The Pipeline module of TrailmakerTM is available for the processing of FASTQ files generated using Parse Biosciences’ EvercodeTM technology. This FASTQ file processing module handles essential tasks such as barcode correction, read alignment, read deduplication, and transcript quantification. These quantified transcripts are then used to generate a cell-by-gene count matrix for each sample that is then used for downstream analysis.
Full instructions on how to set up a Pipeline Run are provided in the user guide. The goal of this article is to provide a brief guided walkthrough of the files and information you’ll need to start a Pipeline Run and to address the FAQs on Pipeline Run set-up.
What you need to set up a Pipeline Run
To set up a Pipeline Run in Trailmaker, you need the following information and files:
- Knowledge of the Evercode kit type and chemistry version
- Knowledge of how many sublibraries you want to run
- One or more pairs of FASTQ files (R1 and R2) per sublibrary from whole transcriptome and/or immune libraries
- A sample loading table Excel (.xlsm) or text (.txt) file in the Parse Biosciences official format that can be accessed by customers on our Support Suite or via the Sample Loading Table in Trailmaker
- For WT runs, knowledge of the reference genome
- For TCR/BCR runs, knowledge of the reference database
When a new Pipeline Run is created, our step-by-step wizard guides you through the information input and file upload process. Full instructions are available in the user guide. Once all required information and files have been input/uploaded successfully, the ‘Run the pipeline’ button becomes enabled and the run can be initiated.
FAQs on Pipeline set-up
Note that many of the articles linked below 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.
Q: Which Parse Biosciences kits are supported by Trailmaker’s Pipeline module?
A: The Trailmaker Pipeline module currently supports Evercode Whole Transcriptome, Evercode BCR and Evercode TCR data. The command-line Parse Pipeline can be used to analyze all Parse kits. Note that FASTQ files generated using other technologies are not supported by either Trailmaker or the Parse Pipeline.
Q: What is the difference between the command-line Parse Pipeline and Trailmaker’s Pipeline module?
A: Trailmaker Pipeline module runs the latest version of the Parse Pipeline. Running your Pipeline in Trailmaker or using the command line Pipeline will give identical results.
Q: My FASTQ files are big. What’s the best way to upload them to Trailmaker?
A: For speed and reliability, we recommend using the command line to upload FASTQ files to Trailmaker. Full instructions are provided in our article How to upload FASTQ files to Trailmaker using command line.
Q: Is there a limit on the size of FASTQ files that can be uploaded to Trailmaker?
A: The limit is approximately 5TB per FASTQ file. To date, no customer has come close to reaching this limit.
Q: What do I do if my genome of interest is not available in Trailmaker?
A: You can build your own custom genome in Trailmaker by following the instructions in our user guide. You'll need your reference genome FASTA and GTF annotation files. If you are working with mixed species, reach out to support@parsebiosciences.com for help.
Q: Where can I find out more about the technical details of the Parse Pipeline?
A: The Pipeline is available to customers via our Support Suite.
Q: How long will my pipeline take to run?
A: The duration of your Pipeline Run depends on the number of cells in your experiment as well as the sequencing depth. A typical WT Mini pipeline run time is 6-12 hours; for a WT kit it’s 12-24 hours; and for a WT Mega kit it could take 24+ hours. Immune profiling (TCR or BCR) runs with paired WT data will take longer than WT only runs.
Q: I don’t have data yet but I’d like to explore the Pipeline Output reports. How can I do this?
A: Pipeline output reports are available on the datasets page of our website. You can filter the datasets by product in order to see the type of reports that will be generated for your kit type.
Q: My Pipeline Run failed. What should I do?
A: The most common causes of Pipeline Run failures are explained in our article How to troubleshoot Pipeline failures in Trailmaker. If your pipeline failure error message is not covered in the article or you need further support, contact us at support@parsebiosciences.com.
Key links
- Access Trailmaker
- Trailmaker user guide
- Introduction to Trailmaker video
- Free Mastering Single Cell RNA-seq Data Analysis course