Coverage track not working

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Coverage track not working

Zipixx

Hi,

I am working with two databases in my configuration file and need to display coverage tracks for both of them.
One works fine, the other does not. When using feature = match and glyph = segments however, the correct data is shown.

The track definitions for databases rnaseqall and ovarysam:

#This coverage track works
[READ_COVERAGE]
feature                 = coverage
glyph                   = wiggle_xyplot
database             = rnaseqall
height                  = 50
fgcolor                 = black
bicolor_pivot       = 20
pos_color             = royalblue
neg_color             = red
key                     = Coverage from RNA-Seq
citation                = RNA-Seq reads combined from all developmental stages.
category                = RNA-Seq

#This track does not work correctly. A scale with and a few non-matching peeks are shown.
[READ_COVERAGE_OVARY]
feature                 = coverage
glyph                   = wiggle_xyplot
database                = ovarysam
height                  = 50
fgcolor                 = black
bicolor_pivot           = 20
pos_color               = royalblue
neg_color               = red
key                     = Coverage ovary
citation                = RNA-Seq reads combined from all developmental stages.
category                = RNA-Seq

#Correct data is shown
[Reads]
feature        = match
glyph          = segments
database       = ovarysam
draw_target    = 1
show_mismatch  = 1
mismatch_color = red
bgcolor        = royalblue
fgcolor        = black
height         = 5
label density  = 50
feature_limit  = 5000
bump           = fast
connector      = solid
key            = RNA-Seq reads match ovary
category       = Experimental Data

 

Is there a method to verify the bam files are correct?
What could be causing the coverage track to be wrong?

Best Regards
Stefan Pfeiffer

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Re: Coverage track not working

Scott Cain
Hi Stefan,

I know that there can be problems associated with sampling the bam data when drawing coverage plots. Have you tried zooming in and out to see if the coverage plot changes to look more "right".  Similarly, have you tried opening the bam file in other software like IGV to see if it looks right? 

Scott


On Mon, May 8, 2017 at 11:59 AM, Stefan Pfeiffer <[hidden email]> wrote:

Hi,

I am working with two databases in my configuration file and need to display coverage tracks for both of them.
One works fine, the other does not. When using feature = match and glyph = segments however, the correct data is shown.

The track definitions for databases rnaseqall and ovarysam:

#This coverage track works
[READ_COVERAGE]
feature                 = coverage
glyph                   = wiggle_xyplot
database             = rnaseqall
height                  = 50
fgcolor                 = black
bicolor_pivot       = 20
pos_color             = royalblue
neg_color             = red
key                     = Coverage from RNA-Seq
citation                = RNA-Seq reads combined from all developmental stages.
category                = RNA-Seq

#This track does not work correctly. A scale with and a few non-matching peeks are shown.
[READ_COVERAGE_OVARY]
feature                 = coverage
glyph                   = wiggle_xyplot
database                = ovarysam
height                  = 50
fgcolor                 = black
bicolor_pivot           = 20
pos_color               = royalblue
neg_color               = red
key                     = Coverage ovary
citation                = RNA-Seq reads combined from all developmental stages.
category                = RNA-Seq

#Correct data is shown
[Reads]
feature        = match
glyph          = segments
database       = ovarysam
draw_target    = 1
show_mismatch  = 1
mismatch_color = red
bgcolor        = royalblue
fgcolor        = black
height         = 5
label density  = 50
feature_limit  = 5000
bump           = fast
connector      = solid
key            = RNA-Seq reads match ovary
category       = Experimental Data

 

Is there a method to verify the bam files are correct?
What could be causing the coverage track to be wrong?

Best Regards
Stefan Pfeiffer

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Ontario Institute for Cancer Research

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Re: Coverage track not working

Zipixx

Hi Scott,

thank you for the suggestion. IGV does show the correct and expected coverage.
Zooming in GBrowse does not change anything.
You can see the result at https://vm19002.virt.gwdg.de/gb2/gbrowse/tribolium/?name=id:469064

If it is helpful, we could also upload the bam file.
samtools view wrong_coverage.bam returns lines like the following.  The position/length integers in the 8th/9th column remain 0 throughout the entire file, which is not the case for correct_coverage.bam. Could this be the cause?

ILLUMINA-D0898A_0001:1:94:15808:4394#0/1        0       NW_015452213.1  1447    40      38M     *       0       0       TGTGGTACTCGTGTACACAGCAGAAGAATTATCAGTAT  bbbbbbbbbbbbbbbbbbabbbbbbbbbbbbbbbbbbb  MD:Z:38 NH:i:1  HI:i:1  NM:i:0  SM:i:40 XQ:i:40 X2:i:0
ILLUMINA-D0898A_0001:1:2:16831:1638#0/1 0       NW_015452213.1  1449    40      38M     *       0       0       TGGTACTCGTGTACACAGCAGAAGAATTATCAGTATAA  ]abbbbbbabaabbbbbbbbbbbbabbbbbbbbbbbbb  MD:Z:38 NH:i:1  HI:i:1  NM:i:0  SM:i:40 XQ:i:40 X2:i:0

Best Regards
Stefan

 

On 2017-05-10 16:51, Scott Cain wrote:

Hi Stefan,
 
I know that there can be problems associated with sampling the bam data when drawing coverage plots. Have you tried zooming in and out to see if the coverage plot changes to look more "right".  Similarly, have you tried opening the bam file in other software like IGV to see if it looks right? 
 
Scott
 

On Mon, May 8, 2017 at 11:59 AM, Stefan Pfeiffer <[hidden email]> wrote:

Hi,

I am working with two databases in my configuration file and need to display coverage tracks for both of them.
One works fine, the other does not. When using feature = match and glyph = segments however, the correct data is shown.

The track definitions for databases rnaseqall and ovarysam:

#This coverage track works
[READ_COVERAGE]
feature                 = coverage
glyph                   = wiggle_xyplot
database             = rnaseqall
height                  = 50
fgcolor                 = black
bicolor_pivot       = 20
pos_color             = royalblue
neg_color             = red
key                     = Coverage from RNA-Seq
citation                = RNA-Seq reads combined from all developmental stages.
category                = RNA-Seq

#This track does not work correctly. A scale with and a few non-matching peeks are shown.
[READ_COVERAGE_OVARY]
feature                 = coverage
glyph                   = wiggle_xyplot
database                = ovarysam
height                  = 50
fgcolor                 = black
bicolor_pivot           = 20
pos_color               = royalblue
neg_color               = red
key                     = Coverage ovary
citation                = RNA-Seq reads combined from all developmental stages.
category                = RNA-Seq

#Correct data is shown
[Reads]
feature        = match
glyph          = segments
database       = ovarysam
draw_target    = 1
show_mismatch  = 1
mismatch_color = red
bgcolor        = royalblue
fgcolor        = black
height         = 5
label density  = 50
feature_limit  = 5000
bump           = fast
connector      = solid
key            = RNA-Seq reads match ovary
category       = Experimental Data

 

Is there a method to verify the bam files are correct?
What could be causing the coverage track to be wrong?

Best Regards
Stefan Pfeiffer

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GMOD Coordinator (http://gmod.org/)                     216-392-3087
Ontario Institute for Cancer Research

 

 

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Re: Coverage track not working

Scott Cain
Hi Stefan,

Unfortunately, I don't have anything more to offer--my guess is that you are just particularly unlucky :-/

Have you considered making a bigwig of the coverage data?  In general, bigwig data work better and are more compact (though you don't get individual read data, so if you need that, then you'd have both the BAM and the bigwig file).

Scott


On Wed, May 31, 2017 at 12:01 PM, Stefan Pfeiffer <[hidden email]> wrote:

Hi Scott,

thank you for the suggestion. IGV does show the correct and expected coverage.
Zooming in GBrowse does not change anything.
You can see the result at https://vm19002.virt.gwdg.de/gb2/gbrowse/tribolium/?name=id:469064

If it is helpful, we could also upload the bam file.
samtools view wrong_coverage.bam returns lines like the following.  The position/length integers in the 8th/9th column remain 0 throughout the entire file, which is not the case for correct_coverage.bam. Could this be the cause?

ILLUMINA-D0898A_0001:1:94:15808:4394#0/1        0       NW_015452213.1  1447    40      38M     *       0       0       TGTGGTACTCGTGTACACAGCAGAAGAATTATCAGTAT  bbbbbbbbbbbbbbbbbbabbbbbbbbbbbbbbbbbbb  MD:Z:38 NH:i:1  HI:i:1  NM:i:0  SM:i:40 XQ:i:40 X2:i:0
ILLUMINA-D0898A_0001:1:2:16831:1638#0/1 0       NW_015452213.1  1449    40      38M     *       0       0       TGGTACTCGTGTACACAGCAGAAGAATTATCAGTATAA  ]abbbbbbabaabbbbbbbbbbbbabbbbbbbbbbbbb  MD:Z:38 NH:i:1  HI:i:1  NM:i:0  SM:i:40 XQ:i:40 X2:i:0

Best Regards
Stefan

 

On 2017-05-10 16:51, Scott Cain wrote:

Hi Stefan,
 
I know that there can be problems associated with sampling the bam data when drawing coverage plots. Have you tried zooming in and out to see if the coverage plot changes to look more "right".  Similarly, have you tried opening the bam file in other software like IGV to see if it looks right? 
 
Scott
 

On Mon, May 8, 2017 at 11:59 AM, Stefan Pfeiffer <[hidden email]> wrote:

Hi,

I am working with two databases in my configuration file and need to display coverage tracks for both of them.
One works fine, the other does not. When using feature = match and glyph = segments however, the correct data is shown.

The track definitions for databases rnaseqall and ovarysam:

#This coverage track works
[READ_COVERAGE]
feature                 = coverage
glyph                   = wiggle_xyplot
database             = rnaseqall
height                  = 50
fgcolor                 = black
bicolor_pivot       = 20
pos_color             = royalblue
neg_color             = red
key                     = Coverage from RNA-Seq
citation                = RNA-Seq reads combined from all developmental stages.
category                = RNA-Seq

#This track does not work correctly. A scale with and a few non-matching peeks are shown.
[READ_COVERAGE_OVARY]
feature                 = coverage
glyph                   = wiggle_xyplot
database                = ovarysam
height                  = 50
fgcolor                 = black
bicolor_pivot           = 20
pos_color               = royalblue
neg_color               = red
key                     = Coverage ovary
citation                = RNA-Seq reads combined from all developmental stages.
category                = RNA-Seq

#Correct data is shown
[Reads]
feature        = match
glyph          = segments
database       = ovarysam
draw_target    = 1
show_mismatch  = 1
mismatch_color = red
bgcolor        = royalblue
fgcolor        = black
height         = 5
label density  = 50
feature_limit  = 5000
bump           = fast
connector      = solid
key            = RNA-Seq reads match ovary
category       = Experimental Data

 

Is there a method to verify the bam files are correct?
What could be causing the coverage track to be wrong?

Best Regards
Stefan Pfeiffer

------------------------------------------------------------------------------
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--
------------------------------------------------------------------------
Scott Cain, Ph. D.                                   scott at scottcain dot net
GMOD Coordinator (http://gmod.org/)                     <a href="tel:(216)%20392-3087" value="+12163923087" target="_blank">216-392-3087
Ontario Institute for Cancer Research

 

 



--
------------------------------------------------------------------------
Scott Cain, Ph. D.                                   scott at scottcain dot net
GMOD Coordinator (http://gmod.org/)                     216-392-3087
Ontario Institute for Cancer Research

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Re: Coverage track not working

Timothy Parnell
In reply to this post by Zipixx
Hi Stefan,

If I navigate to the link you provided below, and dump your bam file, I noticed that virtually all of the alignments are marked as “not primary alignment” (flag bit value of 256 or 272). In fact, only 36 alignments are marked as primary alignments. That can easily account for the sparse coverage in the coverage_xyplot graph.

Evidently the Bio::DB::Sam adapter coverage method employs a low level filter to skip non-primary alignments, which I never realized before. Low level as in this is not the Perl stuff but in either the XS code or the samtools C library.

In IGV, you can filter secondary alignments. It should recreate what you see in GBrowse. Considering Bio::DB::Sam has now been superseded by Bio::DB::HTS (which is not exactly a drop-in replacement), there’s not much you can do about this except to use bigWig generated by a different program.

Tim


On May 31, 2017, at 10:01 AM, Stefan Pfeiffer <[hidden email]<mailto:[hidden email]>> wrote:


Hi Scott,

thank you for the suggestion. IGV does show the correct and expected coverage.
Zooming in GBrowse does not change anything.
You can see the result at https://vm19002.virt.gwdg.de/gb2/gbrowse/tribolium/?name=id:469064

If it is helpful, we could also upload the bam file.
samtools view wrong_coverage.bam returns lines like the following.  The position/length integers in the 8th/9th column remain 0 throughout the entire file, which is not the case for correct_coverage.bam. Could this be the cause?

ILLUMINA-D0898A_0001:1:94:15808:4394#0/1        0       NW_015452213.1  1447    40      38M     *       0       0       TGTGGTACTCGTGTACACAGCAGAAGAATTATCAGTAT  bbbbbbbbbbbbbbbbbbabbbbbbbbbbbbbbbbbbb  MD:Z:38 NH:i:1  HI:i:1  NM:i:0  SM:i:40 XQ:i:40 X2:i:0
ILLUMINA-D0898A_0001:1:2:16831:1638#0/1 0       NW_015452213.1  1449    40      38M     *       0       0       TGGTACTCGTGTACACAGCAGAAGAATTATCAGTATAA  ]abbbbbbabaabbbbbbbbbbbbabbbbbbbbbbbbb  MD:Z:38 NH:i:1  HI:i:1  NM:i:0  SM:i:40 XQ:i:40 X2:i:0

Best Regards
Stefan



On 2017-05-10 16:51, Scott Cain wrote:

Hi Stefan,

I know that there can be problems associated with sampling the bam data when drawing coverage plots. Have you tried zooming in and out to see if the coverage plot changes to look more "right".  Similarly, have you tried opening the bam file in other software like IGV to see if it looks right?

Scott


On Mon, May 8, 2017 at 11:59 AM, Stefan Pfeiffer <[hidden email]<mailto:[hidden email]>> wrote:

Hi,

I am working with two databases in my configuration file and need to display coverage tracks for both of them.
One works fine, the other does not. When using feature = match and glyph = segments however, the correct data is shown.

The track definitions for databases rnaseqall and ovarysam:

#This coverage track works
[READ_COVERAGE]
feature                 = coverage
glyph                   = wiggle_xyplot
database             = rnaseqall
height                  = 50
fgcolor                 = black
bicolor_pivot       = 20
pos_color             = royalblue
neg_color             = red
key                     = Coverage from RNA-Seq
citation                = RNA-Seq reads combined from all developmental stages.
category                = RNA-Seq

#This track does not work correctly. A scale with and a few non-matching peeks are shown.
[READ_COVERAGE_OVARY]
feature                 = coverage
glyph                   = wiggle_xyplot
database                = ovarysam
height                  = 50
fgcolor                 = black
bicolor_pivot           = 20
pos_color               = royalblue
neg_color               = red
key                     = Coverage ovary
citation                = RNA-Seq reads combined from all developmental stages.
category                = RNA-Seq

#Correct data is shown
[Reads]
feature        = match
glyph          = segments
database       = ovarysam
draw_target    = 1
show_mismatch  = 1
mismatch_color = red
bgcolor        = royalblue
fgcolor        = black
height         = 5
label density  = 50
feature_limit  = 5000
bump           = fast
connector      = solid
key            = RNA-Seq reads match ovary
category       = Experimental Data



Is there a method to verify the bam files are correct?
What could be causing the coverage track to be wrong?

Best Regards
Stefan Pfeiffer

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GMOD Coordinator (http://gmod.org/)                     216-392-3087
Ontario Institute for Cancer Research


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Re: Coverage track not working

Keiran Raine

Hi,

 

We created a C program for generating bigwig files following the deprecation of Bio::DB::Sam.  This has the added advantage that you can select which read flags are used in the coverage calculations:

 

https://github.com/cancerit/cgpBigWig#bam2bw

 

You can also use bam2bw in a per-chromosome mode (by sepecifying '--region') and then merge the data with bwjoin for faster turn around.  This is simplified using bamTobw.pl (part of PCAP-core) to handle the multiple threads and merging for you but that is a much larger install.

 

Regards,

 

Keiran Raine

Principal Bioinformatician

Cancer Genome Project

Wellcome Trust Sanger Institute

[hidden email]

Tel:+44 (0)1223 834244 Ext: 4983

Office: H104

 

On 01/06/2017, 00:20, "Timothy Parnell" <[hidden email]> wrote:

 

    Hi Stefan,

   

    If I navigate to the link you provided below, and dump your bam file, I noticed that virtually all of the alignments are marked as “not primary alignment” (flag bit value of 256 or 272). In fact, only 36 alignments are marked as primary alignments. That can easily account for the sparse coverage in the coverage_xyplot graph.

   

    Evidently the Bio::DB::Sam adapter coverage method employs a low level filter to skip non-primary alignments, which I never realized before. Low level as in this is not the Perl stuff but in either the XS code or the samtools C library.

   

    In IGV, you can filter secondary alignments. It should recreate what you see in GBrowse. Considering Bio::DB::Sam has now been superseded by Bio::DB::HTS (which is not exactly a drop-in replacement), there’s not much you can do about this except to use bigWig generated by a different program.

   

    Tim

   

    

    On May 31, 2017, at 10:01 AM, Stefan Pfeiffer <[hidden email]<mailto:[hidden email]>> wrote:

   

    

    Hi Scott,

   

    thank you for the suggestion. IGV does show the correct and expected coverage.

    Zooming in GBrowse does not change anything.

    You can see the result at https://vm19002.virt.gwdg.de/gb2/gbrowse/tribolium/?name=id:469064

   

    If it is helpful, we could also upload the bam file.

    samtools view wrong_coverage.bam returns lines like the following.  The position/length integers in the 8th/9th column remain 0 throughout the entire file, which is not the case for correct_coverage.bam. Could this be the cause?

   

    ILLUMINA-D0898A_0001:1:94:15808:4394#0/1        0       NW_015452213.1  1447    40      38M     *       0       0       TGTGGTACTCGTGTACACAGCAGAAGAATTATCAGTAT  bbbbbbbbbbbbbbbbbbabbbbbbbbbbbbbbbbbbb  MD:Z:38 NH:i:1  HI:i:1  NM:i:0  SM:i:40 XQ:i:40 X2:i:0

    ILLUMINA-D0898A_0001:1:2:16831:1638#0/1 0       NW_015452213.1  1449    40      38M     *       0       0       TGGTACTCGTGTACACAGCAGAAGAATTATCAGTATAA  ]abbbbbbabaabbbbbbbbbbbbabbbbbbbbbbbbb  MD:Z:38 NH:i:1  HI:i:1  NM:i:0  SM:i:40 XQ:i:40 X2:i:0

   

    Best Regards

    Stefan

   

    

    

    On 2017-05-10 16:51, Scott Cain wrote:

   

    Hi Stefan,

   

    I know that there can be problems associated with sampling the bam data when drawing coverage plots. Have you tried zooming in and out to see if the coverage plot changes to look more "right".  Similarly, have you tried opening the bam file in other software like IGV to see if it looks right?

   

    Scott

   

    

    On Mon, May 8, 2017 at 11:59 AM, Stefan Pfeiffer <[hidden email]<mailto:[hidden email]>> wrote:

   

    Hi,

   

    I am working with two databases in my configuration file and need to display coverage tracks for both of them.

    One works fine, the other does not. When using feature = match and glyph = segments however, the correct data is shown.

   

    The track definitions for databases rnaseqall and ovarysam:

   

    #This coverage track works

    [READ_COVERAGE]

    feature                 = coverage

    glyph                   = wiggle_xyplot

    database             = rnaseqall

    height                  = 50

    fgcolor                 = black

    bicolor_pivot       = 20

    pos_color             = royalblue

    neg_color             = red

    key                     = Coverage from RNA-Seq

    citation                = RNA-Seq reads combined from all developmental stages.

    category                = RNA-Seq

   

    #This track does not work correctly. A scale with and a few non-matching peeks are shown.

    [READ_COVERAGE_OVARY]

    feature                 = coverage

    glyph                   = wiggle_xyplot

    database                = ovarysam

    height                  = 50

    fgcolor                 = black

    bicolor_pivot           = 20

    pos_color               = royalblue

    neg_color               = red

    key                     = Coverage ovary

    citation                = RNA-Seq reads combined from all developmental stages.

    category                = RNA-Seq

   

    #Correct data is shown

    [Reads]

    feature        = match

    glyph          = segments

    database       = ovarysam

    draw_target    = 1

    show_mismatch  = 1

    mismatch_color = red

    bgcolor        = royalblue

    fgcolor        = black

    height         = 5

    label density  = 50

    feature_limit  = 5000

    bump           = fast

    connector      = solid

    key            = RNA-Seq reads match ovary

    category       = Experimental Data

   

    

    

    Is there a method to verify the bam files are correct?

    What could be causing the coverage track to be wrong?

   

    Best Regards

    Stefan Pfeiffer

   

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    --

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    Scott Cain, Ph. D.                                   scott at scottcain dot net

    GMOD Coordinator (http://gmod.org/)                     216-392-3087

    Ontario Institute for Cancer Research

   

    

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