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My library is unstranded and the code I'm trying to use is this:

# Run alignment with unstranded method
hisat2 -q --rna-strandness none -x HISAT2/grch38/genome_index_grch38 -U hipsci/Kolf-1_R1_001.fastq | samtools sort -o HISAT2/Kolf-1_R1_001.bam

echo "HISAT2 finished running!"

However, I get this error:

(base) ~ % RNASeq_pipline/data/script/RNASeqpipline.sh

Error: should be one of F, R, FR, or RF 
Error: Encountered internal HISAT2 exception (#1)
Command: /usr/local/bin/hisat2-align-s --wrapper basic-0 -q --rna-strandness none -x 
HISAT2/grch38/genome_index_grch38 -U hipsci/Kolf-1_R1_001.fastq 
(ERR): hisat2-align exited with value 1
[W::hts_set_opt] Cannot change block size for this format
samtools sort: failed to read header from "-"
HISAT2 finished running!
0 minutes and 0 seconds elapsed.

Looking at the HISAT2 Manual:

For single-end reads:

Use 'F' if the read corresponds to a transcript.
Use 'R' if the read corresponds to the reverse complemented counterpart of a transcript.

For paired-end reads:

Use 'FR' if the first read in the pair corresponds to a transcript on the forward strand, and the second read corresponds to the reverse strand.
Use 'RF' if the first read in the pair corresponds to a transcript on the reverse strand, and the second read corresponds to the forward strand.

When you use the --rna-strandness option with either 'FR' or 'RF' for paired-end reads, HISAT2 will assign an XS attribute tag to each read alignment, indicating whether the read belongs to a transcript on the '+' (plus) or '-' (minus) strand of the genome.

It doesn't say what to input in the case of an unstranded library. Any help would be greatly appreciated!

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1 Answer 1

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According to the help produced when I type hisat2 --help, the default strandedness when running HISAT2 is unstranded:

  --rna-strandness <string>          specify strand-specific information (unstranded)

So taking out the rna-strandedness option should map the reads as if they were unstranded:

# Run alignment with unstranded method
hisat2 -q -x HISAT2/grch38/genome_index_grch38 -U hipsci/Kolf-1_R1_001.fastq | samtools sort -o HISAT2/Kolf-1_R1_001.bam

If this is a problem for you, I recommend you create an issue about it on the HISAT2 github page:

https://github.com/DaehwanKimLab/hisat2/issues/new

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  • $\begingroup$ Kind of a bad move to not have a way of explicitly recreating the default behavior. There should be an option you can pass to rna-strandness that replicates not specifying that parameter at all. The usage reads like specifying --rna-strandness unstranded will work but it exits if the arg is not one of those 4 allowed values. $\endgroup$
    – Ram RS
    Nov 3, 2023 at 18:13

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