selecting Advanced Options. Execute the alignments by clicking the Execute bottom. A list of read alignments (BAM files)
and tracks of junctions (BED files) are produced as output files.
6. Assemble and measure the relative transcript abundance using
the Cufflinks tool located on the tools panel, Genomic Analysis
section, folder RNA-seq. Use the BAM output file of TopHat as
Input file. Use the reference annotation file in GFF3 format as a
guide of the assembly process. If your reads are from a stranded
library, choose the appropriate setting under Specify strand
information. The output includes all expressed reference transcripts and any novel transcripts and splice isoforms that are
assembled based on TopHat read alignments. Cufflinks produces three outputs: a GTF file containing the assembled transcripts and two files containing the estimated level of expression
for each transcript and gene, respectively.
7. Merge assemblies using Cuffmerge tool located on the tools
panel, Genomic Analysis section, folder RNA-seq. Use the
GTF output files of Cufflinks and the GFF3 reference genome
annotation file. Cuffmerge merges annotation files to include
novel genes or alternative splicing isoforms that were not
included in the reference genome annotation and produces a
GTF output file that contains an assembly that merges together
the input assemblies.
8. Find significant changes in transcript expression, splicing, and
promoter usage between two conditions using the Cuffdiff
tool located on the tools panel, Genomic Analysis section,
folder RNA-seq. Use the GTF file containing the merged
assemblies produced by Cuffmerge and the BAM output files
of TopHat for the samples. Cuffdiff produces output files such
as transcript and gene FPKM expression tracking, transcript
and gene differential FPKM, and differential splicing and promoter use tests.
9. Visualize the read alignment at the genome level using the
Integrative Genomics Viewer (IGV). This software can be
downloaded from the website https://software.bro
adinstitute.org/software/igv/download. IGV allows the visualization of TopHat outputs, such as read alignment data (BAM
files) and track intron/exon junction data (BED files) as well as
a wide variety of data types.
(a) If the genome is not included in the software database,
create a genome file using the menu Genomes, Create
genome file.
(b) Upload the genome using the menu Genomes, Load
genome from file, or use the menu Load genome from
URL if it is available online.
TRAP-SEQ Analysis of Long Noncoding RNAs
465
and tracks of junctions (BED files) are produced as output files.
6. Assemble and measure the relative transcript abundance using
the Cufflinks tool located on the tools panel, Genomic Analysis
section, folder RNA-seq. Use the BAM output file of TopHat as
Input file. Use the reference annotation file in GFF3 format as a
guide of the assembly process. If your reads are from a stranded
library, choose the appropriate setting under Specify strand
information. The output includes all expressed reference transcripts and any novel transcripts and splice isoforms that are
assembled based on TopHat read alignments. Cufflinks produces three outputs: a GTF file containing the assembled transcripts and two files containing the estimated level of expression
for each transcript and gene, respectively.
7. Merge assemblies using Cuffmerge tool located on the tools
panel, Genomic Analysis section, folder RNA-seq. Use the
GTF output files of Cufflinks and the GFF3 reference genome
annotation file. Cuffmerge merges annotation files to include
novel genes or alternative splicing isoforms that were not
included in the reference genome annotation and produces a
GTF output file that contains an assembly that merges together
the input assemblies.
8. Find significant changes in transcript expression, splicing, and
promoter usage between two conditions using the Cuffdiff
tool located on the tools panel, Genomic Analysis section,
folder RNA-seq. Use the GTF file containing the merged
assemblies produced by Cuffmerge and the BAM output files
of TopHat for the samples. Cuffdiff produces output files such
as transcript and gene FPKM expression tracking, transcript
and gene differential FPKM, and differential splicing and promoter use tests.
9. Visualize the read alignment at the genome level using the
Integrative Genomics Viewer (IGV). This software can be
downloaded from the website https://software.bro
adinstitute.org/software/igv/download. IGV allows the visualization of TopHat outputs, such as read alignment data (BAM
files) and track intron/exon junction data (BED files) as well as
a wide variety of data types.
(a) If the genome is not included in the software database,
create a genome file using the menu Genomes, Create
genome file.
(b) Upload the genome using the menu Genomes, Load
genome from file, or use the menu Load genome from
URL if it is available online.
TRAP-SEQ Analysis of Long Noncoding RNAs
465