiment and qc plot data.
Click a link in the Serial Number column to see all data on this server acquired from this instrument. For example, clicking the "Exactive Series slot #2384" link in the image above:

Linked Experiment and QC Folders

A Panorama folder of type "Experimental Data" provides a repository of Skyline documents, useful for collaborating, sharing and searching across multiple experiments.

A Panorama folder of type "QC" contains quality control metrics of reagents and instruments.

When there is QC data for the instruments used in the experiments, users will find it helpful to navigate directly to the QC data around the time of experimental data acquisition to assess the performance of the instrument at the time.

Navigate the Combined Information

The experiment date range is shown in blue. Any guide sets applied or in the same time range will be shown in gray (overlapping sections will be a mixed darker shade).

In the blue bar above the plot, click Return to Experiment to return to the experiment folder.

Related Topics




Working with Small Molecule Targets


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Using Panorama, you can import, parse, and view small molecule data, such as lipids or other non-amino-acid compounds, inside of Skyline documents. Skyline documents containing mixed sets of peptides and small molecules can be imported. Panorama will separate the mixed peptides and small molecules into their respective views.

Views provided inside Panorama include:

  • Small Molecule Precursor List
  • Small Molecule Summaries
  • Small Molecule Details, including Chromatograms
All of these views are similar to the analogous peptide views, though spectrum graphs are not shown for small molecules.

Importing Small Molecule Documents

  • Create or navigate to a Panorama type Folder.
  • Configure the Panorama folder for Experimental Data. (For details see Configure Panorama Folder.)
  • Click the Data Pipeline tab. In the Data Pipeline web part, click Process and Import Data.
  • Drag and drop the individual Skyline documents or .zip file into the Files web part. This walkthrough uses sample test data; your column names and results may vary.
  • When the documents have been uploaded, select the documents and click Import Data.
  • In the Import Data popup menu, confirm that Import Skyline Results is selected, and click Import.
  • When the import job is complete, click the Panorama Dashboard tab.
  • In the Targeted MS Runs web part, click a Skyline document for views and details on the contents.

Available Views

The Small Molecule Precursor List shows a summary of the document contents.

  • Under Molecule List you will see sections for each molecule group. In the above screenshot, "Library Molecules" is circled and shown expanded.
  • The expanded grid column names may vary. In this screenshot, under Molecule you see links (such as "Cer(d14:1/22:0)"). Click for a details page.
  • Click the molecule group name, here Library Molecules to see a summary page which includes charts showing peak area and retention time information.
  • Click the small PDF and PNG icons next to the plot titles to export to either format.

Go back to the Document Summary. Clicking the ## transitions link will switch the page to show you the Small Molecule Transitions List.

Ion Details

The following screen shot shows details and chromatograms for an ion, here "Cer(d14:1/22:0)" clicked from the Small Molecule Precursor List.

Related Topics

  • Use a Panorama QC folder to track the performance of instruments and reagents using Levey-Jennings plots, Pareto plots, and other tools for both proteomics and small molecule data.

Other Resources




Panorama: Heat Maps


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Heat maps are a powerful way to visualize expression matrix data. Clustergrammer is a free visualization service and open source project provided by the Ma'ayan Lab at the Icahn School of Medicine at Mount Sinai. A heat map for data from runs in Panorama can be generated using the free web service version of Clustergrammer.

Generate a Clustergrammer Heat Map

  • Navigate to the Panorama runs list of interest.
  • Select the runs of interest and click Clustergrammer Heatmap.
  • Adjust the auto-generated title and description if desired, or accept the defaults.
  • Click Save.
  • You'll be asked to confirm that you consent to publish the information to Clustergrammer. Clustergrammer is a third-party service, so all data sent will be publicly accessible. Click Yes if you wish to continue.
  • The heat map will be generated and shown; depending on your run content, it might look something like this:

When you generate a Clustergrammer heat map, the server auto-generates a Link Report giving you a way to access it later. To see the link report, add a Data Views web part:

  • Enter > Page Admin Mode.
  • Select Data Views from the Add Web Part dropdown in the lower left.
  • Click Add.
  • Click Exit Admin Mode.



Panorama Multi-Attribute Method Folder


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Multi-Attribute Method (MAM) analysis improves simultaneous detection, identification, quantitation, and quality control (monitoring) of molecular attributes. Panorama includes MAM-specific reporting with its analytics, offering immediate results and data sharing to any imported data. Additionally, Panorama and AutoQC’s automated workflow provide longitudinal tracking of MAM-related metrics for QC purposes.

Poster

A poster detailing how Panorama features support for MAM-related reports was presented at the ASMS (American Society for Mass Spectrometry) Conference in 2020:

Topics

Multi-Attribute Method (MAM) Folder

The Multi-Attribute Method folder is the same as the Experiment Data folder with additional reports provided.

To set one up:

  • Create a new project or folder, choosing folder type "Panorama".
  • On the Configure Panorama Folder page, select Multi-Attribute method (MAM)

You can also change an existing Experiment folder to add the MAM reports by going to (Admin) > Folder > Management > Module Properties and setting TargetedMS Folder Type to "ExperimentMAM" for the folder.

You'll see links to the additional reports in the Document Summary section.

Post-translational Modification (PTM) Report

The Post-translational Modification (PTM) Report shows the proportion for each peptide variant’s peak area across samples. Panorama automatically groups peptides with identical unmodified sequences, but a user can configure alternative groupings within the Skyline document, to group splice variants, for example.

Peptide Map Report

The Peptide Map Report shows the observed peptides in the MAM analysis, sorted by retention time.

  • It compares the observed and expected masses, and the modifications for each peptide variant.
  • Shows the protein chain and the peptide location as two separate columns instead of combining into a single column.
  • Includes the next and previous amino acids in the sequence column.
  • Shows the location of modifications on the protein/peptide sequence in addition to just the modification name.

Related Topics




Panorama Chromatogram Library Folder


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Chromatogram libraries capture representative chromatograms for a given target in a given set of instrumentation. Users can upload additional documents and then curate which version of a chromatogram is considered the most representative and should be the one stored in the library. A completed library can be downloaded as a .clib file, which is a SQLite database. That can then be imported into Skyline to inform and score future assays that use the same small molecules, peptides, or proteins.

Choose the Chromatogram Library folder type to include the tools for creating libraries. The Rank peptides within proteins by peak area box should be checked when you have targeted results containing relative peptide peak areas for a given protein. Leave this box unchecked if you do not yet have quantitative data from proteolytically digested proteins, such as when you are working with synthetic peptides.

Tutorial

The Panorama Chromatogram Libraries Tutorial shows you how to build a chromatogram library for peptides. The same type of functionality is available for small molecules. In this tutorial you will:

  • Build a chromatogram library in Panorama
  • Use it in Skyline to select peptides and product ions to measure in a new experimental setting
  • Compare library chromatograms with new data to validate peptide indentifications.

Topics




Using Chromatogram Libraries


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

The Panorama Chromatogram Libraries Tutorial shows you how to build a chromatogram library for peptides. The same type of functionality is available for small molecules, as shown in the example on this page.

Highlights and attributes for small molecules include ion-mobility types of data like cross-sectional profiles and mobility, collision energy, and more.

Topics

Upload Documents

Obtain your Skyline file. Within Skyline, you will publish to Panorama, or you can use the Data Pipeline to import within Panorama. You can find a few examples in the LabKey test data on github if you want to demo these tools before you have your own data.

In this walkthrough, we start with the "SmMolLibA.sky.zip" and later import the "SmMolLibB.sky.zip" file to show how to resolve conflicts. While this example shows small molecule data, the features are by no means limited to that type of data.

Follow the steps in the Panorama Chromatogram Libraries Tutorial to create a place to work.
  • Create a new folder of type Panorama, selecting Chromatogram library.
  • Click the Data Pipeline tab.
  • Click Process and Import Data.
  • Drop your Skyline file(s) into the target area to upload them.
  • Select the desired file, then click Import Data.
  • In the popup, confirm that Import Skyline Results is selected, then click Import.
  • You will see import progress in the pipeline. When complete, continue.

All Skyline files uploaded to a library folder will be eligible to include in the .clib file. Whether or not the user has to resolve conflicts, Panorama will choose the molecules based on their Q values, if available. Panorama will choose the one with the minimum Q value as the reference trace. If Q values do not exist, Panorama will then pick the one with the most intense peak. Options for the user to pick which trace to use in the library will not be supported in this iteration.

The Quantitative/non-quantitative flag for transitions shall be added to the .clib file.

See Summary Data

Click the Panorama Dashboard tab to see the overview of the data available in this library. Note the Molecules tab on the right, shown when small molecule data is present.

Note that if you checked the box to "Rank peptides within proteins by peak area" you will see a different dashboard combining information about proteins and peptides with your small molecule data. If you see the tabs Peptides and Proteins, you may want to recreate the folder without that box checked to work with small molecule data.

Tour Page Layouts and Folder Tabs

As shown above, there are three web parts:

  • Chromatogram Library Download: Summary information, statistics, and a button to download this revision.
  • Mass Spec Search: Search for proteins, peptide sequences, or peptides with specific modifications.
  • Targeted MS Runs: This panel lists the Skyline files you have imported, with the counts of molecule lists, small molecules, precursors, transitions, and replicates.
Click the Molecules tab for lists of molecules and precursors.

View Details and Chromatograms

Click the name of a molecule in the Molecules webpart, Molecule column (shown here "C4H9NO3") for a summary, listing of precursors, chromatograms, and panel for generating summary charts at the bottom of the page.

Display Chart Settings

Click the to expand the Display Chart Settings.

  • Set Chart Width and Height.
  • Use checkboxes to Synchronize the X- and/or Y-axis.
  • Multi-select Replicates. (Click Clear to remove current selections.)
  • Multi-select Annotations. (Click Clear to remove current selections.)

Resolve Conflicts

If documents are imported with conflicting information, the dashboard will show a message indicating the number of conflicting molecules. You must resolve all conflicts before you can extend this library. Click Resolve Conflicts to do so.

For each conflict, you will see the Newly Imported Data on the left and the Current Library Data on the right. In each row, select the checkbox for the version you want to include in the library. Click Apply Changes when finished.

Download .clib SQLite Files

To download the .clib SQLite files, click Download on the Panorama Dashboard.

You can import this .clib SQLite file into Skyline and use it to score future experiments.

Note that if you download the library while there are unresolved conflicts, you will download the latest stable version of the library (i.e. it may be missing more recently added information).

Related Topics




Panorama: Reproducibility Report


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

To support the development of robust and reproducible assays, Panorama offers reporting that summarizing analysis of replicates of the same sample multiple times a day over multiple days to assess inter- and intra-day variability. 3 replicates over 3 days or 5 replicates over 5 days are common usages. As long as at least 3 runs are included, Panorama will generate the Reproducibility report for that protein. The report relies on a Replicate annotation in Skyline to identify the day for grouping purposes. The Reproducibility Report visualizes the reproducibility of the assay and which peptides perform best.

The current implementation of the report is specific to protein/peptide data and does not yet support small molecule data.

Configure Skyline Document

For this report, you will load a Skyline document containing multiple replicates for a protein. You must include a Day replicate annotation, and you may want to also captures both NormalizedArea and CalibratedArea values, in order to take advantage of the options available below.

To configure, start in Skyline:

  1. Select Settings > Document Settings
  2. On the Annotations tab, click Add...
  3. Use "Day" as the name, and leave the Editable button toggled
  4. Check Replicates from the "Applies to" list
  5. View->Document Grid and choose "Replicates" from the Reports drop-down
  6. Fill in values for the Day column for the replicates that you want to include in the reporting. You can use whatever values you like to identify the timepoint - "Day 1", "Day 2", "10-08-2021", "Batch 1", etc.
Optionally, add annotations to capture the normalized and calibrated areas.
  1. Select Settings > Document Settings
  2. On the Annotations tab, click Add...
  3. Use "NormalizedArea" or "CalibratedArea" as the name, and choose the Calculated button
  4. Check Precursor Results from the Applies to list
  5. Choose the value to plug in for the annotation. Precursor->Precursor Results->Precursor Quantitation->Precursor Normalized Area or ->Calculated Concentration. Use "Sum" as the aggregate

Create Folder and Load Document

To use this report, start in a Panorama folder of type "Chromatogram library".

When you import a Skyline document containing protein data, the system will automatically add a new Proteins tab.

The protein list on this tab contains Protein/Label, Accession, Preferred Name, Gene, Description, Species, File Name, Protein Note/Annotations and Library State (if available). For proteins where there are multiple replicates included in the document, you will see a link next to the protein / label. Click it to open the Reproducibility Report.

View Reproducibility Report

The report will look for a Replicate annotation named "Day" in the Skyline document to analyze for intra- and inter-day variability. In the absence of this annotation, the report will not be available.

The report contains a series of panels:

  • Protein: This overview section shows some basic metadata about the protein and the file it was loaded from.
  • Sequence Coverage (if available): View the peptide coverage within the protein sequence; highlight various features.
  • Annotations (if available)
  • Precursors: The precursor listing can be filtered and sorted to customize the report.
    • If the Skyline document includes paired heavy and light precursors, you will be able to select either Intensity or Light/Heavy Ratio values for the report. Learn more below.
    • Any CV values (inter-day, intra-day, or total) over 20 in this section are highlighted in red.
    • You can click the link in the second column header to Copy the filtered peptide list to the clipboard.
  • Depending on the selection in the Precursors panel, the first plot is one of these:
    • Hover anywhere on the plots for buttons to print to PDF or PNG.
  • Coefficient of Variation for either intensity or light/heavy ratio.
  • Chromatograms: Clicking on a precursor in the plots or summary table will select that precursor and scroll to its chromatograms at the bottom of the page.
  • Calibration Curve/Figures of Merit: If the imported Skyline file contains calibration data, we will show the Calibration curve on the page. Figures of Merit (FOM) will be displayed below the calibration curve.
    • The Figures of Merit section will only display the Summary table, with the title linking to the detailed FOM page
    • The Calibration Curve title is also a link that would bring the user to a separate Calibration Curve page.

Protein Heading

In the Protein panel at the top of the report, you will see information about the Skyline document, including a link to download it.

Sequence Coverage

If the Skyline document includes a protein sequence, you will see a Sequence Coverage section. The peptide coverage is shown as a shaded bar within the overall protein sequence. A set of Features checkboxes lets you highlight the protein features at each amino acid with assigned colors.

Annotations

If relevant annotations are available, they will be shown in the next panel. For example:

Precursor Listing Options

If the Skyline document includes paired heavy and light precursors, you will have two ways to view the reproducibility report, selectable using the radio button in the Precursors web part. This selection will determine the plots you will see on the page.

  • Intensity: Sort and compare intensity for peak areas and CV of peak areas data.
  • Light/Heavy Ratio: Instead of peak area, analyze the ratio of light/heavy precursor peak area pairs (typically performed by isotope labeling).
  • Sort by lets the user sort the precursors on the plots shown by:
    • Intensity or Light/Heavy Ratio, depending on the radio button selection.
    • Sequence
    • Sequence Location (if two or more peptides are present)
    • Coefficient of Variation
  • Value type: When the document contains calibrated and/or normalized values, you can select among them. The default will depend on what's in the Skyline file: Calibrated, Normalized, and Raw in that order.
    • Calibrated
    • Normalized
    • Raw
  • Sequence Length: Drag the min/max endpoint sliders to filter by sequence length.
Click the link in the second column header to Copy the filtered peptide list to the clipboard.

Intensity (Peak Areas)

When the Intensity radio button is selected in the Precursors web part, the data grid will show:

  • Peptide sequence
  • Charge
  • mZ
  • Start Index
  • Length
  • Inter-day CV (Note that if the report includes 1xN (a single day), the inter-day CV column will still be present, but values will be 0.0%.)
  • Intra-day CV
  • Total CV
  • Mean Intensity
  • Max Intensity
  • Min Intensity
The Intensity web part will plot the peak areas with the selected filters and sorts.

The plot will show each replicate's peak area, divided by run within the day, and day. A box plot will show the full range of all values. Data points will be arranged from left to right within each precursor based on their acquisition time.

Intensity Coefficient of Variation

Based on which Show checkboxes are checked, this plot shows the averages for intra-day and inter-day peak areas, plus the "Total CV", which is the square root of the sum of the squares of the intra and inter-day CVs.

On the CV plot, hover over individual bars for a tooltip:

  • For the inter- and intra-day CVs, see the full peptide sequence (with charge state and mz) and the standard deviation of the CVs, as well as the average CV (which is the value being plotted).
  • For the total CV, you'll see the value and peptide sequence.

Light/Heavy Ratios

If the Skyline document includes paired heavy and light precursors, and Light/Heavy Ratio is selected, the data grid will contain the following information:

  • Peptide sequence
  • Charge (light and heavy)
  • mZ (light and heavy)
  • Start Index
  • Length
  • Inter-day CV of light/heavy ratios of intensity (Inter-day CV)
  • Intra-day CV of light/heavy ratios of intensity (Intra-day CV)
  • Total CV of light/heavy ratios of intensity (Total CV)
  • Mean value of light/heavy ratios (Mean Ratio)
  • Max value of light/heavy ratios (Max Ratio)
  • Min value of light/heavy ratios (Min Ratio)
The CV values will be highlighted in red if the number exceeds 20%. When a heavy ion is not present, the light/heavy ratio for that ion cannot be calculated. In this edge case, the Light/Heavy ratio for this ion will be displayed as "N/A" in the data grid. These ions will also not be shown in the Peak Areas Ratio and CV ratio plots.

Light and heavy ions of the same type will be shown as one type of ion on the chart. For example: ratio data for EAEDL[+7.0]QVGQVE and EAEDLQVGQVE will be shown as EAEDL...+. If the light and heavy ions have different charges, then the ion will be shown on the graph without the charge information.

Light/Heavy Ratio Coefficient of Variation

Based on which Show checkboxes are checked, this plot shows the averages for intra-day and inter-day peak areas, plus the "Total CV", which is the square root of the sum of the squares of the intra and inter-day CVs.

Chromatograms

One chromatogram per replicate is rendered, in a matrix-like layout, with rows that represent the days the data was acquired and columns that represent the indices within the day. Hence, the layout will reflect the MxN nature of the data.

When showing light/heavy ratios, the chromatograms at the bottom of the page will show both the light and heavy precursors plotted together for each replicate.

To see the full layout, expand your browser view. Individual plots are titled based on the day and index.

  • Users can click to download a PDF or PNG of any chromatogram. Hover to see the buttons.

At the bottom of the panel of chromatograms, you can click Show peptide details to switch to the detail page with more adjustable ways to view chromatograms.

Related Topics




Panorama QC Folders


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

The Panorama QC folder is designed to help labs perform QC of their instruments and reagents over time. Runs are uploaded using the data pipeline or directly from Skyline. Panorama QC folders support reviewing and plotting both proteomics (peptide/protein) and small molecule data.

Panorama QC Topics

This page contains details about the dashboard, summary, AutoQC, and plots. More topics related to using the Panorama QC Folder type are:

Panorama Dashboard

The Panorama QC Overview dashboard offers a consolidated way to view good summaries of quality control information. Information from the current folder and immediate subfolders are displayed in a tiled format. For example, subfolders might each represent a specific machine, so you can see instrument conditions at a glance.

The Panorama Dashboard tab shows the QC Summary and QC Plots.

QC Summary

On the Panorama Dashboard, the QC Summary section shows a tile for the current folder and each immediate subfolder the user can access. Typically a folder would represent an individual instrument, and the dashboard gives an operator an easy way to immediately scan all the machines for status. The count of the number of Skyline documents (targetedms.runs) and sample files (targetedms.samplefile) are scoped to the current container. Each tile lists the number of files that have been uploaded, the number of precursors that are being tracked, and summary details for the last 3 sample files uploaded, including their acquired date and whether any outliers were identified. Both proteomics and small molecule data outliers are tracked, and a combined count of outliers is presented. Tiles also include a visual indicator of AutoQC status.

The menu for the web part provides links to:

AutoQC

The TargetedMS module uses the AutoQC tool to ping the server to check if a given container exists, evaluate LC MS/MS performance over time, and quickly identify potential issues.

Learn more about AutoQC on PanoramaWeb here: Quality control with AutoQC

AutoQC Downloads

If you are running Skyline on your instrument computer install AutoQC Loader from this location:

If you are running Skyline-daily, install AutoQC Loader-daily from this location: To learn more about AutoQC, review this documentation: Using AutoQC Results

The QC Summary web part displays a color indicator:

  • Gray ring - AutoQC has never pinged.
  • Red circle - AutoQC has pinged, but not recently.
  • Green check - AutoQC has pinged recently. The default timeout for "recently" is 15 minutes.
Hover over the icon for the time of the last ping.

AutoQC also facilitates the ongoing collection of data in a Panorama QC folder. Each sample is uploaded incrementally and automatically by AutoQC. AutoQC adds new samples to an existing Skyline document, rotating out and archiving old data to prevent the active file from getting too large. As new files are received, they are automatically coalesced to prevent storing redundant copies across multiple files. Whenever a PanoramaQC file receives a new file to import, by the end of that import we have the most recent copy of the data for each sample contained in the file, even if it had been previously imported.

Sample File Details

The display tile shows the acquired date/time for the latest 3 sample files along with indicators of which QC metrics have outliers in the Levey-Jennings report, if any. Hover over the icon for a sample file in the QC Summary web part to see popover details about that file.

The hover details for a sample file with outliers show the per metric "out of guide set range" information with links to view the Levey-Jennings plot for that container and metric.

Delete a Sample File

To delete an unwanted sample file, such as one you imported accidentally, click the link showing the number of sample files in the folder to open a grid, select the relevant row, and click Delete. The data from that sample file will be removed from the plot.

QC Plots

The QC Plots web part shows one graph per precursor for a selected metric and date range. Choose from a variety of different available plot types, sizes, and other options. For more details, see Panorama QC Plots.

Example Plots with guide sets:

Include or Exclude Precursors (Peptides/Small Molecules)

The precursors, whether peptides or small molecules, can be divided into two groups for mass spectrometry analysis:

  • Included group: The precursors driving the plots and AutoQC assessments.
  • Excluded group: Those you want to exclude from AutoQC assessments and from plots (unless you check the box to "Show Excluded Precursors").
From the QC Summary web part, choose Include or Exclude Peptides/Molecules from the menu.

You'll see a grid of the precursors available in the files, with some summary data. By default, all are marked as "Included". Select one or more precursors using the checkboxes, then move them between the two groups by clicking either Mark as Included or Mark as Excluded.

In the QC Plots web part, the Show Excluded Precursors checkbox controls whether to show those marked as being in the "Excluded group".

Related Topics




Panorama QC Plots


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

The Panorama QC folder is designed to help labs perform QC of their instruments and reagents over time. Runs are uploaded using the data pipeline or imported directly from Skyline. QC Plots, covered in this topic, give a visual illustration of performance over time that can make data validation easier and more reliable.

QC Plots Web Part

A given molecule will have many potential precursors. You want to choose the one that gives a robust, consistent signal. In practice, for a given set of instrumentation, you'll wind up being able to best measure certain charge states, and hence, certain precursors of a given molecule.

The QC Plots web part shows one graph per precursor by default. The web part header allows you to specify a number of options, including selecting one or more type of plot using checkboxes. Hover over a plot name to learn more. This topic uses the default Levey-Jennings plot to illustrate features.

QC Plot Types

  • Levey-Jennings: (Default) Plots quality control data to give a visual indication of whether a laboratory test is working well. Points more than three standard deviations from the mean are considered outliers.
  • Moving Range (MR): Plots the moving range over time to monitor process variation for individual observations by using the sequential differences between two successive values as a measure of dispersion.
  • CUSUMm: A CUSUM plot is a time-weighted control plot that displays the cumulative sums of the deviations of each sample value from the target value. CUSUMm (mean CUSUM) plots two types of CUSUM statistics: one for positive mean shifts and one for negative mean shifts.
  • CUSUMv: The CUSUMv (variability or scale CUSUM) plots two types of CUSUM statistics: one for positive variability shifts and one for negative variability shifts. Variability is a transformed standardized normal quantity which is sensitive to variability changes.

QC Plot Features

Select the metric to plot using the pulldown menu in the web part header. Set other plot options to control the display of plots in the web part.

  • Metric: Select the desired metric from the pulldown. Learn more below
  • Date Range: Default is "All dates". Other options range from last 7 days to last year, or you can specify a custom range.
  • Plot Size: When multiple plots are selected, choose:
    • Small: Display 2 plots across the page.
    • Large: Show full width plots, one per row.
  • QC Plot Type: Check one or more boxes for plot type. Options outlined above.
  • Y-Axis Scale: Options: linear, log, percent of mean, or standard deviations. Not all are applicable to every plot type. See below.
  • Click the Create Guide Set button to create a guide set.
  • Group X-Axis Values by Date: Check this box to scale acquisition times based on the actual dates. When this box is not checked, acquisition times are spaced equally, and multiple acquisitions for the same date will be shown as distinct points.
  • Show All Series in Single Plot: Check this box to show all fragments in one plot.
  • Show Excluded Samples: Check to include points for samples you've excluded from your plot(s) as outliers or for other reasons. Note that this is not related to the "Excluded group" of precursors you can designate.
  • Show Referenced Guide Set: Always include the guide set in the plot, even if it is outside the range that would otherwise be shown.
  • Show Excluded Precursors: Check to include precursors (peptides or small molecules) that are marked as in the "Excluded group". By default all are in the "Included group". Note that the "Excluded group" is not related to sample exclusions.

Filter by Replicate Annotations

If the Skyline document you imported included replicate annotations, you will have the option to filter your plots using the Filter: Replicate Annotations drop down that will be present.

Check the box(es) for the annotation(s) to select and click Apply. Selected annotations will be listed in the panel.

Click Clear to remove the filtering.

Y-Axis Scale Options

All plot types support the selection of linear or log scale for the Y-axis. The type selected will be shown along the left sidebar, including a units label for non-CUSUM plots. This labelling does not apply to Transition/Precursor Area plots which use other labelling on the dual Y-axes.

For Levey-Jennings and Moving Range plots, you can also normalize the plots using two additional Y-axis scale types. Note that if you select these scales for CUSUM plots, a linear scale is used instead.

  • Percent of Mean:
    • The mean is considered as 100% and other values are plotted above or below based on the percent over or under the mean they are.
    • Value plotted as (value/mean) * 100%.
    • Levey-Jennings plots will include +/- three standard deviations over and under the mean.
    • Moving Range plots will include upper and lower limits as a percent of mean, showing the variability.
  • Standard Deviations:
    • The mean is plotted as zero on the Y-axis. Values are plotted based on the difference from the mean as a ratio with the standard deviation.
    • Levey-Jennings plots include standard deviation bars of +/- three standard deviations.
For example, this plot of Retention Time has been normalized to percent of mean.

The range shown along the Y-axis is based on the variance of the values. This means the size of the range will be dynamic to fit all normalized data points. Levey-Jennings plots include a default guide set to cover the entire plot when viewing a single series in a single plot.

Metrics

Administrators have the ability to control which metrics are available for plotting, as described in this topic:

By default, each type of plot can be shown for the following metrics:

Isotopologue Metrics

There are four isotopologue metrics that will be available when there is data they can use:

  • Isotopologue Accuracy
  • Isotopologue LOD: Limit of Detection
  • Isotopologue LOQ: Limit of Quantitation
  • Isotopologue Regression RSquared: Correlation coefficient calculated from the calibration curve
Isotopologues are molecules that differ from each other solely by their isotopic composition. They are valuable in mass spectrometry because it’s possible to create variants of the same peptide with different masses. These isotopologues can then be added at different concentrations in a single sample, allowing for a calibration curve for a peptide in a single sample (and therefore single run of the mass spec). Other common calibration curve approaches require separate samples for each concentration of a single peptide.

Researchers can use these isotopologue samples for QC and system suitability purposes. By monitoring the limit of quantitation (LOQ), limit of detection (LOD), and other metrics, a researcher can gain insight into the performance of the instrument and the dynamic range it’s accurately measuring. Commercial kits, such as the Pierce™ LC-MS/MS System Suitability Standard (7 x 5 Mix) are an easy way to implement this kind of monitoring.

Skyline provides the ability to populate attributes on data elements like precursors and peptides with calculated values. The isotopologue metrics are used in conjunction with AutoQC to provide automated system suitability monitoring in Panorama. Panorama QC will look for the following calculated annotations on the Precursor Results elements. The name of each annotation must match exactly:

  • PrecursorAccuracy, populated from Precursor Quantification > Precursor Accuracy
  • RSquared, populated from Peptide Result > Replicate Calibration Curve > Replicate R Squared
  • LOD, populated from Peptide Result > Batch Figures of Merit > Batch Limit of Detection
  • LOQ, populated from Peptide Result > Batch Figures of Merit > Batch Limit of Quantification
When present in the Skyline document, Panorama will import these values as annotations on a PrecursorChromInfo data element (captured in the targetedms.precursorchrominfo table during import) with the names “LOD”, “LOQ”, “PrecursorAccuracy”, and “RSquared”.

Example data from the 7x5 kit is available on PanoramaWeb , as is a template Skyline document for analyzing raw data files from the kit.

Run-Scoped Metrics

Metrics that are not tied to an individual molecule can be tracked and plotted using metrics that produce a single value for the entire run. The built-in metric "TIC Area" (Total Ion Chromatogram Area) shows an example of such a run-scoped metric.

When viewing run-scoped metrics, the "Show all Series in a Single Plot" option is not available since there is only one series to show.

Transition & Precursor Areas

To show both precursor and fragment values in the same plot, select the metric option Transition & Precursor Areas.

The plot is more complex when all fragments are shown. Use the legend for reference, noting that long peptide names are abbreviated. Hover over a truncated name to see the full peptide name.

QC Metric Settings Persistence

Once any user has configured a useful set of metrics and ways of viewing your plots, they will be persisted. The next time that user visits this folder and the plots on this dashboard, they will see the same metric they were most recently viewing with the same settings they were using.

Set Default View

When an administrator has customized the QC Plots web part and metrics settings for a container, they have the option to save these settings as a default view for all other users visiting this folder. From the menu, select Save as Default View.

Users viewing the plots will now see the admin-defined default plot configuration settings, unless they have customized their own view of plots in this container. Users can start from the default and make changes as they work, coming back to their own settings when the return.

All users also have the option to Revert to Default View at any time. If no admin-customized default view is available, the original default settings will be applied.

Hover Details and Outlier Exclusions

Hovering over a point in the graph will open a tool tip with additional information about that data point.

You can click to view either the chromatogram or sample file for this point. In the Status section of the tooltip, you can select an exclusion option if needed, such as for an outlier data point. Elect either of the following options to do so:

  • Exclude sample from QC for this metric
  • Exclude sample from QC for all metrics
Excluded samples are left out of the guide set range calculations and QC outlier counts for the QC summary panel and Pareto plots. Outlier samples can also be excluded from QC by entering the replicate annotation "ignore_in_QC" from the Skyline app, which will cause the sample to be excluded from all metrics.

When a sample has been excluded, the QC dashboard hover information will note "not included in QC".

Export

You can export any of the plots by hovering to expose the buttons, then clicking the icon for:

  • PNG: Export to a PNG image file.
  • PDF: Export as a PDF document.

Small Molecule Data

The same Levey-Jennings, MR, CUSUM, and Pareto plot features apply to both proteomics (peptide/protein) data and small molecule data. Data from both types may be layered together on the same plot when displaying plots including all fragments. Counts of outliers and sample files include both types of data.

When visualizing small molecule data, you are more likely to encounter warnings if the number of precursors exceeds the count that can be usefully displayed. This screenshot shows an example plot with small molecule data.

Note that the legend for this illegibly-dense plot does not list all 50 precursors. You can narrow what the plot displays by marking uninteresting small molecules as in the "Excluded group" to hide them.

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Panorama QC Plot Types


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

A Panorama QC folder offers several plot types useful in quality control:

Levey-Jennings Plots

The default plot in a Panorama QC folder is the Levey-Jennings plot which is helpful in visualizing and analyzing trends and outliers. The distance between a given observation and the mean (expected value) is measured in standard deviations (SD). For a walkthrough of plotting features featuring these plots, see Panorama QC Plots.

Moving Range Plots

The moving range can be plotted over time to monitor process variation for individual observations by using the sequential differences between two successive values as a measure of dispersion. Moving Range (MR) plots can be displayed alongside Levey-Jennings plots for integrated analysis of changes. To create a Moving Range plot, check the box in the QC Plots web part.

In this screencap, both the Levey-Jennings and Moving Range plots are shown side by side. Notice the two elevated points on the moving range plot highlight the one peak (two large changes). Otherwise the value for retention time remained quite consistent.

The plotting features outlined in Panorama QC Plots also apply to Moving Range plots.

CUSUM Plots

A Cumulative Sum (CUSUM) plot is a time-weighted control plot that displays the cumulative sums of the deviations of each sample value from the target value. This can highlight a problem when seemingly small changes combine to make a substantial difference over time.

The legend for the dotted and solid lines is included on a CUSUM plot:

  • CUSUM- is a solid line
  • CUSUM+ is a dotted line
The plotting features outlined in Panorama QC Plots also apply to both types of CUSUM plots.

CUSUMm (Mean CUSUM)

The CUSUMm (mean CUSUM) plots two types of CUSUM statistics: one for positive mean shifts and one for negative mean shifts.

CUSUMv (Variable CUSUM)

The CUSUMv (variability or scale CUSUM) plots two types of CUSUM statistics: one for positive variability shifts and one for negative variability shifts. Variability is a transformed standardized normal quantity which is sensitive to variability changes.

A sample CUSUMv plot, shown with no other plot type selected:

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Panorama QC Annotations


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Annotations can be added to Panorama QC plots to assist in quality control. Marking a specific event can help identify causes of changes. If there are replicate annotations present in the imported Skyline data file, they will also be shown and can be used to filter plots.

Quality Control Annotations

Color coded markers can be used to annotate the QC plots with information about the timing of various changes. The annotated plot will show colored Xs noting the time there was a change in instrumentation, reagent, etc. The legend shows which color corresponds to which type of annotation.

Hovering over an annotation pops up a tooltip showing information about when the annotation event occurred, the description of the event, and who added it.

Add Annotations

Select the Annotations tab to define and use annotations.

Define Types of Annotations

Each type of annotation has a name, description, and color to use. There are three built-in categories which are shared by all Panorama folders on the server. You may change them or the colors they use, but be aware that other projects may be impacted by your changes. Annotation Types defined in the "Shared" project are available throughout the server. Types defined at the project level are available in all subfolders.

  • Instrumentation Change
  • Reagent Change
  • Technician Change
You can also define your own annotation types as required. For example, you might want to note changes to environment like addition of building HVAC or power outages.

If you wish to Add new categories of annotations, use (Insert data) > Insert New Row in the QC Annotation Types section.

To Edit existing annotation types, hover over the row and click the (Edit) icon that will appear in the leftmost column.

Add New Annotations to Plots

To enter a new annotation, use (Insert data) > Insert New Row in the QC Annotations section. Select the type of event (such as Reagent Change), enter a description to show in hover text ("new batch of reagent"), and enter when it occurred. Dates that include a time of day should be of the form "2013-8-21 7:00", but a simple date is sufficient. Return to the Panorama Dashboard tab to view the plots with your new annotation applied.

The annotation symbol is placed on the x-axis above the tick mark for the date on which the event occurred. If there are multiple tickmarks for that date, the annotation will appear above the leftmost one. If an annotation occurred on a date for which there is no other data, a new tick mark will be added to the x-axis for that date.

Replicate Annotations

If the Skyline document included replicate annotations, you will see them listed in the ReplicateAnnotation panel at the bottom of the page. To learn about filtering QC plots using replicate annotation information, see this topic

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Panorama QC Guide Sets


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

The Panorama QC folder is designed to help labs perform QC of their instruments and reagents over time. Runs are uploaded using the data pipeline or directly from Skyline. This topic describes how quality control guide sets can help track performance of metrics over time.

Quality Control Guide Sets

Guide sets give you control over which data points are used to establish the expected range of values in a QC plot. Instead of calculating the expected ranges based on all data points in the view, you can specify guide sets based on a subset of data points.

You create a guide set by specifying two dates:

  • training start date
  • training end date
The training start and end dates (called the "training period") establish the period to calculate the expected range of values. The data points within the training period are used to calculate the mean and standard deviation for that guide set's expected range.

Standard deviations are shown as colored bars: green for +/-1, blue for +/-2, and red for +/-3 standard deviations from the mean. The expected range calculated by a training period is applied to all future data points, until a new training period is started.

Data points for a given guide set share a similar shape (circle, square, triangle, etc.). A different shape is introduced when a new guide is started.

The training periods are shown with a grey background -- hover over the training area to see detailed information about that guide set. You can also see these details on the Guide Sets tab.

Define Guide Sets

You can create guide sets directly from the QC plot. To add a new guide set, first uncheck Group X-Axis Values by Date and Show All Series in a Single Plot. Next click Create Guide Set, drag to select an area directly on the graph, and click the Create button that appears over the selected area.

Note: a warning will be given if fewer than 5 data points are selected. You cannot create overlapping guide sets.

Alternatively, you can create guide sets by entering the start and end dates manually: click the Guide Sets tab and click Insert New to manually enter a new guide set. Note that you must have been granted the Editor role or greater to create guide sets from either of these two methods.

Edit or Delete Guide Sets

To edit or delete guide sets, click the tab Guide Sets. To edit, hover over a row to reveal buttons for a guide set that has already been created. Click the (pencil) icon to edit. To delete, check the target guide set and click the (Delete) button.

Show Reference Guide Set

In any QC Plot display, use the Show Reference Guide Set checkbox to always include the guide set data in QC plots. This box is checked by default.

When zooming in to view QC data for a specific date range, the guide set data currently being used to identify outliers will always be shown, even if it is outside the plot date range.

The guide set is shaded in gray, and separated from the "selected" plot range by a vertical line.

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Panorama: Pareto Plots


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Pareto Plots

Pareto plots combine a bar plot and a line plot, and are used to quickly identify which metrics are most indicative of a quality control problem. Each bar in the plot represents a metric (see metric code below). Metric bars are ordered by decreasing incidence of outliers, where outliers are defined as the number of instances where each metric falls outside of the +/- 3 standard deviation range. The line shows the cumulative outliers by percentage.

There are separate pareto plots for each guide set and plot type (Levey-Jennings, Moving Range, CUSUMm, and CUSUMv) combination.

Other items to note in Pareto plots:

  • Hover over dots in the line plot to show the cumulative %.
  • Hover over a metric bar to show the number of outliers.
  • Click a metric bar to see the relevant QC plot and guide set for that metric.

Print Plots

Hover over any plot to reveal PDF and PNG buttons. Click to export the Pareto plot for that guide set.

Metric Codes

  • FWB - Fill Width at Base
  • FWHM - Fill Width at Half Maximum
  • MA - Mass Accuracy
  • PA - Peak Area
  • P Area - Precursor Area
  • RT - Retention Time
  • T Area - Transition Area
  • T/PA Ratio - Transition/Precursor Area Ratio

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Panorama: iRT Metrics


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

Indexed Retention Time (iRT) is a scheme for calibrating retention times across runs. For example, you might use an iRT scale like this set of 11 synthetic peptides from Biognosys. Based on these, peptides, the set of iRT anchor points can be extended to other peptides. The iRT of a peptide is a fixed number relative to a standard set of reference iRT-peptides that can be transferred across labs and chromatographic systems. iRT can be determined by any lab and shared transparently.

This topic describes how you can use metrics related to the iRT calculations as system suitability metrics in Panorama. Monitoring the slope, intercept, and R-squared correlation values for the regression line can help mass spectrometrists confirm that equipment is performing as expected.

iRT Metrics

The iRT metrics (slope, intercept, and R-squared correlation) are scoped to each sample file. Panorama will detect whether to include these metrics alongside other metrics available in QC plots based on whether the Skyline document is configured to monitor iRT. Learn more about plot options in this topic: Panorama QC Plots

iRT Correlation

iRT Intercept

iRT Slope

Hover Details

As with other plotted metrics, hover over any point in the plot to see details including the value and acquired date.

From the hover panel, you can also choose to exclude the sample from QC if desired. Learn more in this topic: Panorama QC Plots

Notifications

Users can subscribe to receive notifications when iRT metric values are out of range, as for other outlier values. Learn about outlier notifications in this topic: Panorama: Outlier Notifications.

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Panorama: Configure QC Metrics


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

This topic describes how Panorama folder administrators can customize metrics, enable or disable them, and define new metrics of their own.

Enable and Disable Metrics

An administrator can control which QC metrics are available for quality control plots on a folder-by-folder basis. Disabling metrics may be practical for a variety of reasons, including if they produce many outliers without being diagnostic of real problems. For each metric, you can select:

  • Enabled: Always show the metric in this folder.
  • Auto: (Default) Show the metric when relevant data is present. For custom metrics, you provide your own query to control when to enable the metric.
  • Disabled: Never show the metric in this folder.
The configuration menu is available on the QC Plots web part.

  • In the upper right of the QC Plots web part, open the (triangle) pulldown menu.
  • Select Configure QC Metrics.
  • Use the pulldowns to select Enabled/Auto/Disabled as desired.
  • The Type column indicates whether a metric is scoped to a single molecule (precursor) or to a run as a whole.
  • Click Save.

Disabled metrics are hidden from the main QC plot page, QC summary web part, and pareto plot.

Define Custom Metrics

An administrator can create their own custom metric by first defining a query, typically in the targetedms schema, with the following columns:

NameTypeDescriptionNotes
MetricValueNUMERICThe value to be plotted 
SampleFileIdINTValue of the related Id in targetedms.SampleFile 
PrecursorChromInfoIdINTValue of the related Id in targetedms.PrecursorChromInfo. May be NULL, in which case there will be click handler for the point.
SeriesLabelVARCHARThe peptide sequence, custom ion name, etc
DataTypeVARCHARHow to label the series in the overall list (typically either “Peptide”, “Fragment”, etc)
PrecursorIdINTValue of the related Id in the targetedms.Precursor or targetedms.MoleculePrecursor table.
mzNUMERICmass to charge ratio

The fields PrecursorChromInfold must be included in a query for run-scoped metrics but should return NULL. The fields SeriesLabel, DataType, PrecursorId, and mz are optional. They will be automatically calculated if they are missing, but you can use them to customize the display.

Once your query is defined, reopen the Configure QC Metrics page from the web part menu and click Add New Custom Metric.

In the popup:

  • Enter a Name
  • Select the Schema and Query for Series 1.
  • Give provide an Axis Label.
  • If you would like a second Y-axis tracking a different schema and query, specify those in Series 2.
  • Select the Metric Type:
    • Precursor: This metric varies for each individual molecule.
    • Run: This metric will have a single value per run, such as a maximum value of a pressure trace during a run.
  • If you would like to be able to use the Auto option to enable your metric based on the presence of data, supply the following:
    • Enabled Schema: Schema in which to find the "Enabled" query.
    • Enabled Query: Query to determine when to enable the metric.
  • Click Save.
The new metric will appear on the metric configuration list as a clickable link. Click to see the definition. You can edit and save or delete if if necessary. As with predefined metrics, you can enable or disable your custom metric using the same menu in the Status column.

When enabled, your new metric will also be available in the QC Plots pulldown menu for plotting. If you created a molecule-scoped metric, each series will be titled with the value of the "SeriesLabel" column from the backing query.

Define Pressure Trace Metrics

Pressure traces, total ion current, and other chromatograms help monitor the performance of the mass spectrometry equipment. For example, a higher than normal value in system back pressure during the acquisition probably indicates the system is overpressure or starting to leak. Skyline will automatically extract pressure traces and other sample-scoped chromatograms from the raw files. When they're present, you can can monitor this at specific time points by configuring a metric to pull the pressure trace within the chromatogram at specific points in time, or track the time that a specified pressure value is reached.

There are two options for these metrics:

  • Track trace value at user defined time (in minutes)
  • Track time of user defined trace value
Open the Configure QC Metrics page from the web part menu and click Add New Trace Metric. In the popup, specify:
  • Metric Name: You can use the name to give users details about the metric.
  • Use Trace: Select the trace to use. If this box reads "No trace can be found", you will not be able to create a trace metric.
  • Y Axis Label: You can use this label to give information about units (PSI, etc) as needed.
  • Select the type of trace with the radio buttons:
Use trace value when time is greater than or equal to: Enter the number of minutes in the box.

Use time when the trace first reaches a value greater than or equal to: Enter the desired value in the box.

  • Click Save to add the new metric.
  • Use the Status dropdown to set it to Enabled/Auto/Disabled.
  • Click Save to return to the QC Plots dashboard.
You can now view your new metric(s) in QC plots, outlier calculations, and automated emails.

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Panorama: Outlier Notifications


Premium Feature — Available only with Premium Editions of LabKey Server through the Panorama Partners Program. Learn more about Panorama Partners or contact LabKey.

In this topic, learn how any non-guest Panorama user can customize the notifications they receive. Opt in to notifications when any data is uploaded, or only when the number of outliers in a specified number of files exceeds a custom threshold.

Subscribe to Notifications

Any non-guest user can configure their own subscription to notifications about imports and outliers. An unusual number of outliers or pattern sustained over multiple runs may indicate a problem with an instrument that should be corrected.

To subscribe to notifications:
  • Open the (triangle) menu on the QC Summary web part.
  • Click Subscribe Outlier Notifications.
  • Use the radio buttons to control whether email notifications are sent to you.
  • If you select Email me when... you can also specify the threshold for triggering email:
    • Number of runs to consider, from 1 to 5. For one run, select most for two runs, select two most, etc.
    • Number of outliers per file that will trigger the notification. Valid values from 0 to 999. To be notified of every upload regardless of presence of outliers, enter 0 (zero).
  • Click Save.

To unsubscribe, reopen the panel, select Do not email with notifications about newly imported data, and click Save.

Notification Content

The notification will be sent immediately following a triggering upload and will have the email subject:

  • Panorama QC Outlier Notification - <##> outliers in file <filename> in <containername>
The body of the email will be the table shown in the QC Summary plot for a single sample file.

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Panorama and Sample Management


Premium Integration — These features are available when you are using both Panorama and LabKey Sample Manager. Learn more or contact LabKey.

Associating sample metadata with results data, including targeted mass spectrometry data, is made simple when you use both Panorama and LabKey Sample Manager on the same server.

Users can perform deeper analyses and quickly gain insight into which samples have data associated. Additionally, users can export sample data to help configure their instrument acquisition and Skyline documents.

Prerequisites

Set up a LabKey Sample Manager folder on the server. To show Skyline/Panorama data, enable the targetedms module in that folder. Other Panorama projects and folders on the same server will be able to connect to this shared sample repository.

Note that the terms "Sample Name" and "Sample ID" are interchangeable in this topic, and this walkthrough assumes that you already have Panorama data and want to link it to additional Sample metadata.

Switch between the Sample Manager interface and Panorama (LabKey Server) interface using the product selection menu.

Link Panorama Data to Sample Metadata

To link samples in Panorama to metadata about them, first create the Sample Type with desired fields in Sample Manager. Your Sample Type definition can include as much information as you have or will need. For example, you might create a "Panorama Samples" type that included a project name and collection date, shown in the examples on this page.

In any Panorama folder on the server, load your Skyline document(s) as usual. Click the Runs tab, click the name of the Skyline file, then click on the replicates link to get a list of Sample Names.

Return to Sample Manager, and create new Samples for each of the Sample Names you want to link, populating the metadata fields you defined. You can do this in bulk or by file import, but for this small example, we add the metadata in a grid.

Note that the sample metadata and Skyline documents can be imported in any order.

Once you've registered the samples, return to the replicate list in Panorama and you will now see the sample metadata for your samples.

Click the name of a linked sample here and you will return to the Sample Manager interface for that sample, where you could find it in storage, review assay data, etc.

Link from Samples to Panorama Data

When viewing the details for a sample in Sample Manager, if there is linked data in Panorama, you will see a tab for Skyline Documents on the Assays tab.

If there were additional assay data about these samples, it would be shown on other tabs here. Notice you can also track lineage, aliquots, workflow jobs, and the timeline for any sample linked in this way.

Click the name of the File to go to the Panorama data for that sample, which could be in another project or folder on your server.

Provide Sample List to Acquisition Software

Users may want to export a list of samples to their acquisition software. This will reduce errors and improve efficiency.

Within Sample Manager:

  • Open the Panorama sample type you created and identify the desired samples by filtering, sorting, or creating a picklist.
  • Select the desired samples to include in your acquisition list.
  • Export the grid of samples to Excel, csv, or tsv file.

If the default set of exported fields does not match your requirements, you could edit it offline to remove extraneous columns.

Using the LabKey Server interface, an administrator could also create a custom grid view of samples specifically tailored to the requirements. When present, such a grid could be selected from the Grid Views menu:

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