Latest improvements for QIAGEN RNA-seq Analysis Portal

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QIAGEN RNA-seq Analysis Portal 4.0

Released February 2023

New Features and improvements

• The QIAseq FastSelect Lib Kit has been added with three protocol options, one for each DNA synthesis protocol available for the sample kit:
  • QIAseq FastSelect RNA Lib Kit (N6-T RT primer)
  • QIAseq FastSelect RNA Lib Kit (ODT-T RT primer)
  • QIAseq FastSelect RNA Lib Kit (N6-T RT + ODT-T RT primers)No well-picker option is presented for these options.
• QIAseq UPX’3 Transcriptome kit and UPXome RNA Lib kits can now be Align and counted with demultiplexed data.
  • If you select a single input sample the well-picker option is activated, and multiple samples are selected the “Use already demultiplexed sample data” is selected
• UPXome RNA Lib and QIAseq FastSelect RNA Lib kits that have been run with two primers will have sections for both hexamer and combined sequences in the Quality Control report
• Onboarding pop-ups have been added to assist first-time users in getting started using the Portal.
  • Clicking “Don’t show again” will hide these pop-ups
  • If you want to see them again, they can be re-activated from the ‘Help’-menu

Bugfixes and other changes

• Added a more stable uploader that works in a separate browser tab. This allows for improved stability for upload sessions spanning hours – including automatic and manual retries if connectivity fails during upload.
• It is now possible to click the ‘Start now’ in the lower-right and see the selected features in GeneGlobe’s What’s Next feature without saving the filter first if the selection contains less than 1000 features. The Portal will then ask for the name of the filter set before moving to GeneGlobe
• Improved support for sample lists in various dialogs on smaller screens
• The label on the p-value y-axis in the experiment volcano plot now changes with the selection between FDR p-value and p-value
• Fixed an error causing renamed experiments to not display the new name. The name is now always updated correctly
• Fixed a bug where the sorted order of the taxonomic profiling of unmapped reads when detecting contamination was not correct. They now order consistently with higher values at the bottom in the stacked bar chart.
• Fixed a bug where sorting of features table in differential expression views now is persisted between grid and table views
• Fixed an issue that prevented experiments using % as part of the attribute names from being enriched with insights from IPA
• Barchart plots now have a minimum width relative to the number of samples. This means that in an experiment containing many samples, the bar charts in the QC report will be very wide and have a horizontal scrollbar on the screen. In PDF/print view, it will be scaled down proportionally.
• Help now links to the external product page for RNA-seq Analysis Portal with the label “About QIAGEN RNA-seq analysis”
• Help on the demo version now contains a “Reach out to an expert” menu item that will let users contact our support/sales team with questions or comments
• Now “Strand specific setting” in the Quality control tab shows as empty when no reads are mapped in the Align and count process. Previously it displayed “0” which is not correct
• Updated the volcano plot to show two decimals on the y-axis for smaller p-values
• Implemented an updated cookie acceptance and management feature

 

QIAGEN RNA-seq Analysis Portal 3.0.1

Released November 2022

Improvements

• In the sample kit dropdown, the QIAseq UPXome Lib Kit item has been replaced by three new QIAseq UPXome Lib Kit entries, one for each DNA synthesis protocol available for the sample kit:
  • QIAseq UPXome Lib Kit (N6-T RT primer)
  • QIAseq UPXome Lib Kit (ODT-T RT primer)
  • QIAseq UPXome Lib Kit (N6-T RT + ODT-T RT primers)If you analyzed data with the previous QIAseq UPXome Lib Kit element, we recommend that you rerun your data with the relevant new sample kit item.

Bugfixes and other changes

• Fixed an issue where fold change values for differential expressions with experimental setup All group pairs would sometimes be the opposite direction to that intended, see
  https://digitalinsights.qiagen.com/technical-support/faq/important-clc-notifications/qiagen-rna-seq-analysis-portal-differential-expression-fold-change-values-for-all-group-pairs-comparisons-may-be-the-opposite-direction-to-that-intended/

QIAGEN RNA-seq Analysis Portal 3.0

Released July 2022

New features

• Integrated upload. Upload of data now happens via a dialog within the existing browser tab. Previously, you were taken to a separate tab. You can monitor progress of your file uploads via the Uploads icon next to the Help menu. Once an upload is done, you can proceed to the Align and count dialog with the sample data pre-selected.
• Connected dialogs for improved analysis flow. You can proceed from the Align and count dialog to the Create experiment dialog with your new samples pre-selected.
• Contamination analysis. For RNA samples with more than 25% unmapped reads, an additional taxonomic profiling analysis is performed to test for potential contamination from bacteria and archaea.
• lncRNA analysis. For RNA experiments, the differential expression analysis will include both protein coding and lnc_RNA genes. On the Differential expression view, the feature table will indicate the biotype. You can filter on biotype using the new Advanced filters functionality.
• Advanced filtering. On the Differential expression view, the Advanced filters functionality allows you to filter on biotype and CPM expression values.
• A demo version of the RNA-seq Analysis Portal can be accessed without the need for login. This allows users, who do not have a My QIAGEN account, to get a feel for how you would upload data and start analysis, and how analysis results are visualized.

Improvements and changes

• For RNA sample kit data, the expression value downloads will contain CPM values.
• The application will support creation of experiments with up to 2x 384 samples.
• The Align sample data analysis time has been reduced by approximately 50%.
• In the Align and count dialog, BaseSpace access will no longer trigger browser pop-up blockers.
• On the Experiment summary and QC report, the Biotype distribution chart and table will have biotypes listed in alphabetical order.
• On the Differential expression and Comparison views, the feature tables are sorted on Name. Also, the ‘Sort by’ dropdown will reflect sorting on table headers, and vice versa.
• On the Differential expression view, a Reset button allows you to set filtering to default values. Previously, this was done via the filter dropdown where you would select Default filter.
• For QIAseq miRNA Library Kit differential expression results, the Biological Insights category Upstream regulators that for miRNA analyses was always left empty has been removed.

Bugfixes and other changes

• Fixed an issue for the Comparison view where values on the Venn diagram sections would sometimes be over- or underestimated.
• Fixed an issue for the Comparison view where manual selection of features in the table would prevent subsequent feature selection via the Venn diagram.
• Fixed an issue for the qPCR normalization gene view of the differential expression feature table where the message for cases where no genes met the requirements for qPCR normalization genes would incorrectly state that the absence of qPCR normalization genes was due to the samples having been analyzed on an older analysis workflow version. The message for such cases now correctly reads ‘Zero features meet the criteria for qPCR normalization genes’.

 

QIAGEN RNA-seq Analysis Portal 2.5.1

Released April 2022

Improvements

• Experiment summary and QC report: On the Quality control tab, the biotype distribution plot and table will report on all tested biotypes regardless detection. Previously, biotypes would be listed only if detected in at least one sample.

Bugfixes and changes

• Fixed an issue that could cause CPM values exported from QIAseq miRNA Library Kit experiments to be associated with the wrong sample name in the Excel file.
  The issue, including information on how to spot whether your downloaded expression values are affected, is documented at https://digitalinsights.qiagen.com/technical-support/faq/important-clc-notifications/qiagen-rna-seq-analysis-portal-mirna-expression-value-downloads-may-give-wrong-cpm-value/.
• Fixed an issue with the Differential expression view where the Start now button for sending results to GeneGlobe was enabled for the Default filter results. Clicking the button resulted in an error. Only saved filter results can now be sent to GeneGlobe.
• To improve the user experience, the Download expression value ‘multi-sample file’ options are no longer available on the Experiment summary and QC report. The amount of data involved in the download caused the browser to become unresponsive for several minutes and sometimes even crash. The ‘One file per sample’ options remain.

QIAGEN RNA-seq Analysis Portal 2.5

Released March 2022

New features and improvements

• Support for QIAseq UPXome RNA Lib Kit.
• Download graphics. You can download the various plots and charts as .png.
• Download Experiment summary and QC report. You can print or save the report as PDF.
• 15 additional reference species: Thale cress, cow, roundworm, dog, zebra fish, fruit fly, horse, chicken, crab-eating macaque, rhesus macaque, rainbow trout, rabbit, baker’s yeast, fission yeast, and pig. All species are supported for RNA sample kits, 11 of them also for the QIAseq miRNA Library Kit.
• qPCR normalization genes. The differential expression view lists candidate qPCR normalization genes.
• Upstream regulating miRNAs: For RNA sample kit analysis workflows, the Biological insights results Upstream regulators has been split in two: Upstream regulators and Upstream regulators (miRNA). The content of the latter will be sent to GeneGlobe alongside differentially expressed features and Canonical pathways when you click the ‘Start now’ button.
• Changes to existing analysis workflows
  • QIAseq miRNA Library Kit: To ensure considerably faster data analysis and enhance support for large datasets, the Quality control report will report on miRBase and, when available, piRNA annotation records only. If you are interested in knowing what other RNAs are present in your library, you may process your sample with the Legacy analysis pipelines on geneglobe.qiagen.com/analyze.
  • QIAseq UPX 3′ Transcriptome Kit: The demultiplex step will apply the cell ID as suffix for sample naming. Previously, the suffix was based on the well plate row and column. Cell IDs and well plate layout is given in the sample kit handbook https://www.qiagen.com/us/resources/download.aspx?id=28512222-986f-4027-bb45-2c7e65d3fd2b&lang=en .
  • QIAseq UPX 3′ Transcriptome Kit: The demultiplex step allows 1 mismatch per barcode. Previously, no mismatches were allowed.
  • RNA Stranded sample kits: On the Quality control tab of the Experiment summary and QC report, the metric ‘Reads after trim’ covers paired reads only. Reads from broken pairs are not counted as these are not used in downstream analysis.
• Data upload: Lower case file names are supported and will be appropriately grouped alongside upper case file names.
• Differential expression view
  • The Save and go to My projects functionality has been split in two. You now save your filter and send results to GeneGlobe My projects in separate steps.
  • On the heatmap, you can select whether you want to see sample and feature labels.
• Experiment summary and QC
  • The report was renamed from previously Sample and quality control.
• On the Project overview, if a project contains failed samples this is indicated in the Project details panel.
• Project and experiment view
  • Information on the Project view has been rearranged. If a project contains failed samples, the number of failed samples is posted both for the project as a whole and for the individual experiments.
  • On the experiment view, the analysis tiles were made smaller for better overview and navigation.
• Samples overview
  • Names in the Project column are now links. Click a project name to navigate to project view for that project.
  • Improved messaging for failed samples. A tooltip on failed samples will specify if the failure was caused by corrupt data or, for UPX sample kits that no reads have been assigned to the sample during the demultiplexing analysis step. The latter could indicate that the wrong wells were selected in the Align and count dialog.
• Create experiment dialog
  • The dropdown ‘While controlling for’ only allows selection of attributes that are independent of the attribute applied for ‘Test differential expression due to’. This restriction is imposed as the effects of dependent attributes cannot be estimated separately. Previously, selecting dependent attributes was allowed but resulted in an error.
• Link Support submit to existing support case. When you use the Share experiment with Support functionality, you can enter a support case number to link the experiment to an existing support case.
• On smaller screens, certain elements will be modified or reduced in size to improve navigation and optimize use of the limited screen real estate.
• Easier selection of samples. In the various dialogs, instead of having to click the checkboxes you can select samples by clicking anywhere in the respective rows.
• Scientific naming. References are named by their scientific name, for example homo sapiens instead of human.
• Ensembl IDs included in downloads. When downloading gene lists from differential expression and comparison views, or gene and transcript expression values from the Experiment summary and QC report, the downloads will contain the Ensembl IDs.
• For RNA sample kit data processed with the most recent analysis workflow version, the expression value downloads will contain genes and transcripts of all available biotypes as well as a biotype column. For older analysis workflows, only protein coding features are included.
• You find a link to the RNA-seq Analysis Portal Latest improvements by clicking the ‘i’ icon at the bottom of the user interface.

Bugfixes

• Fixed an issue where differential expression results could not be saved and sent to GeneGlobe if the raw p-value was selected for filtering instead of the default FDR p-value.
• Fixed an issue for the Comparison view where values on 3-circle Venn diagrams were overestimated. Genes found in more than one group would be counted towards multiple sections of the plot. As an example, a gene found in both group 1, 2, and 3 were included in the counts for both the appropriate intersection 1-2-3, as well as intersections 1-2, intersection 2-3, unique area 1, and unique area 2. Values on the plot legends were correct. 2-circle Venn diagrams were not affected.
• Fixed an issue with the Rat QIAseq miRNA Library Kit Quality control report where metrics for annotation with alternate databases piRNA, tRNA, rRNA, mRNA, snoRNA, and lnc_RNA/scRNA/ncRNA/snRNA were based on mapping to human sequences, not rat. The impacted QC tables were ‘Annotation records found’, ‘Unique search sequences’, and ‘Reads’. miRBase counts and therefore the downstream differential expression results for miRNA were not affected.
• Fixed an issue with the Create experiment dialog for projects with mixed Illumina sample kit samples. If a project contained both Illumina Stranded Total RNA Prep and TruSeq Stranded Total RNA Library Prep samples, in the Create experiment dialog the presented samples would sometimes be of the opposite Illumina sample kit type than what was selected in the sample kit dropdown.
• Fixed an issue where the ‘You have received one or more project copies’ notification pop-up would duplicate as you migrated between pages. As a result, if you wanted to close the pop-up before it disappeared automatically, you would need to click the ‘x’ multiple times.
• Fixed an issue on the Differential expression view where most entries in the main Biological Insights table would be missing once you had opened the expanded table by clicking the View details button.
• Fixed a bug where Venn diagram labels would contain the text “Fold change”.

 

QIAGEN RNA-seq Analysis Portal 2.0.1

Released December 2021

Improvements and bugfixes

• Improved performance of the Send copy of project functionality for larger projects.
• Fixed an issue with the Send copy of project functionality where case sensitivity on the recipient email address caused the project to not always show up in the recipient’s RNA-seq Analysis Portal account.
• Fixed an issue where Align and count analysis would fail for RNA Stranded data if ‘Spike-ins’ was selected.
• Fixed an issue for the QIAseq miRNA Library Kit Quality control report where the Summary column ‘UMI reads annotated’ listed the values for ‘Total UMI reads’ instead of ‘Total UMI reads annotated’.
• Fixed an issue where coloring on the volcano plot was opposite of what was indicated by the legend.
• Fixed an issue with the Quality control report for RNA sample kits where the Summary column ‘Total rRNA’ would incorrectly show ‘-%’ when Mt_rRNA reads, but no cytoplasmic rRNA reads were detected.

 

QIAGEN RNA-seq Analysis Portal 2.0

Released November 2021

New features and improvements

• Send copy of project. You can send a copy of a project including samples and experiments to colleagues. The copy will be an independent copy sitting in the recipient’s RNA-seq Analysis Portal account.
• Notification email when analyses are done. The system will notify you via email when an RNA-seq Analysis Portal job has finished, and when you receive a copy of a project from a colleague. You can opt out of these emails.
• Move samples. You can move samples between projects.
• Download expression values. You can download sample expression values from the Samples and Quality control report. For mRNA experiments, you will get counts, RPKM, and TPM values. For miRNA experiments, counts, CPM and IsomiR counts are available.
• Samples overview page
  • The Sample kit and Reference columns were replaced by one combined column: Sample kit analysis workflow. Apart from sample kit and reference info, this column displays the analysis workflow version number.
  • The Sample kit analysis workflow and Project columns support filtering. In the filter dropdown, you can enter text to limit the list of options. The Project column can be sorted.
• On the project view, the list of experiments is sorted by Created date, newest experiment on top.
• Data upload: Warnings relating to the selected fastq files are presented in a modal dialog to increase visibility, and messaging has been improved.
• In the Align and count dialog, you will get a warning if you are about to produce samples with same name as samples already in the selected project.
• In the Create experiment dialog, the Sample kit vendor, Sample kit, and Reference dropdowns will display only values that apply to the samples in the selected project. When only one value applies, this will be pre-selected.
• Differential expression view
  • You can choose between raw p-value and FDR p-value for filtering and display of the volcano plot.
  • If you apply a functional filter by selecting e.g. a canonical pathway, on the volcano plot items that are not associated with the pathway will be colored differently, leaving green-yellow colors only on the filtered feature subset.
• Samples and Quality report
  • The report will be generated also for experiments where all samples were successfully created, but the subsequent differential expression analysis failed.
  • The use of integers vs. decimal numbers has been improved, and values are now rounded correctly.
  • On the PCA plot you have the choice of turning labels on or off, and the plot scaling was improved.
• For miRNA samples, reference information will include the applied miRBase version.
• Support for copying text and table contents to the clipboard was improved.
• Various minor enhancements to the user interface.

Bugfixes

• Fixed an issue with the Differential expression view where applying a functional filter from the biological insights table would sometimes exclude too many genes from the feature table.
• Fixed an issue with the Comparison view where features with ‘-‘ values across the underlying differential expressions would be included in table and plot.
• Fixed an issue with the Comparison view where the Venn diagram circles would sometimes overlap. This could happen if the attributes applied for the experiment contained hyphens and gave rise to differential expression analyses names that were identical up until the first hyphen.
• Fixed an issue in the Create experiment dialog where the ‘Experimental setup (comparisons)’ dropdown did not allow you to select the enabled options if the attribute selected for ‘Test differential expression due to’ had 8 or more values.
• Fixed an issue in the Create experiment dialog where the ‘Select all’ functionality sometimes did not work as expected.
• Fixed an issue in the Create experiment dialog where experiments would sometimes fail if for the applied metadata the same attribute value was used for more than one attribute.
• Fixed an issue with visualization on the Samples and quality control report where a vertical scroll bar would be added and tables truncated even when not needed.
• Fixed an issue where “Share experiment with Support” would sometimes be enabled by mistake and result in an error if you attempted to use the functionality.
• Fixed an issue where “Share experiment with Support” would fail for large experiments.

 

QIAGEN RNA-seq Analysis Portal 1.1.1

Released August 2021

Improvements

• Fastq files up to 50 GB in size can be uploaded. The limit was previously 8 GB.
• The number of fastq files that can be uploaded at a time was increased from 50 to 500.
• The date format DD-MM-YYYY HH:MM is used consistently across the application.
• On the Samples page, the number of selected samples is indicated.
• Various minor user interface improvements.

Bugfixes

• Fixed an issue where dots in fastq file names would result in truncated sample names.
• Fixed an issue with permissions for BaseSpace sample data.
• Fixed an issue with the Align and count dialog were Created date was not displayed for BaseSpace sample data.
• Fixed an issue with the Align and count dialog well-picker (QIAGEN QIAseq UPX 3′ Transcriptome Kit) where check boxes for full rows and columns didn’t always work as intended.
• Fixed an issue for the Create experiment dialog where selections in step 2 would be deleted when going back to step 1.
• Fixed an issue for the Create experiment dialog where the warning message for missing attribute values was not always shown.
• Fixed an issue in the Create experiment dialog where attribute name uniqueness was not enforced.
• Fixed an issue where the volcano plot threshold lines would sometimes jump unintendedly.
• Fixed an issue where the functionality “Share experiment with Support” did not work if the experiment had been accessed through a direct link.

 

QIAGEN RNA-seq Analysis Portal 1.1

Released July 2021

New features and improvements

• The name of the application was changed to QIAGEN RNA-seq Analysis Portal.
• Support for additional RNA sample kits
  • TruSeq^® Stranded Total RNA Library Prep (Human/Rat, Gold, Globin), Illumina^®
  • Illumina^® Stranded Total RNA Prep with Ribo-Zero Plus, Illumina^®
  • NEBNext^® Ultra^TM II Directional RNA Library Prep Kit for Illumina, New England Biolabs^®
  • KAPA^® RNA HyperPrep Kit, Roche Sequencing solutions^®
  • SMARTer^® Stranded Total RNA Sample Prep Kit – HI Mammalian, Takara Bio^®
  • SMARTer^® Stranded Total RNA Sample Prep Kit – Low Input Mammalian, Takara Bio^®
  • Collibri^TM Stranded RNA Library Prep Kit for Illumina Systems, Thermo Fisher Scientific^®

   

• It is possible to Delete projects, experiments, analyses and samples.
• If you have questions relating to the results of a particular experiment and would like to share these results with QIAGEN Support, this is possible via the Help menu item Share experiment with Support.
• Analysis credits are required for running Align and count analyses. Analysis credits can be redeemed or purchased from the QIAGEN GeneGlobe Data Analysis Center. The number of available analysis credits is visible from the top of the Analysis Portal page.
• If you paste a QIAGEN RNA-seq Analysis Portal result direct link into your browser address line without being logged into My QIAGEN, you will be taken to the specified result upon login.
• Improved speed for sample data upload.
• Improved trimming for QIAseq Stranded mRNA Select/Total RNA Lib Kits, in particular for single read data sets. The Align and count analysis workflow now includes trimming of specific adapters. Previously, adapter trimming was limited to automatic read-through adapter trimming of paired data.
• Align and count dialog changes
  • A Sample kit vendor drop-down menu has been introduced.
  • The specific spike-in set supported for the individual sample kits is indicated.
  • The adapter list used for processing samples is now determined using a Custom R2 primer checkbox instead of a drop down menu of possible sequencing sources.
• Create experiment dialog changes
  • The sample selection section includes a ‘select all’ checkbox as well as a counter for number of samples selected.
  • Improved validation of the selected combination of sample grouping, attributes and experimental design.
• Samples and Quality control report changes
  • The Experiment summary tab displays the analysis workflow version.
  • On the Samples tab PCA plot, samples names are displayed as tooltips instead of labels on the plot, and the plot includes a legend.
  • On the Quality control tab, coloring of rows and number of values was adjusted for improved readability.
  • In the Quality control report summary section, the metric Mapped to rRNA was replaced by Mapped to total rRNA, which represents the sum of the corresponding cytoplasmic rRNA and mitochondrial rRNA (Mt_rRNA) values.
  • The Quality control tab will only display a ‘Spike-ins’ section if the ‘Spike-ins’ box was checked for ‘Align and count’ analysis of one or more of the samples.
• Differential expression view changes
  • The heatmap labels include attribute values and the plot legends have been enriched.
  • On the volcano plot, it is no longer possible to drag the vertical fold change bars across value zero.
  • Improved messaging for missing Biological Insight table results.

Bugfixes

• Fixed an issue where spike-in values would not be included in the Quality control report for QIAseq Stranded mRNA Select/Total RNA Lib Kit.
• Fixed an issue where colors of plots would sometimes change.
• Fixed an issue where project and experiment names could be left empty.

 

QIAGEN RNA Analysis Portal 1.0

Released May 2021

The first release of this application.