Hisat2 error with single strand (Error: Encountered internal HISAT2 exception (#1) )

Question

[Gaye]

Hi, I'm new to RNASeq pipeline and biostars. So I apologize in advance if I say anything incomplete or incorrect.

For C.elegans , after downloading the index file via the link and unzip/untar, I ran the .sh file with the bash command. Then I ran the hisat code below.

For the single strand:

hisat2 \
  -p 16 \
  --rg-id=SRR006519 \
  --rg SM:L3 \
  --rg LB:SRR006519-L3 \
  --summary-file SRR006519.txt \
  --rg PL:ILLUMINA \
  -x $RNA_REF_INDEX_NEW \
  --dta \
  --rna-strandness RF \
  -u $RNA_DATA_TRIM_DIR_NEW/SRR006519.fastq.gz \
  -S ./SRR006519.sam

For the reverse strand:

hisat2 \
  -p 16 \
  --rg-id=SRR360125 \
  --rg SM:L3 \
  --rg LB:SRR360125-DAUER \
  --summary-file SRR360125.txt \
  --rg PL:ILLUMINA \
  -x $RNA_REF_INDEX_NEW \
  --dta \
  --rna-strandness RF \
  -1 $RNA_DATA_TRIM_DIR_NEW/SRR360125_1.fastq.gz \
  -2 $RNA_DATA_TRIM_DIR_NEW/SRR360125_2.fastq.gz \
  -S ./SRR360125.sam

And I got the following error from both of them:

Error: Encountered internal HISAT2 exception (#1)

Then I tried to create index with hisat2-build by the following code:

hisat2-build -p 8 Caenorhabditis_elegans.WBcel235.86.fa Caenorhabditis_elegans.WBcel235.86

Here is the links for assembly and annotation files.

After re-running the same codes for single strand and reverse strand which is above, the code for reverse strand worked properly while getting the same error for single strand. I couldn't solve the problem, I would be grateful if you could help.

Answer 1

[GenoMax]

You need to use a capital U for single strand so change

-u $RNA_DATA_TRIM_DIR_NEW/SRR006519.fastq.gz

to

-U $RNA_DATA_TRIM_DIR_NEW/SRR006519.fastq.gz

[Gaye]

thank you very much everything is working fine now