Introduction

Bases2Fastq Software processes sequencing data and converts base calls into FASTQ files. During a sequencing run, the Element AVITI™ System records base calls and associated quality scores (Q-scores) in bases files. Using Bases2Fastq, you can convert the bases files into the FASTQ file format for secondary analysis with the FASTQ-compatible software of your choice.

The software offers the following features:

• Demultiplexing libraries
• Paired-end and single-end adapter trimming, including the detection of adapter sequences
• Base masking to include or exclude cycles from FASTQ files
• Optional index sequence orientation detection
• HTML quality control (QC) reports that you can review in a browser
• Unique molecular identifier (UMI) support

Figure 1: Converting bases into FASTQ files

Software Workflow

Bases2Fastq operates off-instrument through a command-line interface (CLI). A command prompt executes the software. Bases2Fastq starts with demultiplexing, a process that 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 the execution uses the --detect-adapters optional argument. To support individually addressable lanes, automatic detection leverages data from each lane.

The trimming process depends on the adapter trimming settings in the run manifest. For single-end trimming, the software determines where the adapter starts by matching the expected adapter sequences to each position in a read. 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.

You can execute Bases2Fastq with the output run manifest from a sequencing run or a prepared corrected version. You can also use optional arguments in Bases2Fastq to override run manifest settings. For example commands, see Example Execution Commands.

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.