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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. Bases2Fastq converts 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
  • Quality control (QC) reports in an HTML format
  • Unique molecular identifier (UMI) support

Figure 1: Converting bases into FASTQ files

Bases2Fastq

Execution Environments​

Bases2Fastq software can execute in local or cloud compute environments.

The following options and resources for cloud-based execution are available:

Software Workflow​

The local version of Bases2Fastq operates off-instrument through a command-line interface (CLI). Bases2Fastq starts with demultiplexing, which identifies each sample by the index sequences and assigns polonies to that sample. 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.

Run Manifest​

A run manifest is a comma-separated values (CSV) file that specifies demultiplexing settings, FASTQ file settings, and sample information. By default, Bases2Fastq uses the run manifest that the AVITI System outputs into the run folder (RunManifest.csv).

You can execute Bases2Fastq with the original 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 more information, see --run-manifest and --settings on the Optional Arguments page.

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

Adapter Trimming​

Bases2Fastq trims adapters using the adapter values (R1Adapter and R2Adapter) and trimming settings in the run manifest or through automatic detection. Automatic detection occurs when the run manifest contains no adapter values or the execution uses the --detect-adapters optional argument. The detection uses a passing filter (PF) rate threshold of > 70% to select reference regions. If you are using the Individually Addressable Lanes add-on, automatic detection leverages data from each lane.

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 comparing Read 1 and Read 2 to determine where the adapters start. If the run manifest contains no adapter information, the software automatically detects adapters using the best estimate from comparing Read 1 and Read 2.

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

License Agreement​

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

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