Visualisation of mapped reads - Revision history

Rf at 20:35, 11 May 2017

2017-05-11T20:35:25Z

Rf at 10:11, 9 May 2017

2017-05-09T10:11:01Z

Rf at 21:28, 8 May 2017

2017-05-08T21:28:47Z

Rf at 20:25, 8 May 2017

2017-05-08T20:25:06Z

Rf at 20:23, 8 May 2017

2017-05-08T20:23:00Z

Rf at 18:38, 8 May 2017

2017-05-08T18:38:33Z

Rf: Created page with "= Introduction = In contrast to the other more quantitative stages, this exercise is qualitative in the sense that we get a visual feel for a certain area of interest. = Aim..."

2017-05-08T18:34:02Z

Created page with "= Introduction = In contrast to the other more quantitative stages, this exercise is qualitative in the sense that we get a visual feel for a certain area of interest. = Aim..."

New page

= Introduction =

In contrast to the other more quantitative stages, this exercise is qualitative in the sense that we get a visual feel for a certain area of interest.

= Aims =
In this part you will learn to:
visualise mapped reads

= Software to be used =
* samtools v0.1.19: http://samtools.sourceforge.net/
* Integrated Genomics Viewer (IGV) 2.3.92: http://www.broadinstitute.org/igv/

To load these up:

module load samtools IGV

The data set you'll be using is downloaded from ENA (http://www.ebi.ac.uk/ena/data/view/SRP019027). The reads belong to
samples SRR769314 and SRR769316. The data set is tailored with respect to the time allocated for the workshop. Reads were alig
ned to the first 20 Mb of chromosome 19 of the mouse reference genome (GRCm38/mm10) using TopHat and duplicates marked
using Picard MarkDuplicates.
You will use the following files:
SRR769314_duplicates_marked.bam: aligned reads (without and with using gene annotation)
SRR769316_duplicates_marked.bam: aligned reads (without and with using gene annotation)
mm10_chr19-1-20000000.fasta: mouse reference genome sequence
mm10_chr19-1-20000000_Ensembl.gtf: Ensembl mouse gene models

Type text like this in the terminal at the $ command prompt, then press the
[Enter] key to run the command.

Data
The data is available in the directory 06_Visualisation_of_mapped_reads:

cd /home/training/Data/06_Visualisation_of_mapped_reads

Indexing BAM files
To enable fast access to any part of the BAM files we need to create an index using samtools:

samtools index SRR769314_duplicates_marked.bam
samtools index SRR769316_duplicates_marked.bam

Visualising mapped reads
Start IGV:

igv.sh &

To load the mouse genome:

Page 1

�Edinburgh Genomics - Introduction to RNA-seq Data Analysis 19 & 20 May 2016

Select Genomes -> Load Genome from File...
Navigate to home -> training -> Data/ -> 06_Visualisation_of_mapped_reads -> Reference
Select the mm10_chr19-1-20000000.fasta file
Click [Open]

To load the alignments:
Select File -> Load from File...
Navigate to home -> training -> Data/ -> 06_Visualisation_of_mapped_reads
Select the SRR769314_duplicates_marked.bam and SRR769316_duplicates_marked.bam files
Click [Open]

To load the Ensembl gene models:
Select File -> Load from File...
Navigate to home -> training -> Data/ -> 06_Visualisation_of_mapped_reads -> Reference
Select the mm10_chr19-1-20000000_Ensembl.gtf file
Click [Open]

Zoom in until you start seeing reads.

1. Navigate to chr19:3715000-3718000 (note that you don't have to include commas in the base coordinates, as IGV will add these)
and identify reads spanning exon-exon junctions
2. Navigate to chr19:5748800-5751100. Zoom in to observe each end of this exon-exon junction.
What do you think of the alignment? How would you fix it?

Add the reads aligned using gene annotation data:
Select File -> Load from File...
Navigate to home -> training -> Data/ -> 06_Visualisation_of_mapped_reads -> with_gtf
Select the SRR769314_duplicates_marked.bam and SRR769316_duplicates_marked.bam files
Click [Open]

3. Navigate to chr19:5748800-5751100. Verify that the alignment looks better.
How accurate do you think TopHat would be to detect novel (unannotated) junctions?
4. Navigate to chr19:4709000-4756000. Right click on the track names and select Collapsed.
What do you think of the difference in coverage between the SRR769314 and SRR769316 samples?
5. Navigate to chr19:6982100-6987800. Right click on the track names and select Sashimi Plot.
Can you identify which isoform is more expressed in each sample?

Page 2

�

@@ Line 17: / Line 17: @@
   module load samtools IGV
-We'll be using the same data as before, but this time we will have two alignment files (i.e. two samples) from the same study. They are samples SRR769314 and SRR769316. The are tailored with respect to the time allocated for the workshop. They were aligned to the first 20 Mb of chromosome 19 of the mouse reference genome (GRCm38/mm10) using TopHat and duplicates have already been marked using Picard MarkDuplicates.
+We'll be using the same data as before, but this time we will have two alignment files (i.e. two samples) from the same study. They are samples <code>SRR769314</code> and <code>SRR769316</code>. The are tailored with respect to the time allocated for the workshop. They were aligned to the first 20 Mb of chromosome 19 of the mouse reference genome (GRCm38/mm10) using TopHat and duplicates have already been marked using Picard MarkDuplicates.
 We shall will use the following files:
 * <code>SRR769314_duplicates_marked.bam</code>: aligned reads (without and with using gene annotation)
 * <code>SRR769316_duplicates_marked.bam</code>: aligned reads (without and with using gene annotation)
-* <code>mm10_chr19-1-20000000.fasta</code>: mouse reference genome sequence
+* <code>mm10_chr19-1-20000000.fasta</code>: mouse reference genome sequence. Has has already been indexed.
 * <code>mm10_chr19-1-20000000_Ensembl.gtf</code>: Ensembl mouse gene models
@@ Line 42: / Line 42: @@
 == To load the mouse genome ==
-  Select Genomes -> Load Genome from File...
+  Select <code>Genomes</code> -> <code>Load Genome from File...</code>
-  Navigate to <yourhomedirectory> -> i2rda_data -> 06_Visualisation_of_mapped_reads -> Reference
+  Navigate to <code><yourhomedirectory></code> -> <code>i2rda_data</code> -> <code>04_Visualisation_of_mapped_reads</code> -> <code>Reference_files</code>
-  Select the mm10_chr19-1-20000000.fasta file
+  Select the <code>mm10_chr19-1-20000000.fasta</code> file
-  Click [Open]
+  Click <code>Open</code>
 == To load the alignments ==
- Select File -> Load from File...
+* Select <code>File</code> -> <code>Load</code> from File...
- Navigate to <yourhomedirectory> -> i2rda_data -> 06_Visualisation_of_mapped_reads
+* Navigate to <code><yourhomedirectory</code>> -> <code>i2rda_data</code> -> <code>04_Visualisation_of_mapped_reads</code>
- Select both the SRR769314_duplicates_marked.bam and SRR769316_duplicates_marked.bam files by Ctrl+Click
+* Select both the <code>SRR769314_duplicates_marked.bam</code> and <code>SRR769316_duplicates_marked.bam</code> files by <code>Ctrl</code>+Click
- Click [Open]
+* Click <code>Open</code>
 == To load the Ensembl gene models ==
- Select File -> Load from File...
+* <code>Select File</code> -> <code>Load from File...</code>
- Navigate to <yourhomedirectory> -> i2rda_data -> 06_Visualisation_of_mapped_reads -> Reference
+* Navigate to <code><yourhomedirectory></code> -> <code>i2rda_data</code> -> <code>04_Visualisation_of_mapped_reads</code> -> <code>Reference_files</code>
- Select the mm10_chr19-1-20000000_Ensembl.gtf file
+* Select the <code>mm10_chr19-1-20000000_Ensembl.gtf</code> file
- Click [Open]
+* Click <code>Open</code>
 = Zoom into target areas =
@@ Line 63: / Line 63: @@
 . Navigate to <code>chr19:3715000-3718000</code>. Copy-paste for best accuracy. Note that IGV adds thousand separators to your location afterwards.
-. Navigate to chr19:5748800-5751100. Zoom in to observe each end of this exon-exon junction.
+. Navigate to <code>chr19:5748800-5751100</code>. Zoom in to observe each end of this exon-exon junction.
 <ins>Question</ins>:
@@ Line 69: / Line 69: @@
 Add the reads aligned using gene annotation data:
- Select File -> Load from File...
+* <code>Select File</code> -> <code>Load from File...</code>
- Navigate to home -> training -> Data/ -> 06_Visualisation_of_mapped_reads -> with_gtf
+* Navigate to <code><yourhomedirectory></code> -> <code>i2rda_data/</code> -> 04_Visualisation_of_mapped_reads -> with_gtf
- Select the SRR769314_duplicates_marked.bam and SRR769316_duplicates_marked.bam files
+* Select the <code>SRR769314_duplicates_marked.bam</code> and <code>SRR769316_duplicates_marked.bam</code> files
- Click [Open]
+* Click <code>Open</code>
 . Navigate to <code>chr19:5748800-5751100</code>. Verify that the alignment looks better.
 . Navigate to <code>chr19:4709000-4756000</code>. Right click on the track names and select Collapsed.

@@ Line 6: / Line 6: @@
 In this part you will learn to:
 * Use a genome browser, the Broad Institute's IGV, to visualise mapped reads
 = Software to be used =
@@ Line 23: / Line 25: @@
 * <code>mm10_chr19-1-20000000_Ensembl.gtf</code>: Ensembl mouse gene models
-Data
+= Data location =
-The data is available in the directory 04_Visualisation_of_mapped_reads:
+The data is available in the directory <code>04_Visualisation_of_mapped_reads</code>, i.e.
   cd ~/i2rda_data/04_Visualisation_of_mapped_reads

@@ Line 34: / Line 34: @@
   samtools index SRR769316_duplicates_marked.bam
-= Starting IGV =
+= Starting and using IGV =
   igv.sh &
-To load the mouse genome:
+== To load the mouse genome ==
   Select Genomes -> Load Genome from File...
@@ Line 45: / Line 45: @@
   Click [Open]
-To load the alignments:
+== To load the alignments ==
   Select File -> Load from File...
   Navigate to home -> training -> Data/ -> 06_Visualisation_of_mapped_reads
@@ Line 51: / Line 51: @@
   Click [Open]
-To load the Ensembl gene models:
+== To load the Ensembl gene models ==
   Select File -> Load from File...
   Navigate to home -> training -> Data/ -> 06_Visualisation_of_mapped_reads -> Reference
@@ Line 57: / Line 57: @@
   Click [Open]
 Zoom in until you start seeing reads.