Hello everyone, I have aligned my RNA seq paired read end by HISAT2 because Tophat is deprecated on the galaxy main server, then face this problem when try to assembly transcripts by cufflink depending on BAM file produced by HISAT2. return code = 1 Command line: cufflinks -q --no-update-check -I 300000 -F 0.100000 -j 0.150000 -p 10 -g /jetstream/scratch0/main/jobs/19270512/inputs/dataset_24819040.dat --3-overhang-tolerance 600 --intron-overhang-tolerance 50 -u -b /cvmfs/data.galaxyproject.org/byhand/galGal4/sam_index/galGal4.fa /jetstream/scratch0/main/jobs/19270512/inputs/dataset_24909996.dat [19:26:23] Loading reference annotation. [19:26:23] Inspecting reads and determining fragment length distribution. BAM record error: found spliced alignment without XS attribute BAM record error: found spliced alignment without XS attribute Any explanation will help more Fat
Tophat is a deprecated tool and should be avoided. HISAT2 is the replacement.
When using HISAT2, setting the strand and the alignment reporting type both need to be specified for the resulting BAM to be used with certain downstream tools (including Cufflinks/Cuffdiff).
How-to is covered in the Galaxy tutorials: https://galaxyproject.org/learn/
- RNA-seq: Discovering and quantifying new transcripts - an in-depth transcriptome analysis example. https://galaxyproject.org/tutorials/nt_rnaseq/
- Specifically here: https://galaxyproject.org/tutorials/nt_rnaseq/#spliced-mapping-with-hisat
For that tutorial, an output for Stringtie is chosen, but the option for Cufflinks is available in the same tool form section.
Thanks! Jen, Galaxy team