Introduction

Learn how to convert AVITI Dx System base calls into FASTQ files off-instrument for IVDR workflows with the Bases2FastqDx CLI.

During a run, the Element AVITI Dx™ System records base calls and associated quality scores (Q scores) in .bases files. Bases2FastqDx operates off-instrument through a command-line interface (CLI) and converts the bases files into the FASTQ file format for secondary analysis with the FASTQ-compatible software of your choice.

Note:

Bases2FastqDx converts sequencing output into FASTQ files for downstream analysis. In IVDR workflows, perform this step as part of the validated data processing pipeline and use only approved parameters.

For sequencing runs, analysis begins with demultiplexing, which identifies each sample by the index sequences and assigns polonies to that sample. If samples are not indexed, Bases2FastqDx skips demultiplexing and assigns all polonies to one sample. The software converts the demultiplexed bases into FASTQ files and generates one FASTQ file per read per sample.

Bases2FastqDx includes the following features for sequencing data:

• Demultiplexing: Identify sequencing libraries by index sequence and generate FASTQ files
• Native Adapter Trimming: Trim adapter sequences during FASTQ generation, including the automated detection of adapter sequences
• QC Report: HTML Quality control (QC) reports to summarize run and sample quality
• Unique molecular identifier (UMI) Generate UMI FASTQ files

Run manifest

A run manifest is a .csv file that specifies demultiplexing settings (when applicable), FASTQ file settings, and sample information for sequencing runs. By default, Bases2FastqDx uses the run manifest that the AVITI Dx System outputs into the run folder (RunManifest.csv).

You can execute Bases2FastqDx with the original run manifest or an alternate corrected manifest. Optional arguments provided at execution time override run manifest settings.

For more information on run manifests that includes preparation instructions for sequencing and use cases for a corrected run manifest, see the Run Manifest Documentation.

Adapter trimming

During sequencing library prep, Read 1 and Read 2 adapters are added to each sample. When the length of Read 1 or Read 2 exceeds the length of the DNA insert, the run sequences into the adapter. Adapter trimming removes the adapter sequences from the 3' end of each read to prevent adapter-based errors in certain analyses.

Run manifest settings enable adapter trimming and specify adapter sequences. When adapter trimming is enabled in the run manifest, Bases2FastqDx automatically detects and trims adapter sequences if the run manifest contains no adapter values or if the execution uses the --detect-adapters optional argument. Adapter sequence detection requires a passing filter (PF) rate threshold of > 70% to select reference regions during detection. If you are using the Individually Addressable Lanes add-on, automatic detection leverages data from each lane. Alternately, the adapter can be explicitly specified using the adapter values (R1Adapter and R2Adapter) in the run manifest.

Visual Representation of Trimming adapter sequences from Read 1 and Read 2

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

Paired-end versus single-end trimming

Bases2FastqDx includes paired-end and single-end adapter trimming. When a sample includes insertions and deletions (indels), the software accurately trims adapters that are as short as one base.

• For single-end trimming, the software determines where the adapter starts by matching the expected adapter sequences to each position in a read. Single-end adapter trimming individually processes each read, removing the adapter sequences without alignment.

• For paired-end trimming, the software considers data from the expected adapter sequences and compared Read 1 and Read 2 data to determine where the adapters start. Paired-end adapter trimming aligns the Read 1 and Read 2 inserts to accurately trim short adapters.

Paired-end adapter trimming is more accurate but requires that Read 1 and Read 2 each include at least 17 cycles. Single-end adapter trimming supports applications that do not meet this requirement. Neither type of adapter trimming increases the run time.

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

License agreement

Use of Bases2FastqDx is subject to the license agreement available at the Element Biosciences website.

Intended use

The AVITI Dx System product family delivers precise, high-quality raw DNA sequencing data from a variety of biological samples. The product is intended solely as a data generation tool and does not provide diagnostic interpretations or direct clinical recommendations.

For additional information about the intended use and regulatory information, see About Bases2FastqDx.