For successful Pipeline Runs in TrailmakerTM, the Pipeline Outputs page displays the reports for you to explore, allows you to download the various different output files, and provides a link to the downstream analysis in the Insights module.
Detailed explanations of each of these components of the Pipeline Outputs page are provided in the user guide. The goal of this article is to provide a brief guided walkthrough of the Pipeline Outputs page features, and to provide guidance on how to resolve failed Pipeline Runs.
Pipeline Outputs page for successful Runs
Successful pipeline runs will display the reports in the Pipeline Outputs tab for you to explore. The “all samples” report is shown by default. You can choose to view the reports for individual samples using the dropdown menu at the top of the page.
In the barcode rank plot, you’re looking for a clearly defined ‘knee’ with the threshold in the steepest part of the drop. This threshold is dynamically set and is likely to be different for different samples. The slope of the curve at the cutoff is, more of than not, correlated with sample quality. Steeper slopes tend to reflect a clean cutoff between healthy cells and barcoded “background” transcripts. More details on barcode-rank plots can be found in the article How are cell counts determined? (available to customers only).
The QC metrics include the estimated number of cells as well as the median number of genes and transcripts per cell. These metrics can be compared across samples, and can be considered in the context of published data or your previous experiments.
Further metrics are available in the csv file that can be downloaded in the "Combined reports" option.
The plate heatmaps underneath the plots display transcripts and cells per well and are useful for catching pipetting and plate loading errors. Ideally, you'd like to see a homogenous distribution across the plates with no streaks or outliers.
The Pipeline version is stated at the bottom of the Pipeline Outputs page. We recommend that you cite the version as well as Trailmaker in all publications. See our article on How to use and cite Trailmaker in a publication for guidance.
The pipeline outputs are available to download from the Pipeline Outputs page.
- The count matrices can be found in the “Unfiltered matrices” and “Filtered matrices” options. These are useful if you choose to perform downstream analysis outside of Trailmaker. Note that the filtered matrices expire 30 days after creation.
- Unfiltered matrices: This matrix provides a more inclusive dataset for analysis with minimal initial filtering. Barcodes with fewer than 10 transcripts are filtered out, but no other filtering parameters are applied. Trailmaker Insights module automatically uses these unfiltered matrices for downstream analysis.
- Filtered matrices: Further filtered based on the threshold determined from the barcode-rank plot in the Pipeline outputs tab of the Pipeline module. Note that the same threshold is applied in the Data Processing module of Trailmaker Insights. For this reason it is not recommended to use the filtered matrix for analyses in Trailmaker Insights.
- The “Combined reports” option allows you to download the all_summaries.zip file which contains the html reports, QC metrics (as a csv file) and log files that are output from Parse’s pipeline (also known as split-pipe) combine mode.
- The “Sublibrary reports” option allows you to download the html reports, QC metrics (as a csv file) and log files for each independent sublibrary within your pipeline Run.
- The “All files” option contains the full pipeline output, including the alignment BAM files. Downloading the “All files” option might take a long time for large datasets. For users who are comfortable with the command line, the “All files” option can be downloaded by copying the download command. Note that the “All files” download option expires 30 days after the creation of the pipeline outputs following a successful pipeline run.
Detailed explanation of the pipeline output files is available in this article on our support suite. Our article on Content of FASTQ files and BAM files may also be useful. These articles are available to customers only.
The unfiltered count matrices generated by successful pipeline runs are automatically sent to the Insights module for downstream analysis and visualization. Simply click the ‘Go to Insights downstream analysis’ button to navigate to the Data Processing page of the Insights module where you can begin to deep dive into your dataset.
Pipeline Outputs page for failed Runs
Failed pipeline runs give the option to download the logs for troubleshooting purposes:
The most common causes of Pipeline Run failures are explained in our article How to troubleshoot Pipeline failures in Trailmaker (available to customers only). If your pipeline failure error message is not covered in the article or you need further support, contact us at support@parsebiosciences.com.
Note that many of the support articles linked from this article are available to Parse Biosciences customers only. If you are a current customer, and do not have access to the support suite, please fill out your contact information in the Pipeline Download Request Form for access.
Key links
- Access Trailmaker
- Trailmaker user guide
- Introduction to Trailmaker video
- Free Mastering Single Cell RNA-seq Data Analysis course