Introduction

Bases2Fastq Software demultiplexes sequencing data and converts base calls into FASTQ files for secondary analysis with the FASTQ-compatible software of your choice. The Element AVITI™ System records base calls, which are the main output of a sequencing run, with associated quality scores (Q-scores) in bases files. Bases files must be converted into the FASTQ file format for secondary analysis.

The software includes the following features:

• Demultiplexing libraries
• Paired-end and single-end adapter trimming, including detection of adapter sequences
• Base masking that includes or excludes cycles from FASTQ files
• Index sequence orientation detection, which you can enable or disable
• Quality control (QC) reports that open in a browser
• Unique molecular identifier (UMI) support

Figure 1: Converting bases into FASTQ files

Software Workflow

Bases2Fastq runs off-instrument and has a command-line interface (CLI). A command prompt executes the software. Bases2Fastq starts with demultiplexing, which identifies each sample by the index sequence and assigns polonies based on the sequence. If samples are not indexed, Bases2Fastq skips demultiplexing and assigns all polonies to one sample. The software converts the demultiplexed bases into FASTQ files, generating one FASTQ file per read (e.g., Read 1 or Read 2) per sample.

Adapter Trimming

Bases2Fastq trims adapters during execution using the adapter values set in the run manifest (R1Adapter and R2Adapter) or through automatic detection. Automatic detection occurs when the run manifest contains no adapter values or if execution uses the --detect-adapters optional argument.

The trimming process depends on the adapter trimming settings in the run manifest. For single-end trimming, the software matches the expected adapter sequences to each position in a read to determine where the adapter starts. For paired-end trimming, the software considers data from the expected adapter sequences and matching Read 1 and Read 2 against each other to determine where the adapters start. If the run manifest contains no adapter information, the software automatically detects adapters using the best estimate from matching Read 1 and Read 2.

For information on the adapter trimming settings, see the Run Manifest Documentation.

Run Manifest

A run manifest is a comma-separated values (CSV) file that specifies demultiplexing settings, FASTQ file settings, and sample information. Bases2Fastq uses the run manifest to execute the software, by default accessing the output run manifest named RunManifest.csv in the run folder.

Execute Bases2Fastq with the output run manifest or a prepared corrected version. You can also use optional arguments in Bases2Fastq to override run manifest settings. For an example command for a corrected run manifest, see Use a Corrected Run Manifest.

The Run Manifest Documentation provides complete information on run manifests, including preparation instructions and use cases for a corrected run manifest.

License Agreement

Use of Bases2Fastq is subject to the license available at the Element Biosciences website.