Trying to identify low coverage regions (bases) in my sample from a Bam and BED file.

pfeifferr · 03-24-2014, 06:55 AM

I have a bed of my targets (start and stop coordinates of each exon of my genes of interest). I have a BAM file generated from my ION PGM run. I am trying to identify any location identified in my BED file where I have less than 20x coverage so I can fill in these regions with traditional Sanger sequencing.

Basically, if any bases within the exon are covered at less than 20x, I want to know which. (I have heard 20x is a good threshold for germline variants, agreed?)

Also, if I add a descriptive section to the BED file contain “gene-exon” that could be included would be even more helpful.

I have been reading post online for days, and I am lost here. Can anybody help please?

Thanks

Pfeifferr

lindenb · 03-24-2014, 10:56 AM

cross-posted http://www.biostars.org/p/95986/

GenoMax · 03-24-2014, 11:04 AM

If this data was analyzed using Ion reporter then you should be able to get a "coverage" report by including the coverage plugin. Just adding that as another available option.

subtractBed from Bedtools may be an option. But that would not tell you if something is present at 20x or less (just present or absent) so it may not work for you.

pfeifferr · 03-24-2014, 12:01 PM

Thanks Genomax,

I did run the coverage analysis plugin. However, this doesn't solve my real problem. I am trying to find out which bases within a set of coding exons are covered at < 20x. However, the coverage analysis plugin gives you "amplicon coverage summary" which is coverage per amplicon, and since we have multiple amplicons covering an exon and overlapping amplicons, low amplicon coverage does not necessarily mean low coverage of a base in the target. The "base depth of coverage" can tell me how many bases are covered at 1x, 2x, 3x etc, but now where they are located.

GenoMax · 03-24-2014, 12:22 PM

Then bedtools/coverageBed analysis with the "-d" option to get base level coverage would be the way to go (http://bedtools.readthedocs.org/en/l.../coverage.html). Some post-processing will be required to separate positions that have < 20 x coverage.

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03-24-2014, 06:55 AM	#1
pfeifferr Junior Member Location: utica ny, 13501, usa Join Date: Aug 2010 Posts: 7	Trying to identify low coverage regions (bases) in my sample from a Bam and BED file. I have a bed of my targets (start and stop coordinates of each exon of my genes of interest). I have a BAM file generated from my ION PGM run. I am trying to identify any location identified in my BED file where I have less than 20x coverage so I can fill in these regions with traditional Sanger sequencing. Basically, if any bases within the exon are covered at less than 20x, I want to know which. (I have heard 20x is a good threshold for germline variants, agreed?) Also, if I add a descriptive section to the BED file contain “gene-exon” that could be included would be even more helpful. I have been reading post online for days, and I am lost here. Can anybody help please? Thanks Pfeifferr

03-24-2014, 11:04 AM	#3
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,080	If this data was analyzed using Ion reporter then you should be able to get a "coverage" report by including the coverage plugin. Just adding that as another available option. subtractBed from Bedtools may be an option. But that would not tell you if something is present at 20x or less (just present or absent) so it may not work for you. Last edited by GenoMax; 03-24-2014 at 11:11 AM.

03-24-2014, 10:56 AM	#2
lindenb Senior Member Location: France Join Date: Apr 2010 Posts: 143	cross-posted http://www.biostars.org/p/95986/

03-24-2014, 12:01 PM	#4
pfeifferr Junior Member Location: utica ny, 13501, usa Join Date: Aug 2010 Posts: 7	Thanks Genomax, I did run the coverage analysis plugin. However, this doesn't solve my real problem. I am trying to find out which bases within a set of coding exons are covered at < 20x. However, the coverage analysis plugin gives you "amplicon coverage summary" which is coverage per amplicon, and since we have multiple amplicons covering an exon and overlapping amplicons, low amplicon coverage does not necessarily mean low coverage of a base in the target. The "base depth of coverage" can tell me how many bases are covered at 1x, 2x, 3x etc, but now where they are located.

03-24-2014, 12:22 PM	#5
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,080	Then bedtools/coverageBed analysis with the "-d" option to get base level coverage would be the way to go (http://bedtools.readthedocs.org/en/l.../coverage.html). Some post-processing will be required to separate positions that have < 20 x coverage.