A Practical Guide to Cancer Systems Biology

5. Transcriptomic Data Analysis 59


6. Click “Execute” button to start the job.


7. Repeat steps 1–7 for each set of pair-end data.

Assemble transcripts using Cufflinks

The read alignment files produced by TopHat or HISAT are provided to
Cufflinks to generate a transcriptome assembly and quantify the expression
level of each transcript fragment for each sample.^7

1. In the Tools panel, expand “NGS:RNA Analysis” and click “Cufflinks”.


2. In the “SAM or BAM file of aligned RNA-Seq reads”, use “Multiple
   datasets” and select the datasets produced by TopHat (accepthit) or
   HISAT.


3. Click “Execute” button to run the analysis.

Merge assembled transcripts and map to a reference
annotation using Cuffmerge

Because Cufflinks generates assembled transcripts for each sample, it is
required to merge them together using Cuffmerge. In addition, when a
reference genome annotation is available, Cuffmerge can integrate reference
transcripts into the merged assembly.

1. In the Tools panel, click “NGS:RNA Analysis” to expand tool menu.


2. Click “Cuffmerges”.


3. Set “GTF files produced by Cufflinks” to the “assembled transcripts”
   GTF file.


4. Click “Insert Additional GTF inputs” button for each additional “assem-
   bled transcripts” dataset.


5. Choose “Yes” for “Use Reference Annotation” and select the GTF
   annotation files exported from UCSC.


6. Click “Execute” button to run the analysis.

Differential expression analysis with Cuffdiff

Cuffdiff calculates expression in two or more samples and tests the statistical
significance of each observed change in their expression.^7

1. In the Tools menu, expand “NGS:RNA Analysis” and click “Cuffdiff”.


2. Set “Generate SQLite” to “Yes”. This file is used for visualizing the results
   of differential expression analysis.