The demultiplexing step

This step allows to launch demultiplexing using bcl2fastq 2.x. Aozan can use samplesheets in XLS or XLSX format or generated by an external tool in addition of samplesheets in CSV format. Using a samplesheet in XLS or XLSX format allows to use aliases for adapter sequence indexes. A template of bcl2fastq samplesheet file in XLS format can be found Here.

If not using an external tool to build the samplesheet, the samplesheet must be created in a dedicated directory using a standardized name (e.g. samplesheet_SNL177_0153.xls where SNL177 is the name of the sequencer provided by the run id, and 0153 is the run number.

If no samplesheet file is provided in the dedicated directory, Aozan will use if it exists, the SampleSheet.csv file at the root of the run directory. The SampleSheet.csv file is created by the sequencer control software when the user provides a samplesheet at the run startup.

Alongside Aozan, we provide a standalone Bcl2fastq samplesheet validator available on our website that allows you to check your run samplesheet and generate a valid CSV samplesheet file. The validator is available here.

Configuration parameters

The following table contains the parameters for this step.

Aozan property Type Default value description
demux.step boolean True Enable the step string Not set Path of the email template for the end of the demultiplexing step.
demux.tool string bcl2fastq Tool to use for demultiplexing (available values: bcl2fastq or bclconvert)
demux.use.hiseq.output boolean False If this parameter is set to true and if the synchronization step is not enabled, this step will use directly the sequencer output for demultiplexage step double 0.7 Factor to use to get the estimation of the amount of space needed in the fastq directory for the run based on the amount of space used in the bcl directory by the run data
index.sequences string Not set Path to a file containing aliases for adapter sequence indexes. See an example
samplesheet.format string xls Bcl2fastq samplesheet format ("csv", "xls", "xlsx", or "command")
samplesheet.prefix string samplesheet Prefix for Bcl2fastq samplesheet file
samplesheet.generator.command String Not set Command to execute to generate the Bcl2fastq samplesheet in CSV format when the parameter bcl2fastq.samplesheet.format is set to "command"
samplesheet.path string Not set Path to CSV/XLS/XLSX samplesheet files. boolean false Allow "_" character in sample IDs.
samplesheet.allow.sample.ids.max.length boolean true Enable limit of 20 characters for sample IDs.
bcl2fastq.mismatches integer 0 Number of mismatches allowed in adapter indexes. This value is overrided by the MismatchCount setting in the samplesheet
bcl2fastq.adapter.fasta.file.path string Not set Adapter sequence file to use when bcl2fastq is launched
bcl2fastq.path string /usr/local/bcl2fastq Path to Bcl2fastq binary
bcl2fastq.with.failed.reads boolean True Add the reads that do not pass Illumina filter in the FASTQ files
bcl2fastq.additionnal.arguments string Not set Additional arguments for Bcl2fastq
bcl2fastq.threads integer Number of computer cores Number of threads to use for demultiplexing
bclconvert.path string /usr/bin/bcl-convert Path to BCL Convert binary

Configuring Bcl2fastq barcode mismatches in the samplesheet

With Aozan, you can define the number of barcode mismatches to use with Bcl2fastq by adding a entry named MismatchCount in your Settings section of the samplesheet. This value will override the bcl2fastq.mismatches value set in the Aozan configuration file.

Configuration required to run bcl2fastq or BCL Convert with Docker images.

This Aozan version can run Docker image for demultiplexing step. On our platform, we build a new image for each bcl2fastq new version. They can be downloaded from the genomic paris centre repository.

Aozan property Type Default value Description
bcl2fastq.use.docker boolean True Enable to use Docker container to run bcl2fastq for demultiplexing.
bcl2fastq.docker.image string Not set Docker image to use for bcl2fastq.
bclconvert.use.docker boolean True Enable to use Docker container to run BCL Convert for demultiplexing.
bclconvert.docker.image string Not set Docker image to use for BCL Convert.
docker.uri string unix:///var/run/docker.sock Docker deamon URI.

An example of email sent at the end of demultiplexing step.

  • Example on HiSeq 1500

    example email sent at the end of demultiplexing