SEQCOUNT ERROR:: system args failed: 65280

[Former user]

Hi everyone,

I was running a RNA-seq analysis test with miRNAs, everything was going fine but in the Readcount Analysis module, appears the following error "SEQCOUNT ERROR:: system args failed: 65280" and then appears different folder pathways:

(mkdir -p Examples/basic_examples/miRNAs/Known_miRNAs/results//cut_bw2_readcount_results/ ;featureCounts -s 1 -t miRNA -g transcript_id -T 4 -a Examples/basic_examples/miRNAs/Known_miRNAs/Arabidopsis_thaliana/Ensembl/TAIR10/Annotation/Genes/genes.gtf -o Examples/basic_examples/miRNAs/Known_miRNAs/results//cut_bw2_readcount_results/SRR3269277_cut_bw2.tab Examples/basic_examples/miRNAs/Known_miRNAs/results//Bowtie2_results/SRR3269277_cut_bw2.sam 2>> Examples/basic_examples/miRNAs/Known_miRNAs/results//miARma_logfile.8204.log) at lib/CbBio/RNASeq/Readcount.pm line 200, <STAT> line 336.

I'm new in bioformatics so I do not understand what I am missing. I would appreciate any help.

Regards!

[camilarm]

I am the one who wrote above

[Eduardo Andres Leon]

Hi ! Could you be so kind to attach the summary xls file created in the output_dir folder?

Thanks in advance

[camilarm]

Hi Eduardo,

This is my xls ouput file, there are only 2 fasta because it is just a test

[Eduardo Andres Leon]

Hi, Before checking anything else ,,, are you sure that the provided adapter sequence is correct ? Only 3.4 and 0.1% of the reads are processed. Due to tha,t most of your reads (79 and 99%) are discarded because of the size (too long reads).

If you check the alignment, only 750 and 50k reads are aligned so, I guess that the readcount fails because there is no enough read to count

Could you check the adapter sequence ?

Edu

[camilarm]

Ah, ok!

That may be the problem, because I assumed that library data was made with truseq small RNA kit, where exists two adapter sequences, and I had pick one out of that two. I could try with the other one and see what happens.

Thank you very much!