<p>According to the help produced when I type <code>hisat2 --help</code>, the default strandedness when running HISAT2 is unstranded:</p>
<pre><code>  --rna-strandness &lt;string&gt;          specify strand-specific information (unstranded)
</code></pre>
<p>So taking out the <code>rna-strandedness</code> option should map the reads as if they were unstranded:</p>
<pre><code># 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
</code></pre>
<p>If this is a problem for you, I recommend you create an issue about it on the HISAT2 github page:</p>
<p><a href="https://github.com/DaehwanKimLab/hisat2/issues/new" rel="nofollow noreferrer">https://github.com/DaehwanKimLab/hisat2/issues/new</a></p>

CC BY-SA 4.0
2023-11-03T18:13:43.403Z
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.
Ram RS
CC BY-SA 4.0
2023-11-03T03:58:40.420Z
2023-11-04T04:31:59.240Z
2023-11-04T04:31:59.240Z
gringer
<p>My library is unstranded and the code I'm trying to use is this:</p>
<pre><code># 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 &quot;HISAT2 finished running!&quot;
</code></pre>
<p>However, I get this error:</p>
<pre><code>(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 &quot;-&quot;
HISAT2 finished running!
0 minutes and 0 seconds elapsed.
</code></pre>
<p>Looking at the HISAT2 Manual:</p>
<p>For single-end reads:</p>
<blockquote>
<p>Use 'F' if the read corresponds to a transcript.<br />
Use 'R' if the read corresponds to the reverse complemented counterpart of a transcript.</p>
</blockquote>
<p>For paired-end reads:</p>
<blockquote>
<p>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.<br />
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.</p>
<p>When you use the <code>--rna-strandness</code> 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.</p>
</blockquote>
<p>It doesn't say what to input in the case of an unstranded library. Any help would be greatly appreciated!</p>

<rna-seq><rna><rna-alignment><strandedness>
HISAT2: RNA strandedness
# HISAT2: RNA strandedness

(Asked by: username_0 on 2023-11-02)

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!

**Tags:** rna-seq, rna, rna-alignment, strandedness

---

## 1 answers

### Answer by username_1 on 2023-11-03. Score: 2

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

> **Comments:**
> **username_2**: 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.  

---

