7. Data Processing and Analysis

This section provides information to process your data using the Salmon Software Package (available online). The process begins with extracting reads from FASTQ files generated by an Illumina sequencer. Before proceeding, ensure that your sample information is entered correctly into the Illumina SampleSheet. The SampleSheet file must include all indexing details and the sample names associated with your experiment.

Convert Sequencing Data to FASTQ Format

1. To convert Illumina BCL intensity reads into FASTQ files, use the following command line with bcl2fastq:

$ bcl2fastq -R /path/to/BCL_folder --sample-sheet /path/to/Samplesheet/SampleSheet.csv --output-dir /path/to/output_folder --create-fastq-for-index-reads --ignore-missing-bcls --minimum-trimmed-read-length 0 --mask-short-adapter-reads 0 --no-lane-split --use-bases-mask=Y*,I10,I10,Y*

After the conversion is complete, the specified output directory (/path/to/output_folder) will contain a set of demultiplexed FASTQ files. These files will be used as input for the Salmon software and should resemble the following:

filename_S0_I1_001.fastq.gz
filename_S0_I2_001.fastq.gz
filename_S0_R1_001.fastq.gz
filename_S0_R2_001.fastq.gz

Make sure to verify that the output files match your experimental setup before proceeding with Salmon analysis.