AUGUSTUS can 1) integrate evidence from RNAseq to genome alignment and 2) predict atypical splice sites from evidence if you define the putative patterns when calling AUGUSTUS.

What type of organism are you working with? Is it something that doesn't currently have annotation data available, or is there some other reason you're trying to identify intron boundaries?

You could try something like BRAKER1:

https://github.com/Gaius-Augustus/BRAKER

But you need to provide the AUGUSTUS argument for the atypical splice site pattern to BRAKER (that is possible).

PASA (https://github.com/PASApipeline/PASApipeline) can used to predict gene model from RNA-seq/EST data, but RNA-seq raw reads should be assembled first by Trinity (https://github.com/trinityrnaseq/trinityrnaseq).

Dear Katharina Hoff Cameron J Grisdale Thank you, Your answers was helpful. I want to annotate splice site or intron of the novel species. For that species annotation is not available but it is known that these species predominantly contains non-cannonical splite site ( non-5' GT-3'AG). I have both raw and assembled RNA- and DNA-seq ? I would really appreciate If you guys have any more suggestion or comments ?

Hello Katharina Hoff Cameron J Grisdale ,

I have run command prothint to generate and prothint_augustus.gff used to run braker in next step. Along with bam file obtained from Star aligner using --twopassMode Basic

1.) prothint.py dj.fa protein_homo_database.fasta

2.) braker.pl --genome=genome.masked.fa --species=species_name --hints=prothint_augustus.gff --bam genomestaralign.bam --etpmode --softmasking –AUGUSTUS_ab_initio

genemark.gtf is present, however, augustus.gff file is missing and species folder is also empty.

Could you please suggest, where I am making mistake or it is fine ? Any suggestion, how can I improve the pipeline ?

Prasoon Thakur please make this an issue on Github at https://github.com/Gaius-Augustus/BRAKER for better visibility. I am afraid we are very slow and solving issues at the moment but having it there will help to keep track.

The BRAKER Readme.md file contains some hints at which files contain possible error messages. Please go throught these. The actual error message will be required in order to track this problem. We need to know where BRAKER died ;-)