Workflow for The Drosophila Dicer-2 partner Loquacious-PD is specific to processing of hairpin- and 3' cis-NAT-derived siRNAs
Raw read libraries for each dataset should be stored in their directory under Input/Libraries.
Due to size constraints, With the exception of the Genome fasta file, General Feature Format (GFF) file and RepeatMasker output, all required input files for Bowtie Index generation are provided in Input/Downloads. Refer to Methods for more details on the required input files.
A single config file should be setup as Configfiles/main_config.yaml, which contains essential config parameters for the workflow. These parameters are used in the various Snakefiles and should be modified when appropriate. Further documentation can be found within the file itself.
All helper functions are stored in Scripts/Python/helpers.py. Further documention can be found within the file itself.
All plots are created by the R scripts stored in Scripts/R.
Both Figure-1 and Figure-2 are stitched together using patchwork.R, after creating the subplots using Main_figures.R, Main_figures2.R, hpRNA_figure.R, hpRNA_figure2.R.
Main_figures.R and Main_figures2.R are identical and are used to create the bar plots for counts across the various small RNA features. They should be run separately with the required paramters to generate the sub-plots for both GSE37443Head and GSE37443Ovary.
Similarly hpRNA_figure.R and hpRNA_figure2.R are identical and are used to create the bar plots for hpRNA features that are in Figure-1 and Figure-2.
TE_figure.R generates bar plots for Transposable Element features and Read_position.R generates the read position plots.
Detailed below is the order in which the Snakefiles are run. '' act as placeholders for required config parameters for each run of the Snakefile. More details on individual config parameters can be found within each Snakefile.
- Generate Bowtie indexes
snakemake -s Index_generation.Snakefile\ - Pre-process libraries
snakemake -s Okamuralib_preprocessing.Snakefile
snakemake -s Externallib_preprocessing.Snakefile --config dataset=''\ - Run Sequential Mapping and gather counts for each Bowtie mapping
snakemake -s Okamuralib_Sequential_mapping.Snakefile --config dataset='' runID ='' lengthfilter = '' minlength = '' maxlength= ''
snakemake -s GSE37443_Sequential_mapping.Snakefile --config dataset='' runID ='' lengthfilter = '' minlength = '' maxlength= ''\ - Run Multi-mapped Sequential Mapping
snakemake -s Multi_sequential_mapping.Snakefile --config dataset='' runID ='' minlength = '' maxlength= ''\ - Extract counts for each feature present in target bowtie output
snakemake -s Feature_counts.Snakefile --config dataset='' runID='' lengthfilter='' minlength='' maxlength=''\ - Extract read position output for each feature present in target bowtie output
snakemake -s Read_position.Snakefile --config dataset='' runID ='' lengthfilter ='' minlength = '' maxlength= ''\