Galaxy | Accessible Workflow | Human Splice DB / UMN

Human Splice DB Workflow

Galaxy Workflow ' Human Splice DB'

Annotation: Create a peptide fasta database with novel splice junctions that are inferred from RNAseq data

Step	Annotation
Step 1: Input dataset RNA-Seq left mate pair fastq select at runtime	RNA-Seq left mate pair fastq (These should be in fastqsanger format. If not, convert with "Fastq Groomer" tool.)
Step 2: Input dataset RNA-Seq right mate pair fastq select at runtime	RNA-Seq right mate pair fastq (These should be in fastqsanger format. If not, convert with "Fastq Groomer" tool.)
Step 3: Input dataset Reference Genome FASTA file select at runtime	GRCh37_canon.fa Contains only sequences from canonical chromosomes (chr1-22, X, Y, M)
Step 4: Select first Select first 100000 from Output dataset 'output' from step 1	Limit the sequence count for demonstration and testing purposes (100000)
Step 5: Select first Select first 100000 from Output dataset 'output' from step 2	Limit the sequence count for demonstration and testing purposes (100000)
Step 6: Tophat for Illumina RNA-Seq FASTQ file Output dataset 'out_file1' from step 4 Use a built in reference genome or own from your history Use a genome from history Select the reference genome Output dataset 'output' from step 3 Is this library mate-paired? Paired-end RNA-Seq FASTQ file Output dataset 'out_file1' from step 5 Mean Inner Distance between Mate Pairs 150 TopHat settings to use Full parameter list Library Type FR Unstranded Std. Dev for Distance between Mate Pairs 20 Anchor length (at least 3) 5 Maximum number of mismatches that can appear in the anchor region of spliced alignment 0 The minimum intron length 70 The maximum intron length 500000 Allow indel search Yes Max insertion length. 3 Max deletion length. 3 Maximum number of alignments to be allowed 20 Minimum intron length that may be found during split-segment (default) search 50 Maximum intron length that may be found during split-segment (default) search 500000 Number of mismatches allowed in the initial read mapping 2 Number of mismatches allowed in each segment alignment for reads mapped independently 2 Minimum length of read segments 25 Use Own Junctions Yes Use Gene Annotation Model Yes Gene Model Annotations select at runtime Use Raw Junctions No Only look for supplied junctions No Use Closure Search No Use Coverage Search No Use Microexon Search No	GTF-guided Tophat alignment. Allow for detection of splice junctions not in the GTF file.
Step 7: Tophat for Illumina RNA-Seq FASTQ file Output dataset 'out_file1' from step 4 Use a built in reference genome or own from your history Use a genome from history Select the reference genome Output dataset 'output' from step 3 Is this library mate-paired? Paired-end RNA-Seq FASTQ file Output dataset 'out_file1' from step 5 Mean Inner Distance between Mate Pairs 150 TopHat settings to use Full parameter list Library Type FR Unstranded Std. Dev for Distance between Mate Pairs 20 Anchor length (at least 3) 5 Maximum number of mismatches that can appear in the anchor region of spliced alignment 0 The minimum intron length 70 The maximum intron length 500000 Allow indel search Yes Max insertion length. 3 Max deletion length. 3 Maximum number of alignments to be allowed 20 Minimum intron length that may be found during split-segment (default) search 50 Maximum intron length that may be found during split-segment (default) search 500000 Number of mismatches allowed in the initial read mapping 2 Number of mismatches allowed in each segment alignment for reads mapped independently 2 Minimum length of read segments 25 Use Own Junctions Yes Use Gene Annotation Model Yes Gene Model Annotations select at runtime Use Raw Junctions No Only look for supplied junctions Yes Use Closure Search No Use Coverage Search No Use Microexon Search No	GTF-guided Tophat alignment. Do not allow for detection of splice junctions absent from the GTF file.
Step 8: Filter BED on splice junctions BED file Output dataset 'junctions' from step 6 reference bed file Output dataset 'junctions' from step 7 Extend the start position 66 Extend the end position 66	Filter out known splice junctions, thereby only keeping the novel ones.
Step 9: Extract Genomic DNA Fetch sequences for intervals in Output dataset 'novel_junctions' from step 8 Interpret features when possible No Source for Genomic Data History Using reference file Output dataset 'output' from step 3 Output data type Interval	Retrieve the DNA sequences for the novel splice junctions.
Step 10: Translate BED Sequences BED file with added sequence column Output dataset 'out_file1' from step 9 Genome reference name The SEQTYPE:STATUS to include in the fasta ID lines pep:splice Add the bed score field fasta ID line with this tag name depth Filter out translations with stop codons before the splice site Yes Stop codon filtering start position base pairs 66 Stop codon filtering end position base pairs 66 Trim translations to stop codons Yes Minimum length of a translation to be reported 10	Translate and output splice-junction peptide sequences from the DNA sequences.