A. Load the supplied example data

1. Go to http://galaxy.fml.mpg.de.
2. Open NGS: Quantitation Tools in the toolbar on the left.
3. Click on Examples and Instructions and choose either "Caenorhabditis elegans (one nGASP training region on chromosome I)" or "Caenorhabditis elegans (nGASP training regions)" as Example data.
4. Press "Execute" to load the example data. Then six new objects will appear in your history panel on the right.
• Example Data: Genome Sequence in FASTA format
• Example Data: Genome Annotation in GFF3 format
• Example Data: Alignments in SAM format
• Example Data: Transcript Profiles in tab limited format
• Example Data: Intron Distances in tab limited format
• Example Data: Information about the datasets

B. Convert SAM to BAM file

1. Click on SAM-to-BAM in NGS: SAM Tools. Select "History" as Source for the reference list. Select the Example SAM file (Example Data: Alignments in SAM format) as SAM file and the Example FASTA file (Example Data: Genome Sequence in FASTA format) as reference file.
2. Press "Execute" and wait until the result file is generated in your history panel.
• SAM-to-BAM

C. Run read statistic tool ReadStats

1. Click on ReadStats and select the Example GFF3 file (Example Data: Genome Annotation in GFF3 format) and your previously generated BAM alignment file from your history as input.
2. Press "Execute" and wait until the result text file is generated in your history panel.
• Read Statistic: The text file contains the number of reads in the alignment file, the read coverage of the given genes, the number of covered introns, and the intron coverage.

D. Run quantitation tool rQuant

1. Click on rQuant and select the Example GFF3 file (Example Data: Genome Annotation in GFF3 format) and your previously generated BAM alignment file from your history as input. For the option Transcript Profiles use either "No profiles" or "Load profiles" or "Learn Profiles". If "Load profiles" is selected, use the Example Data Profiles and Introns Distances as the respective input files.
2. Press "Execute" and wait until the result GFF3 file is generated in your history panel.
• rQuant Transcript Quantification: The GFF3 file contains the two additional attributes ARC and RPKM that describe the abundance of a transcript in ARC (estimated average read coverage) and RPKM (reads per kilobase of exon model per million mapped reads), respectively.