HISAT2 error running both --dta and --min and --max-intronlen

Question

[GLFR]

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

Answer 1

[lakhujanivijay]

This is strange, can you please try this?

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

Ofcourse, omitting --dta will affect the downstream processing. Check out the explanations:

1.    --dta/--downstream-transcriptome-assembly

Report alignments tailored for transcript assemblers including StringTie. With this option, HISAT2 requires longer anchor lengths for de novo discovery of splice sites. This leads to fewer alignments with short-anchors, which helps transcript assemblers improve significantly in computationa and memory usage.

2.     --dta-cufflinks

Report alignments tailored specifically for Cufflinks. In addition to what HISAT2 does with the above option (--dta), With this option, HISAT2 looks for novel splice sites with three signals (GT/AG, GC/AG, AT/AC), but all user-provided splice sites are used irrespective of their signals. HISAT2 produces an optional field, XS:A:[+-], for every spliced alignment