Hello,

I'm currently using trying hisat2-2.0.5 to align RNA-seq reads to the A.thaliana genome, however, I'm running into a couple of problems. Whilst it runs well using default parameters, when I try to include options, depending on which combination I used I encounter some errors.

If I include only the --dta option the program runs just fine. However, if I try to run --dta and --min-intronlen and --max-intronlen I get the following error:

"Extra parameter(s) specified: "--dta" Note that if <mates> files are specified using -1/-2, a <singles> file cannot also be specified. Please run bowtie separately for mates and singles. Error: Encountered internal HISAT2 exception (#1)"

Example input for both:

These run:

hisat2 --dta -x path_to_index -1 S3_R1.fq -2 S3_R2.fq -S S3.sam

or

hisat2 --min-intronlen 40 --max-intronlen 2000 -x path_to_index -1 S3_R1.fq -2 S3_R2.fq -S S3.sam

This does not:

hisat2 --min-intronlen 40 --max-intronlen 2000 --dta -x path_to_index -1 S3_R1.fq -2 S3_R2.fq -S S3.sam

As the default settings for min and max intron length really aren't suitable for A.thaliana I have to change them, but this means I cannot run the --dta option without returning the above error.

Could anyone suggest why the error is happening and how I can fix it? If not, will omitting the --dta option impact my downstream analysis (stringtie and ballgown)?

Many thanks for your help as always