Small RNA Pipeline

Running Small RNA pipeline on fourierseq

Inputs:  35 bp reads files

            filters file

            miRNA gff file

            reference genome

Example run is at /home/daras/small_rna/SA11009_miRNApanel/

1. Trim the 50 pb F3 reads to 35 bp

2. Make a directory for each sample.

3. Alter the config file : /home/daras/small_rna/SA11009_miRNApanel/config2.txt and place it in the directory.

i. If you want to run filtering, mirna matching and genome matching, set all three of these (RUN_FILTERING, RUN_MIRBASE_MATCHING RUN_GENOME_MATCHING) to true.
ii. Provide the miRNA gff file under miRBase_step_gff_reference_file. The first time you are running miRNA matching using this gff file, make sure MAKE_PRECURSOR_FASTA_REFERENCE is set to yes and provide a file name where you want to store the miRNA sequences under miRBase_step_reference_fasta_file. From the second time onwards, you don't have to create this file everytime.

When mapping against the miRNA panel, miRBase_step_gff_reference_file is set to /home/daras/small_rna/reference/hsa_mmu_rno_ambionrefpanel.gff_shorter and miRBase_step_reference_fasta_file is set to /home/daras/small_rna/reference/hsa_mmu_rno_ambionrefpanel_SREK.fa_shorter

iii. Provide the genome reference under genome_step_reference_fasta_file

4. Alter the run_small_rna.sh file : /home/daras/small_rna/SA11009_miRNApanel/bc01 and place it in the sample directory.
i. Provide location of config file
ii. Provide location of F3 cfsasta file
iii. Provide output directory

5. Run run_small_rna.sh

6. In the output folder created, under scripts, run in this order:
i. output_Filter_1.sh
ii. output_miRBase_1.sh
iii. All the output_chr.....sh files (one for each chromosome in the reference)
iv. output_POST_GENOME_MATCHING.sh

These need to run in this order because miRNA matching is done on the reads that passed the filter step, genome matching is done on the reads that passed the miRNA matching step etc.

7. The output will be under output/filter, output/miRBase and ouput/genome