The following steps demonstrate how to generate a wiggle track from an RNASeq sample with Goby (this short demo assumes you have downloaded and installed Goby and bwa, for more information about each of these steps, see our detailed walk-through). For this brief example, we use the sample dataset included with the Goby distribution and chromosome chr1 from the UCSC MM9 assembly.

  • The input data files must be converted to the Goby compact format, one command for the reads and another for the reference dataset.
java -Xmx3g -jar goby.jar --mode fasta-to-compact data/reads/goby-mouse-reads-sample.fasta.gz
java -Xmx3g -jar goby.jar --mode fasta-to-compact --include-identifiers data/reference/mm9/chr1.fa.gz
  • The reference dataset is indexed with the following command

java -Xmx3g -jar goby.jar --mode align --aligner bwa --index --database-name chr1-index \
--reference data/reference/mm9/chr1.compact-reads --database-directory data/reference-index/mm9
  • We then align the reads to the reference with

java -Xmx3g -jar goby.jar --mode align --aligner bwa --search --database-name chr1-index \
                  --reference data/reference/mm9/chr1.compact-reads --database-directory data/reference-index/mm9 \
                  --reads data/reads/goby-mouse-reads-sample.compact-reads --basename goby-sample
  • Now count information can be produced from the alignment

 java -Xmx3g -jar goby.jar --mode alignment-to-counts goby-sample
  • And the counts converted to the wiggle format for visualization on the UCSC genome browser
  • java -Xmx3g -jar goby.jar --mode counts-to-wiggle goby-sample

Try uploading the file produced (i.e., goby-sample-all.wig.gz) to the UCSC Genome Browser.

The process illustrated above is the same with an entire genome sequence. Aligning color-space reads is a matter of specifying the color-space argument in the indexing and search steps. The aligner option allows to switch transparently to the last aligner.

If you prefer to generate histograms in the BED format, use the counts-to-bed mode instead of the counts-to-wiggle-mode.