Association saturation mutagenesis¶
This workflow is about assocation variant calls with barcodes. Variants are introduced by an error-prone PCR. The workflow takes the sequencing of the region, with barcodes in index read and the reference sequence and maps the reads to the reference, calls variants and associates them with the corresponding barcode.
Input files¶
Fastq Files¶
3 Fastq files from library association sequencing –Reference sequencing, 1 forward and 1 reverse read –Barcode sequence, 1 read covering the barcode
Reference file¶
Fasta file of the referencesequence describing the mutated sequence
Example file:
>TERT
GATCTGCGATCTAAGTAAGCCCAGGACCGCGCTTCCCACGTGGCGGAGGGACTGGGGACCCGGGCACCCGTCCTGCCCCT
TCACCTTCCAGCTCCGCCTCCTCCGCGCGGACCCCGCCCCGTCCCGACCCCTCCCGGGTCCCCGGCCCAGCCCCCTCCGG
GCCCTCCCAGCCCCTCCCCTTCCTTTCCGCGGCCCCGCCCTCTCCTCGCGGCGCGAGTTTCAGGCAGCGCTGCGTCCTGC
TGCGCACGTGGGAAGCCCTGGCCCCGGCCACCCCCGCGAAAGCTTGCATGCCCTGCAGG
association_saturationMutagenesis.nf¶
Options¶
With --help or --h you can see the help message.
Usage:
- Mandatory arguments:
--fastq-insert Full path to library association fastq for insert --fastq-insertPE Full path to library association fastq for read2 --fastq-bc Full path to library association fastq for bc --design Full path to fasta of reference sequence (only one reference sequence) --name Name of the association. Files will be named after this. - Optional:
--bc-length Barcode length (default 15) --clipping-penalty bwa mem clipping penalty (default 80) --min-ireads minimum number gapped reads for indel candidates (default: 3) --split Split up the fastq read into chunks with max limit of reads (default: 2000000) --outdir The output directory where the results will be saved and what will be used as a prefix (default outs) --h, --help Print the help message
Processes¶
Processes run by nextflow in the Association Saturation Mutagenesis Utility.
- clean_design
- Removes any illegal characters (defined by Piccard) in the reference file.
- create_BWA_ref
- Creates a BWA reference based on the design file
- get_name
- Recieves the name written in the header of the reference fasta file
- create_BAM
- Merges the forward and reverse reads and stores them in BAM format. This is run for each chunk.
- collect_chunks
- combine the chunks (merge bams)
- PE_mapping
- Map the reads to the reference.
- get_count
- Get the barcodes and count for each barcode. Filter them
- extract_reads
- Create a BAM file for each barcode with the corresponding reads in it. This create a large number of files!
- call_variants:
- Call variants for each barcode seperately.
- combine_variants:
- Combine barcode/variant calls to one final output file.
Output¶
The output can be found in the folder defined by the option --outdir and the subfolder definedby option --name. It is structured in folders of the condition as
Files¶
- design_rmIllegalChars.fa
- Reference file with remove illegal characters
- ${datasetID}.variants.txt.gz
- Barcode to variant association. Named by the header in the reference.
- counts_${datasetID}.filtered.tsv.gz
- Filtered barcode counts. Named by the header in the reference.
- counts_${datasetID}.tsv.gz
- All barcode counts. Named by the header in the reference.