Issues regarding processing_workflow.sh

[Gregory94]

This can be used for posting issues, suggestions and questions regarding the processing_workflow.sh file.

For questions on how to use the software, please read the help text.

This is a processing workflow designed for SAturated Transposon Analysis in Yeast (SATAY).

The program can be used by running the following command: bash [path]/processing_workflow.sh, where [path] is the path to the processing_workflow.sh file.

The program can trim sequencing reads, create quality reports of raw data and of the trimmed data, align the reads to a reference genome (S288C yeast genome, downloaded from the SGD) in .sam- and .bam-format, sort and index the bam file and perform transposon-mapping.
The following tools are used:

• quality report: FASTQC
• trimming: BBDuk or Trimmomatic
• alignment: BWA MEM
• create flagstat report after alignment: SAMTools
• sort and index .bam-files: SAMBamba
• transposon-mapping: Python3 together with custom python software

The program does not take inputs from the command line, except for the help text which can be accessed with the arguments --help or -h. It can handle both single-end data, paired-end data and paired-end interleaved data.

When the program is started, a window appears where the datafile(s) can be selected. The datafiles should be in fastq format and must have the extension .fastq or .fq. They can be either unpacked or gzipped (i.e. having the extension .gz). Select the right extension in the bottom right corner and navigate to the datafile(s). In case of single-end data or paired-end interleaved data select only one file. In case of paired-end data where the pairs are stored in two seperate files, select two files by holding ctrl-button and clicking the two files.

After pressing ok, a new window appears where some options and parameters can be selected.

• file primary reads and file secondary reads: Show the selected file(s). If only one file is chosen, the file secondary reads will show none.
• Data type: Select whether the reads are paired-end or single-end. If two data files were chosen but this setting is set to Single-end, the secondary reads file will be ignored.
• which trimming to use: Select whether to use bbduk or trimmomatic for trimming the reads or select Do_not_trim to prevent trimming of the reads.
• trimming settings: Input trimming settings. See the documentation of the selected trimming software which settings can be applied. When 'which trimming to use' is set to Do_not_trim, this field will be ignored. Sequences that need to trimmed (e.g. adapter or primer sequences) have to be entered in the adapters file which can be accessed using the Open adapters file button on the bottom of the window. NOTE 1: For bbduk do not input interleaved=t when using interleaved data. For trimmomatic do not input SE or PE to indicate single-end or paired-end data. This will all be automatically set depending on your selection in the Data type-field. NOTE 2: For trimmomatic, when using ILLUMINACLIP, do not specify the path to the adapters file as this is inserted automatically (see example settings below)."
  EXAMPLE SETTINGS:
• Trimmmomatic: ILLUMINACLIPPING:1:30:10 TRAILING:20 SLIDINGWINDOW:5:10 MINLEN:15
• bbduk: ktrim=l k=15 mink=10 hdist=1 qtrim=r trimq=10 minlen=30
• alignment settings: Input alignment settings. See the documentation of BWA MEM which settings can be applied. NOTE: Do not set -p for smart pairing (i.e. interleaved paired-end data). This will be automatically set depending on your selection in the 'Data type'-field. [http://bio-bwa.sourceforge.net/bwa.shtml].
• Quality checking raw data: Perform a fastqc quality check on the raw reads.
• Quality checking trimmed data: Perform a fastqc quality check on the trimmed reads. This setting is ignored if which trimming to use it set to Do_not_trim.
• Quality check interrupt: This quits the program after performing the quality report on the raw dataset and creating a temporary file with your settings. This allows you to check the quality report before continuing. To continue the program, restart the program (using bash processing_workflow.sh). It will automatically set the options you have chosen the first time, but these can be changed if this is necessary depending on the outcome of the quality report. This option can be useful if you have no idea how the dataset looks. This requires Quality checking raw data.
• Delete sam file: After alignment the .sam file is converted to its binary equivalent and only this .bam file is used for downstream processing. Since the .sam file typically requires a lot of memory, this is can be deleted. It is recommended to keep the .sam file only for manual checking te alignment results.
• Sort and index bam files.: This is needed for transposon-mapping and for many other downstream processes. It is recommended to always leave this on.
• Transposon mapping: This custom python script requires sorting and indexing of the .bam file and creates the following files:
  • .bed file: Creates list of all insertion locations with the number of reads in each location in bed-format.
  • .wig file: Creates list of all insertion locations with the number of reads in each location in wig-format. Small difference with the bed-file is that here reads from insertions at the same location but with different orientation are added up. In the bed-file these are regarded two separate insertions.
  • 4 .txt-files: List of all genes with the number of insertions and reads in each gene. The files are different in whether they show all genes or all annotated essential genes and whether they also show the distribution of insertions within the genes.
• Create flagstat report: Creates a flagstat report based on the .bam file.
• Open adapters file: Opens the text file where the adapter and primer sequences can be entered that will be trimmed. Enter the sequences in fasta format.

Questions, recommendations and issues can be noted here

Dependencies:

• fastqc
• bbmap
• trimmomatic
• bwa
• samtools
• sambamba
• python3
  • transposonmapping_satay.py
  • numpy
  • pysam
  • python_modules
  • data_files