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Picard by default does +/- 250 bp. I recommend using the same file for baits and targets: you can have baits that extend past targets and hence get more coverage for baits than for target if you had all of your target sequence covered by baits. If you had say only 80% of your targets covered by baits, then you already know this, and it just complicates things to try to consider it again. So, again, I recommend using the actual bait intervals if possible.
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Another vote for Picard's CalculateHsMetrics. It's in the public Galaxy (http://main.g2.bx.psu.edu/ under "NGS: Picard (beta)").
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samtools flagstat my_data.bam
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Originally posted by swbarnes2 View Post...So then use samtools flagstat to count the number of mapped reads of the original .bam, and then of the intersect.bam
(I am using samtools in Cygwin in a Windows 7 system)
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Originally posted by laura View PostYou may find picards CalculateHsMetrics useful
http://picard.sourceforge.net/comman...ulateHsMetrics
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Originally posted by Jon_Keats View PostPicard HsMetrics is designed by the Broad, which helped develop the Agilent in solution capture method. In the case of Agilent they provide positions for both the baits and the target regions.
Think:
Code:1---------Target------------1 ------IIIIIIIIIIII exon IIIIIIIIIIIIII-------- xbaitx ybaity zbaitz
Unfortunately, Illumina only provides the target regions not the actual bait locations. So it is harder to decide if some of the non-exonic reads are uncaptured flow-through or captured regions that are not in the "official" targets. Clearly, in my opinion Illumina has captured entire 5kb regions that include 3 exons totaling 1kb resulting in 4kb of high coverage intronic region. Not sure if the designer was just lazy, has ulterior motives, some internal data that this makes target recovery the best?
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Picard HsMetrics is designed by the Broad, which helped develop the Agilent in solution capture method. In the case of Agilent they provide positions for both the baits and the target regions.
Think:
Code:1---------Target------------1 ------IIIIIIIIIIII exon IIIIIIIIIIIIII-------- xbaitx ybaity zbaitz
Unfortunately, Illumina only provides the target regions not the actual bait locations. So it is harder to decide if some of the non-exonic reads are uncaptured flow-through or captured regions that are not in the "official" targets. Clearly, in my opinion Illumina has captured entire 5kb regions that include 3 exons totaling 1kb resulting in 4kb of high coverage intronic region. Not sure if the designer was just lazy, has ulterior motives, some internal data that this makes target recovery the best?Last edited by Jon_Keats; 01-20-2012, 08:18 PM.
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Originally posted by gwilymh View PostWhat is the difference between the BAIT_INTERVALS and TARGET_INTERVALS files?
I suspect that when you have files from your pull down supplier there may be some subtle differences but I don't think it matters to hugely
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Originally posted by laura View PostYou may find picards CalculateHsMetrics useful
http://picard.sourceforge.net/comman...ulateHsMetrics
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BEDTools intersectBed will work with bam files, and output .bam files. I use it that way all the time. Your command line look something like:
intersectBed -abam yourbam.bam -b paddedExometarget.bed > intersect.bam
So then use samtools flagstat to count the number of mapped reads of the original .bam, and then of the intersect.bam
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You may find picards CalculateHsMetrics useful
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Create a bed file of your Agilent SureSelect targets and use BEDtools to merge adjacent targets and then slopBed to add 50 bps to either side of your merged targets. Then use Bedtools BedtoBam to convert your bam file to a bed file and then use intersectBed to create an intersection of your bam.bed and the target.bed. This will create a bed file illustrating the target regions covered by your bam file which you can then parse for percent on and off target. I think there may be examples of this workflow in the BEDtools manual available online.
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% On-Off target
hello,
I'm looking for a tool (or command line) to determine the % On-Off target + or - 50 bp of exon from my capture file but not annotated (!!!). Capture SureSelect agilent home.
Current pipeline:
GAIIx Illumina
CASAVA1.8
IGV
CNV-seq
SAMtools
BEDtools
GALAXY
NextGENe
Please HELP
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