Preparing read data - Revision history

69.173.108.246 at 15:30, 24 October 2013

2013-10-24T15:30:17Z

69.173.108.246 at 15:30, 24 October 2013

2013-10-24T15:30:02Z

Thomas: /* With Picard (OS independent, recommended) */

2013-10-18T14:57:13Z

With Picard (OS independent, recommended)

69.173.108.246: /* With Picard (OS independent, recommended) */

2013-10-18T14:56:14Z

With Picard (OS independent, recommended)

69.173.108.246: /* With Picard (OS independent, recommended) */

2013-10-18T14:54:59Z

With Picard (OS independent, recommended)

Thomas Admin at 23:17, 17 October 2013

2013-10-17T23:17:33Z

Thomas Admin: /* Sorting and indexing */

2013-10-17T23:16:45Z

Sorting and indexing

Thomas Admin: /* Preparing for visualization */

2013-10-17T23:15:10Z

Preparing for visualization

Thomas Admin at 23:12, 17 October 2013

2013-10-17T23:12:27Z

Thomas Admin: /* Sorting and indexing with Picard (OS independent) */

2013-10-17T22:58:25Z

Sorting and indexing with Picard (OS independent)

@@ Line 67: / Line 67: @@
 == Downsampling data ==
-Some time you don't want to look at all reads, but just at a fraction. This can be done by downsampling you BAM files before indexing.
+Sometimes you don't want to look at all reads, but just at a fraction. This can be done by downsampling you BAM files before indexing.
   java -Xmx500m -jar DownsampleSam.jar I=sorted.bam O=downsample.bam P=0.1

← Older revision		Revision as of 15:30, 24 October 2013
Line 65:		Line 65:

	samtools index sorted.bam (will create sorted.bam.bai, which is read by GenomeView together with the bam file)		samtools index sorted.bam (will create sorted.bam.bai, which is read by GenomeView together with the bam file)
		+
		+	== Downsampling data ==
		+	Some time you don't want to look at all reads, but just at a fraction. This can be done by downsampling you BAM files before indexing.
		+
		+	java -Xmx500m -jar DownsampleSam.jar I=sorted.bam O=downsample.bam P=0.1
		+	[http://genomeview.org/loki/picard-tools-recent/DownsampleSam.jar Download DownsampleSam.jar]
		+
		+
		+
		+	The I parameter is the input file
		+
		+	The O parameter is the output file
		+
		+	The P parameter is the probability that a read is retained, so 0.1 means that 10% of the reads are kept.
		+
		+

	== Summary visualizations ==		== Summary visualizations ==

@@ Line 17: / Line 17: @@
 === With Picard (OS independent, recommended) ===
-You need to have a a recent version of Java installed and you need to run these commands on the [[command-line]]. There are download links with each program where you can fetch the exact version that these manual pages were tested with. Alternatively, you may want to install a more recent version of Picard.
+You need to have a a recent version of Java installed and you need to run these commands on the [[command-line]]. There are download links with each program where you can fetch the exact version that these manual pages were tested with. Alternatively, you may want to install a more recent version of [http://picard.sourceforge.net/ Picard].
 In this example we have a read alignment in BAM format called 'alignment.sam'. We use the program [http://picard.sourceforge.net/command-line-overview.shtml#SortSam SortSam] from Picard to sort the file by coordinates. This also works if your aligner gives you a BAM file as output, i.e. 'alignment.bam'

@@ Line 17: / Line 17: @@
 === With Picard (OS independent, recommended) ===
-You need to have a local copy of [[Install Picard|Picard]] installed and you need to run these commands on the [[command-line]].
+You need to have a a recent version of Java installed and you need to run these commands on the [[command-line]]. There are download links with each program where you can fetch the exact version that these manual pages were tested with. Alternatively, you may want to install a more recent version of Picard.
 In this example we have a read alignment in BAM format called 'alignment.sam'. We use the program [http://picard.sourceforge.net/command-line-overview.shtml#SortSam SortSam] from Picard to sort the file by coordinates. This also works if your aligner gives you a BAM file as output, i.e. 'alignment.bam'

@@ Line 24: / Line 24: @@
 The instruction below will sort the SAM file by coordinate.
   java -Xmx500m -jar SortSam.jar I=aligned.sam O=sorted.bam SO=coordinate
 Important:
@@ Line 39: / Line 40: @@
   java -Xmx500m -jar BuildBamIndex.jar I=sorted.bam
 Important:

← Older revision		Revision as of 23:17, 17 October 2013
Line 1:		Line 1:
−	The best format to present short read alignments to GenomeView is the BAM format. You need to have your read data in this format and it has to be aligned.	+	{{TOC\|align=right}}
		+	The best format to present short read alignments to GenomeView is the SAM/BAM format. You need to have your read data in this format and it has to be aligned to a reference genome.

	== Aligning reads ==		== Aligning reads ==

@@ Line 9: / Line 9: @@
 Before you can visualize read data you need to prepare the data. This needs to be done because those files are generally huge and we do not want to read the complete file if we're only looking at a small portion of it.
-We mainly need to do two things when we get data fresh from the aligner.
+There are two things to prepare data fresh from the aligner.
 # Sort reads based on genomic coordinates
@@ Line 15: / Line 15: @@
+=== With Picard (OS independent, recommended) ===
 You need to have a local copy of [[Install Picard|Picard]] installed and you need to run these commands on the [[command-line]].
@@ Line 49: / Line 48: @@
 # Make sure that you now have a file 'sorted.bam.'''bai'''' and that it's not completely empty, i.e. it has a size > 0. This file will be created in the same directory as the 'sorted.bam' file.
-=== Sorting and indexing with SAMtools (Mac OS and Linux) ===
+=== With SAMtools (Mac OS and Linux) ===
-Steps to get from the various aligner formats to the SAM format are available on the SAMtools website.
+Steps to get from the various aligner formats to the SAM format are available on the [http://samtools.sourceforge.net/ SAMtools website].
 Steps to go from SAM to indexed BAM.
-samtools faidx reference.fasta (will create reference.fasta.fai for the next step)
+ samtools faidx reference.fasta (will create reference.fasta.fai for the next step)
-samtools view -bS -t reference.fasta.fai alignment.sam -o alignment.bam
+ samtools view -bS -t reference.fasta.fai alignment.sam -o alignment.bam
-samtools sort alignment.bam sorted (will create sorted.bam)
+ samtools sort alignment.bam sorted (will create sorted.bam)
-samtools index sorted.bam (will create sorted.bam.bai, which is read by GenomeView together with the bam file)
+ samtools index sorted.bam (will create sorted.bam.bai, which is read by GenomeView together with the bam file)
 == Summary visualizations ==

@@ Line 6: / Line 6: @@
 If you need help aligning your reads, you may want to have a look at the [[Recipe to align reads]] to get some ideas.
-== Preparing for visualization ==
+== Sorting and indexing ==
-To prepare a BAM file straight from the aligner there are a few more steps you may have to take to get your data in the right format.
+Before you can visualize read data you need to prepare the data. This needs to be done because those files are generally huge and we do not want to read the complete file if we're only looking at a small portion of it.
 # Sort reads based on genomic coordinates
 # Index sorted reads.
 === Sorting and indexing with Picard (OS independent) ===
@@ Line 57: / Line 61: @@
 samtools index sorted.bam (will create sorted.bam.bai, which is read by GenomeView together with the bam file)
 == Summary visualizations ==

@@ Line 17: / Line 17: @@
 In this example we have a read alignment in BAM format called 'alignment.sam'. We use the program [http://picard.sourceforge.net/command-line-overview.shtml#SortSam SortSam] from Picard to sort the file by coordinates. This also works if your aligner gives you a BAM file as output, i.e. 'alignment.bam'
-<code lang='bash'>
+'''Sorting'''
-  java -Xmx1g -jar SortSam.jar I=aligned.sam O=sorted.bam SO=coordinate
+The instruction below will sort the SAM file by coordinate.
-</code>
+  java -Xmx500m -jar SortSam.jar I=aligned.sam O=sorted.bam SO=coordinate
 === Sorting and indexing with SAMtools (Mac OS and Linux) ===
@@ Line 42: / Line 66: @@
 === Variants ===
 If you are investigating SNPs or other genetic variants, you also may want to [[Recipe variant calls|create variant calls]].