The demultiplex.nf pipeline demultiplexes a multiplexed FASTQ reads file using barcodes in a provided spreadsheet, and performs basic quality checks on the produced reads using FASTQC.
Required files are:
multiplexed_fastq- a FASTQ file containing reads from multiple samples, each with a barcode sequence at the start.annotation- a CSV or XLSX sheet of data, which needs at least a "Sample Name" column and a "5' Barcode" column.
If the annotation provided is an Excel spreadsheet, this process wil convert it to a CSV file.
Converts a full annotation spreadsheet to one containing just the columns that Ultraplex needs, mostly the barcode information.
Runs the Ultraplex demultiplexer to create multiple demultiplexed reads file from the multiplexed_fastq file, using the generated barcodes file. Output filenames will be the sample names from the spreadsheet with the ultraplex_demux_ prefix added. A log file will also be produced.
The barcode section at the start of the reads that Ultraplex looks for has 'N' bases at the start and end, and the specific barcode sequence in the middle. The barcode sequence is used to demultiplex, the start and end sections are combined and added to the reads header as an rbc, and the whole barcode section is removed.
The FASTQC program is run on all produced FASTQ files, which produces a HTML report of its quality, as well as a zip file of plain text quality information.
The subworkflow emits three output channels:
fastq- outputs a tuple of [reads_meta,reads files] one by one.fastqc_html- outputs a tuple of [reads_meta,fastqc_report] one by one.fastqc_zip- outputs a tuple of [reads_meta,fastqc_zip] one by one.