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Introduction

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.

Workflow Summary​

The onboard instrument software, AVITI Operating Software (AVITI OS), associates every run with a run manifest. AVITI OS either creates a default run manifest or uses a run manifest with index sequences that you upload during run setup.

After sequencing, AVITI OS outputs the run manifest to a run folder in the selected storage location. You can then run Bases2Fastq with either the run manifest output from AVITI OS or a corrected version.

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
  • A third-party workflow with a Cloudbreak Freestyle™ (FS) sequencing kit

For workflow guides, see User Documentation.

Use a Template

For templates to get started with a library prep, see the downloadable templates.

Manifest Sections​

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.

  • Samples: Identifies the libraries sequenced in a run and any corresponding index sequences.
  • Settings: Specifies settings for Bases2Fastq processes, such as adapter trimming and base masking for demultiplexing.
  • Run Values: Adds custom information to Bases2Fastq output so you can annotate the metadata.
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