1. NGS File formats Raw data from various vendors => various formats
Different quality metrics (some more stringent than others)
As data analysis proceeds, end up with even more formats:
GenBank formats (SRA)
Alignments are in SAM/BAM
Genome Browser formats (wig, bed, gff, etc)
Variants in vcf files (SNPs, indels, etc)

2. What, if there is no Galaxy, …?
Looking for Sequence reads data (SRA) ,
for example:

3. We’ve got read data in sra format.
Now what?
We need to convert to FASTQ format
to use TopHat, STAR, etc. on Pegasus2.

4. SRA toolkit

5. SRA toolkit fastq-dump –X 5 –Z SRR390728
fastq-dump –I --split-files SRR390728
fastq-dump --split-files –fasta 60 SRR390728

6. FASTQ file format

7. Working on pegasus2 We’ve got our fastq file(s).
To align the reads with the reference genome we will use TopHat on pegasus2
transfer data through
scp yourfile.fastq into your home directory
scp yourfiel.fastq
into scratch directory

8. Preparing to use TopHat on Pegasus2
Tophat was built on top of the non-splice-aware aligner Bowtie. So in order to use Tophat, you must also have Bowtie available. Tophat and Bowtie are both available as modules on Pegasus2 to simply load and use.
To load Tophat and Bowtie for use on pegasus, simply type the following commands:
To see a complete list of all available modules, type:
To confirm that the modules have been loaded properly, type:
module load bowtie2/2.2.2
module load tophat/2.0.11
module avail
which tophat
which bowtie

9. Preparing data for TopHat use
We need to give it information about the genome to which we want to align our data: Assuming we are in our accounts on Pegasus
1.) First, we need to download the genome sequence as a fasta file
For human: ftp://ftp.ensembl.org/pub/release-80/fasta/homo_sapiens/ dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz :
wget -O GRCh38.fa.gzftp://ftp.ensembl.org/pub/release-80/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz
2.) Second, we need to download (or construct) an indexed version of the
genome for bowtie2 to work with. Pre-built indexes for Bowtie2 can be
found on

10. Preparing data for TopHat use
However, bowtie2 has a command to build this index out of the genome
sequence file.
bowtie2-build -f GRCh38.fa GRCh38
considering GRCh38.fa contains the genome sequences in fasta format.
This will create a series of files called GRCh38.1.bt2, GRCh38.rev.1.bt2,
GRCh38.2.bt2, etc.

11. Preparing data for TopHat use
3.) Third, we need to download an annotation file containing all the known genes and transcripts for this genome. This third step is technically optional, but it helps to improve the accuracy of splice junction calling and is generally recommended. We are also going to need this file when we quantify the transcripts present, so might as well use it now too.
We need to give it information about the genome to which we want to align our data:
Genome annotation: Human: ftp://ftp.ensembl.org/pub/release-80/gtf/homo_sapiens/Homo_sapiens.GRCh38.80.gtf.gz)
wget -O GRCh38.gtfftp://ftp.ensembl.org/pub/release-80/gtf/homo_sapiens/Homo_sapiens.GRCh38.80.gtf.gz)
We are ready to run TopHat!

12. Running TopHat on Pegasus2
To run a job on Pegasus2 in the background we need to create a shell script (
#!/bin/bash
#BSUB –J Tophat_job1
#BSUB –e Tophat_job1.err
#BSUB –o Tophat_job1.out
#BSUB –n 4
#BSUB -q general
#BSUB -W 72:00 #
Your actual commands for the job are going to be placed here.
which shell?
job name
file for stderr
file for stdout
number of cores
queue, more info
time allocation

13. Running TopHat
The complete list of TopHat parameters and their official descriptions are
Optional parameters:
-G … specify a genomic annotation to use
-o … locate the output directory
Required parameters:
- 'base name' of the genome sequence/index data so that it can go and find it.
- fastq files to use

14. Running TopHat Simplest example: ‘base name’ one fastq file
#!/bin/bash
#BSUB –J Tophat_job1
#BSUB –e Tophat_job1.err
#BSUB –o Tophat_job1.out
#BSUB –n 4
#BSUB -q general
#BSUB -W 72:00 #
tophat -G ~/RNA-Seq/hg38/hg38.gtf -p4 -o . ~/RNA-Seq/hg38/hg38 ~/RNA-seq/Sample1.R1.fastq
‘base name’
one fastq file

15. Running TopHat Paired-ended reads #!/bin/bash #BSUB –J Tophat_job1
#BSUB –e Tophat_job1.err
#BSUB –o Tophat_job1.out
#BSUB –n 4
#BSUB -q general
#BSUB -W 72:00 #
tophat -G ~/RNA-Seq/hg38/hg38.gtf -p4 -o . ~/RNA-Seq/hg38/hg38 ~/RNA-seq/Sample1.R1.fastq ~/RNA-seq/Sample1.R2.fastq
Paired-ended reads

16. Running TopHat multiple fastq files
#!/bin/bash
#BSUB –J Tophat_job1
#BSUB –e Tophat_job1.err
#BSUB –o Tophat_job1.out
#BSUB –n 4
#BSUB -q general
#BSUB -W 72:00 #
tophat -G ~/RNA-Seq/hg38/hg38.gtf -p4 -o . ~/RNA-Seq/hg38/hg38 ~/FastQFiles/Sample1.Lane1.R1.fastq,~/FastQFiles/Sample1.Lane2.R1.fastq,~/FastQFiles/Sample1.Lane3.R1.fastq
tophat -G ~/RNA-Seq/hg38/hg38.gtf -p4 -o . ~/RNA-Seq/hg38/hg38 ~/FastQFiles/Sample1.Lane1.R1.fastq,~/FastQFiles/Sample1.Lane2.R1.fastq,~/FastQFiles/Sample1.Lane3.R1.fastq ~/FastQFiles/Sample1.Lane1.R2.fastq,~/FastQFiles/Sample1.Lane2.R2.fastq,~/FastQFiles/Sample1.Lane3.R2.fastq
multiple fastq files
multiple fastq files wit paired-ended reads

17. Finally submit your TopHat jobs
submit a job with bsub < script.sh
bjobs
bkill <jobid>
returns the status of current jobs
kills job with <jobid>
A successful run of Tophat will return the following files
accepted_hits.bam
junctions.bed
insertions.bed
deletions.bed
holds our results
To view a BAM file you need:
module load samtools/0.1.19
samtools view accepted_hits.bam