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=====================================================
Information about miRNA-seq profiling pipeline
=====================================================
Pipeline uses miRDeep2 and/or mirAligner to annotate and quantify human miRNAs
Dependencies for miRpipe:
1) miRDeep2 from: https://github.com/rajewsky-lab/mirdeep2
2) mirAligner from: https://github.com/lpantano/seqbuster/tree/master/modules/miraligner
1) Bioperl from: http://www.bioperl.org/wiki/Installing_BioPerl_on_Ubuntu_Server
2) Blast+ from: ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/
3) Blast from: ftp://ftp.ncbi.nlm.nih.gov/blast/executables/LATEST/
4) EDirect from: http://www.ncbi.nlm.nih.gov/books/NBK179288/#chapter6.Examples
Prepare databases and references for miRpipe
=====================================================
To prepare all needed databases and references for the pipeline USE update_db.sh script which updates the databases used in IKMB miRNA pipeline.
Script generates directory (in your current working directory) which has the following structure and files (||- shows from where files were downloaded or how they were generated):
|--databases_date
| |
| |-- human_RNA-- |
| | |-- Rfam.fa ||- ftp://ftp.ebi.ac.uk/pub/databases/Rfam/CURRENT/fasta_files/*.fa.gz
| | |-- Rfam.db.* ||- formatdb -i virus_hairpin.fa -pF -oT -n virus_hairpin.db
| |
| |
| |-- viral_RNA-- |
| | |-- viral_genome_RefSeq.fa ||- esearch -db nucleotide -query "Viruses [PORG] AND srcdb_refseq [PROP] AND complete genome [WORD]"
| | |-- viral_genome_RefSeq.db* ||- formatdb -i viral_genome_RefSeq.fa -pF -oT -n viral_genome_RefSeq.db
| | |
| | |-- virus_hairpin.fa ||- ftp://mirbase.org/pub/mirbase/CURRENT/hairpin.fa.gz
| | |-- virus_hairpin.db.* ||- formatdb -i virus_hairpin.fa -pF -oT -n virus_hairpin.db
| |
| |
| |-- mature-- |
| | |-- hsa_mature_mirbase_vX.fa ||- ftp://mirbase.org/pub/mirbase/VERSION_X/mature.fa.gz
| | |-- hominidae_mature_mirbase_vX.fa ||- ftp://mirbase.org/pub/mirbase/VERSION_X/hairpin.fa.gz
| |
| |
| |-- hairpin-- |
| | |-- hsa_hairpin_vX.fa ||- ftp://mirbase.org/pub/mirbase/VERSION_X/hairpin.fa.gz
| |
| |
|
==================================================================================
Structure of miRpipe
==========================================
miRpipe.sh is the main script of the pipeline, please add necessary paths inside the script before starting to run the pipeline.
miRpipe.sh calls scripts in the following order (only 3 levels are shown):
|-- miRpipe.sh
| |
| |-> process_reads.sh
| |
| |-> cutadapt
| |-> fastq2fasta.pl
| |-> collapse_reads_md.pl
|
|
|-> filter_reads.sh
| |
| |-> find_read_count.pl
| |-> blastn
| |-> fastacmd
|
|
|---|--> run_miraligner.sh
| | |-> miraligner.jar
| | AND/OR
| |
| |--> run_quantifier.sh
| |-> quantifier.pl
|
|
|-> predict_reads.sh
| |
| |-> mapper.pl
|-> miRDeep2.pl
For further explanations and options of the pipeline, please call 1st and 2nd level scripts by using -h option e.g. "miRpipe.sh -h".
Please describe the options of 2nd level scripts inside the miRpipe.sh script. Otherwise the default options will be used.
-------------------------------------------------------------------------------------------------------------------------------------------
Input and output stucture for miRpipe
==========================================
example of path to the imput files: /home/user/samples/project_ID_samples/
Please make sure that your provided samples are arranged in the following structure:
|-- project_ID_samples
| |
| |-- run_ID_samples
| |
| |-- sample_1_ID
| | |
| | |-- sample_1.fastq.gz
| |
| |-- sample_2_ID
| | |
| | |--sample_2.fastq.gz
| |
------------------------------------------------------------
example of path to the output files: /home/user/samples/project_ID_results/
For the output files you only need to make a directory "project_ID_r_outesults" and the following directories will be generated automatically:
|-- project_ID_results
| |
| |-- run_ID_results
| |
| |-- sample_1_ID
| | |
| | |-- miRpipe_out_date
| | |
| | |-- dir_blastn_date (optional, output of filtering step)
| | |-- dir_mapper_date (optional, output of mirdeep2 prediction step)
| | |-- for_general_statistics
| | |-- expression_analyses (optional, output of quantifier.pl)
| | |-- mirdeep_runs (optional, output of mirdeep2 prediction step)
| | |-- reads.fa
| | |-- reads_colapsed.fa
| | |-- reads_filtered.fa (optional, created in filtering step)
| | |-- reads_collapsed_vs_genome.arf (optional, output of mirdeep2 prediction step)
| | |-- miRNAs_expressed_all_samples_*.csv (optional, output of quantifier.pl)
| | |-- isomir_sampleID.mirna (optional, output of miraligner.jar)
| | |-- other additional files
| |
--------------------------------------------------------------------------------------------
==============================================================================================