SortmeRNA command of the latest version does not work

Question

[eimanpharmacist]

Hi,

I am new to sortmerna, and I need to use it to sort out rRNA and mRNA from a total RNA sequenced sample. I installed sortmerna using biconda and followed its posted tutorial (https://sortmerna.readthedocs.io/en/latest/manual4.0.html#databases)

According to the tutorial, I used the following command:

sortmerna \
  -ref ~/Documents/metagenomics/fourth_part/sortmerna/rRNA_databases_v4/smr_v4.3_default_db.fasta \
  -reads ~/Documents/metagenomics/fourth_part/sortmerna/sequences/B_rRNA_F 2.fastq \
  -reads ~/Documents/metagenomics/fourth_part/sortmerna/sequences/B_rRNA_R 1.fastq \
  -workdir ~/Documents/metagenomics/fourth_part/sortmerna/working_dir \
  -fastx fastq \
  -paired_in \
  -log \
  -a \
  -blast 1 \
  -num_alignments 1 \
  -v

I got this error:

[process:1393] === Options processing starts ... ===
Found value: sortmerna
Found flag: -ref
Found value: /home/raizada/Documents/metagenomics/fourth_part/sortmerna/rRNA_databases_v4/smr_v4.3_default_db.fasta of previous flag: -ref
Found flag: -reads
Found value: /home/raizada/Documents/metagenomics/fourth_part/sortmerna/sequences/B_rRNA_F of previous flag: -reads
Found value: 2.fastq

[process:1434] ERROR: the value provided without a flag/option. Note that e.g. '-ref' or '-reads' have to be used with Each file. See 'sortmerna -h'

I check sortmerna help and did not find any difference between my code and the tutorial.

Any assistance is much appreciated!

[eimanpharmacist]

One more question, how can I split the aligned.fastq file into forward and reverse sequence files for downstream analysis?

[GenoMax]

See: How To Separate Illumina Based Strand Specific Rna-Seq Alignments By Strand

Answer 1

[GenoMax]

B_rRNA_F 2.fastq

and

B_rRNA_R 1.fastq

Do you have a space in the file names? That will not work. You should change the spaces to an _ to keep the filename intact.

So rename B_rRNA_R 1.fastq to B_rRNA_R_1.fastq.

[eimanpharmacist]

Hello,

I did both, I removed fastq from the code and adjusted the sequence name by replacing spaces with underscores. Then, it works! Thanks so much for your help and suggestions.

[Ram]

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Answer 2

[Mensur Dlakic]

If you follow the order of statements in the printout, it gets to the error after reading the reference and reads files. Then you compare it to the manual where it says:

[-fastx]  Boolean  Output aligned reads into FASTA/FASTQ file

The Boolean argument is usually true or false, not fastq as you entered.