Hello. I'm trying to use the miarma pipeline for a miRNAseq project, but when I tried to use the ReadCounts module it returned an error as if the program wasn't able to locate the sam files to use.

I'm not using a whole integrated pipeline but a step by step one. This is the INI file I created to run the ReadCounts module:

;General parameters [General] ; type of analysis (miRNA, mRNA or circRNA) type=miRNA ;0 for no verbose, otherwise to print "almost" everything verbose=1 ; Folder for miRNA reads read_dir=/home/rayner/smb4k/WD-MARCEL/usb/Emilio/results/alignment_results_run_concatenated/Bowtie2_results/ ; Number of process to run at the same time threads=16 ; label for the analsysis label=microRNA_project ; Folder where miARma has been instaled miARmaPath=. ; Folder to store results output_dir=/home/rayner/smb4k/WD-MARCEL/usb/Emilio/results/readcounts_results/ ; organism used organism=Sus scrofa ; Whether the data is from a strand-specific assay (yes, no or reverse, yes by default) for featureCounts analysis strand=yes stats_file=/home/rayner/smb4k/WD-MARCEL/usb/Emilio/results/readcounts_results//miARma_stat.17456.log logfile=/home/rayner/smb4k/WD-MARCEL/usb/Emilio/results/readcounts_results//miARma_logfile.17456.log

[ReadCount] ; GFF file used to calculate the number of reads in featureCounts analysis database=/home/emilio/NGS/Genome_Reference/Sus_scrofa/Ensembl/Sscrofa10.2/Annotation/Archives/archive-2015-07-17-14-37-09/SmallRNA/ss c.gtf ;GFF attribute to be used as feature ID (default: gene_id) for featureCounts analysis seqid=ID ; Quality value to avoid counting low quality reads quality=10 ;Feature type (3rd column in GFF file) to be used, all features of other type are ignored (default:exon) for featureCounts analysis featuretype=miRNA

I realised that when using the aligner module, the files created are in sam format, not in bam format, although the script creates some files ending in .bam. I also created bam formats from each sam by using samtools view -bS

When running this script.ini to create the readcounts table, the program returns an error:

###################################################################

miARma, miRNA and RNASeq Multiprocess Analysis

miARma v 1.5 (Apr-2016)

Created at Computational Biology and Bioinformatics Group (CbBio)

Institute of Biomedicine of Seville. IBIS (Spain)

Copyright (c) 2016 IBIS. All rights reserved.

mail : miARma-devel@cbbio.es

[Mon Sep 5 16:23:13 2016] Starting a miARma analysis for miRNA [Mon Sep 5 16:23:13 2016] Checking provided parameters for: ReadCount. [Mon Sep 5 16:23:13 2016] Checking General-output_dir parameter ... Exists! [Mon Sep 5 16:23:13 2016] The folder specified in (output_dir=/home/rayner/smb4k/WD-MARCEL/usb/Emilio/results/readcounts_results/) already exists. [Mon Sep 5 16:23:13 2016] Wait 5 seconds to overwrite the folder. Cancel otherwise: 0 [Mon Sep 5 16:23:19 2016] Continue. [Mon Sep 5 16:23:19 2016] All parameters are correct. ERROR :: You are requesting a miRNA readcount analysis, but no aligned files are found (Neither Bowtie1 nor Bowtie2)

Mandatory parameters:

[ReadCount] database=miRNAs_miRBase20.gft

for circRNAs: [ReadCount] database=HS_nbci_37.2.gft bwaindex=index/bwa emilio@pc269:~/NGS/miarma$ gedit readcounts.ini

It seems to me that the program is not recognizing any sam/bam format in the specified read-dir path in the configuration .ini file.

I also found intriguing that in the examples, the read-dir variable for ReadCounts module redirects to the initial raw reads instead of the aligned sam files after bowtie alignment. As far as I'm concerned, I should put the path to alignment results in the read-dir variable to execute the ReadCounts module, but as seen above, it seemed to not recognize any file to process...

Problem with ReadCounts module