A run manifest is a comma-separated values (CSV) file that stores information for a sequencing run on the Element AVITI™ System. The file also supports the analysis of results after a run completes. Bases2Fastq Software uses a run manifest to convert the bases files into FASTQ files and demultiplex pooled libraries based on index sequences.
This documentation provides information on preparing a run manifest for sequencing and FASTQ file generation, including demultiplexing.
Every run is associated with a run manifest. The onboard instrument software, AVITI Operating Software (AVITI OS), either creates a default run manifest or uses an uploaded run manifest with index sequences.
After sequencing, AVITI OS outputs the run manifest to a run folder in the selected storage location. Run Bases2Fastq with either the output run manifest or a corrected version.
Figure 1: Steps to prepare a run manifest
Default Run Manifest
If you do not upload a prepared run manifest during run setup, AVITI OS creates a default run manifest based on the run parameters entered. A default run manifest does not contain index sequences and assigns all reads to one sample per lane during FASTQ generation.
Demultiplexing indexed libraries is not possible with a default run manifest. If sequencing indexed libraries, you must perform one of the following options for demultiplexing:
- Prepare and upload a run manifest with index sequences to the AVITI System.
- Create a corrected run manifest with all samples and the associated index sequences to use with Bases2Fastq after sequencing completes.
Corrected Run Manifest
To prepare a corrected run manifest, update the run manifest used for the sequencing run. Save it in a Bases2Fastq-accessible location.
The following cases require a corrected run manifest:
- You sequenced indexed libraries with a run manifest that does not include index sequences.
- During sequencing, you did not upload a run manifest with indexed samples. Use a corrected run manifest with all samples and their associated index sequences to demultiplex.
- A Bases2Fastq execution failed or reported incorrect results due to incorrect settings or indexes sequences in the run manifest, requiring reprocessing.
Library Prep Workflows
A run manifest supports the following library prep workflows. Metadata in the Settings and Samples sections can vary by library prep workflow. LoopSeq™ for AVITI libraries include Elevate™ indexes and adapters and therefore are considered Elevate libraries.
- 16S LoopSeq for AVITI
- Amplicon LoopSeq for AVITI
- Element Adept™ Library Compatibility Workflow
- Element Elevate Library Prep Workflow
For workflow guides, see User Documentation.
For templates to get started with a library prep, see the downloadable templates.
A run manifest file includes the following sections. Each section starts with a bracketed section heading followed by a row of column headings. Section and column headings are case insensitive. All subsequent rows list metadata for the section.