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  • mhmtgenc
    Junior Member
    • Nov 2016
    • 7
    #1

    How to get a report like stuff of a bam file how many percent of the exons are cover

    Dear Collegues,

    Lets say I have a miseq run and have the .bam file from the squencer and I would like to know how many percent of the exons (specific genes) are coverred in these .bam files?

    Is it possible?

    Thanks in advance
    Tags: bam, coverage, exon, miseq, ngs
  • Gopo
    Member
    • Nov 2013
    • 41
    #2
    Try CollectHSmetrics - part of Picard Tools
    For details see:
    Tool documentation
    https://broadinstitute.github.io/picard/command-line-overview.html

    Comment

    • mhmtgenc
      Junior Member
      • Nov 2016
      • 7
      #3
      Originally posted by Gopo View Post
      Try CollectHSmetrics - part of Picard Tools
      For details see:
      https://broadinstitute.github.io/pic...-overview.html
      So how coul I be able to get a BED file or prepare it by myself? Could you give me a clue or a tutorial of this. Cause I have trouble on this. I know this is hard to expalin it here but can I get any file or manual for this?

      Comment

      • Gopo
        Member
        • Nov 2013
        • 41
        #4
        So, you will have to generate the BED file yourself based on the coordinates of the genes or exons that you are interested in.

        Code:
        # generate fasta index for genome (genome is AmexG_v3.0.0.fa)
samtools faidx AmexG_v3.0.0.fa

# create sequence dictionary
java -Xmx64g -jar ~/bin/picard-2.18.10.jar CreateSequenceDictionary \
R=AmexG_v3.0.0.fa \
O=AmexG_v3.0.0.dict

# convert Bed to interval list
java -jar ~/bin/picard-2.18.10.jar BedToIntervalList \
I=rfs.immunome.bed \
O=rfs.immunome.interval.list \
SD=AmexG_v3.0.0.dict

# run CollectHsMetrics
java -Xmx64g -jar ~/bin/picard-2.18.10.jar CollectHsMetrics \
BAIT_INTERVALS=/ssdwork/jelber2/rfs/rfs.immunome.interval.list \
BAIT_SET_NAME=Immunome \
TARGET_INTERVALS=/ssdwork/jelber2/rfs/rfs.immunome.interval.list \
METRIC_ACCUMULATION_LEVEL=SAMPLE \
R=/ssdwork/jelber2/rfs/AmexG_v3.0.0.fa \
I=ALL-samples.bam \
O=ALL-samples-coverage-metrics.txt
        # note that you might have to add readgroups to the file ALL-samples.bam (whatever your BAM file is named) with

        Code:
        java -Xmx64g -jar ~/bin/picard.jar AddOrReplaceReadGroups \
    I=ALL-samples-recal-no-read-groups.bam \
    O=ALL-samples-recal-no-read-groups-for-callableloci.bam \
    SORT_ORDER=coordinate \
    RGPL=illumina \
    RGPU=barcode \
    RGLB=Lib1 \
    RGID=all \
    RGSM=all \
    VALIDATION_STRINGENCY=LENIENT

        Comment

        • Gopo
          Member
          • Nov 2013
          • 41
          #5
          Code:
          # install picard
cd ~/bin/
wget https://github.com/broadinstitute/picard/releases/download/2.18.10/picard.jar
mv picard.jar picard-2.18.10.jar

# reference genome is AmexG_v3.0.0.fa
# generate fasta index
samtools faidx AmexG_v3.0.0.fa

# create sequence dictionary
java -Xmx64g -jar ~/bin/picard-2.18.10.jar CreateSequenceDictionary \
R=AmexG_v3.0.0.fa \
O=AmexG_v3.0.0.dict

# convert bed to interval list
java -jar ~/bin/picard-2.18.10.jar BedToIntervalList \
I=rfs.immunome.bed \
O=rfs.immunome.interval.list \
SD=AmexG_v3.0.0.dict

# run CollectHsMetrics
java -Xmx64g -jar ~/bin/picard-2.18.10.jar CollectHsMetrics \
BAIT_INTERVALS=/ssdwork/jelber2/rfs/rfs.immunome.interval.list \
BAIT_SET_NAME=Immunome \
TARGET_INTERVALS=/ssdwork/jelber2/rfs/rfs.immunome.interval.list \
METRIC_ACCUMULATION_LEVEL=SAMPLE \
R=/ssdwork/jelber2/rfs/AmexG_v3.0.0.fa \
I=ALL-samples.bam \
O=ALL-samples-coverage-metrics.txt

# if you need to add read groups
java -Xmx64g -jar ~/bin/picard-2.18.10.jar AddOrReplaceReadGroups \
I=ALL-samples.bam \
O=ALL-samples.RG.bam \
SORT_ORDER=coordinate \
RGPL=illumina \
RGPU=barcode \
RGLB=Lib1 \
RGID=all \
RGSM=all \
VALIDATION_STRINGENCY=LENIENT

java -Xmx64g -jar ~/bin/picard-2.18.10.jar CollectHsMetrics \
BAIT_INTERVALS=/ssdwork/jelber2/rfs/rfs.immunome.interval.list \
BAIT_SET_NAME=Immunome \
TARGET_INTERVALS=/ssdwork/jelber2/rfs/rfs.immunome.interval.list \
METRIC_ACCUMULATION_LEVEL=SAMPLE \
R=/ssdwork/jelber2/rfs/AmexG_v3.0.0.fa \
I=ALL-samples.RG.bam \
O=ALL-samples-coverage-metrics.txt

          Comment

          • Gopo
            Member
            • Nov 2013
            • 41
            #6
            [CODE]
            # install picard
            cd ~/bin/
            wget https://github.com/broadinstitute/picard/releases/download/2.18.10/picard.jar
            mv picard.jar picard-2.18.10.jar

            # reference genome is AmexG_v3.0.0.fa
            # generate fasta index
            samtools faidx AmexG_v3.0.0.fa

            # create sequence dictionary
            java -Xmx64g -jar ~/bin/picard-2.18.10.jar CreateSequenceDictionary \
            R=AmexG_v3.0.0.fa \
            O=AmexG_v3.0.0.dict

            # convert bed to interval list
            java -jar ~/bin/picard-2.18.10.jar BedToIntervalList \
            I=rfs.immunome.bed \
            O=rfs.immunome.interval.list \
            SD=AmexG_v3.0.0.dict

            # run CollectHsMetrics
            java -Xmx64g -jar ~/bin/picard-2.18.10.jar CollectHsMetrics \
            BAIT_INTERVALS=/ssdwork/jelber2/rfs/rfs.immunome.interval.list \
            BAIT_SET_NAME=Immunome \
            TARGET_INTERVALS=/ssdwork/jelber2/rfs/rfs.immunome.interval.list \
            METRIC_ACCUMULATION_LEVEL=SAMPLE \
            R=/ssdwork/jelber2/rfs/AmexG_v3.0.0.fa \
            I=ALL-samples.bam \
            O=ALL-samples-coverage-metrics.txt

            # if you need to add read groups
            java -Xmx64g -jar ~/bin/picard-2.18.10.jar AddOrReplaceReadGroups \
            I=ALL-samples.bam \
            O=ALL-samples.RG.bam \
            SORT_ORDER=coordinate \
            RGPL=illumina \
            RGPU=barcode \
            RGLB=Lib1 \
            RGID=all \
            RGSM=all \
            VALIDATION_STRINGENCY=LENIENT

            java -Xmx64g -jar ~/bin/picard-2.18.10.jar CollectHsMetrics \
            BAIT_INTERVALS=/ssdwork/jelber2/rfs/rfs.immunome.interval.list \
            BAIT_SET_NAME=Immunome \
            TARGET_INTERVALS=/ssdwork/jelber2/rfs/rfs.immunome.interval.list \
            METRIC_ACCUMULATION_LEVEL=SAMPLE \
            R=/ssdwork/jelber2/rfs/AmexG_v3.0.0.fa \
            I=ALL-samples.RG.bam \
            O=ALL-samples-coverage-metrics.txt
            [\CODE]

            Comment

            • Gopo
              Member
              • Nov 2013
              • 41
              #7
              You will have to make the BED file yourself. Here is a guide:

              Code:
              # install picard
cd ~/bin/
wget https://github.com/broadinstitute/picard/releases/download/2.18.10/picard.jar
mv picard.jar picard-2.18.10.jar

# index reference (Reference is AmexG_v3.0.0.fa)
samtools faidx AmexG_v3.0.0.fa

# create sequence dictionary
java -Xmx64g -jar ~/bin/picard-2.18.10.jar CreateSequenceDictionary \
R=AmexG_v3.0.0.fa \
O=AmexG_v3.0.0.dict

# Convert BED to interval list
java -jar ~/bin/picard-2.18.10.jar BedToIntervalList \
I=rfs.immunome.bed \
O=rfs.immunome.interval.list \
SD=AmexG_v3.0.0.dict

# run CollectHsMetrics
java -Xmx64g -jar ~/bin/picard-2.18.10.jar CollectHsMetrics \
BAIT_INTERVALS=/ssdwork/jelber2/rfs/rfs.immunome.interval.list \
BAIT_SET_NAME=Immunome \
TARGET_INTERVALS=/ssdwork/jelber2/rfs/rfs.immunome.interval.list \
METRIC_ACCUMULATION_LEVEL=SAMPLE \
R=/ssdwork/jelber2/rfs/AmexG_v3.0.0.fa \
I=ALL-samples.bam \
O=ALL-samples-coverage-metrics.txt

# if needed, add readgroups
java -Xmx64g -jar ~/bin/picard.jar AddOrReplaceReadGroups \
I=ALL-samples.bam \
O=ALL-samples-RG.bam \
SORT_ORDER=coordinate \
RGPL=illumina \
RGPU=barcode \
RGLB=Lib1 \
RGID=all \
RGSM=all \
VALIDATION_STRINGENCY=LENIENT

# run CollectHsMetrics with ReadGroups added to BAM
java -Xmx64g -jar ~/bin/picard-2.18.10.jar CollectHsMetrics \
BAIT_INTERVALS=/ssdwork/jelber2/rfs/rfs.immunome.interval.list \
BAIT_SET_NAME=Immunome \
TARGET_INTERVALS=/ssdwork/jelber2/rfs/rfs.immunome.interval.list \
METRIC_ACCUMULATION_LEVEL=SAMPLE \
R=/ssdwork/jelber2/rfs/AmexG_v3.0.0.fa \
I=ALL-samples-RG.bam \
O=ALL-samples-coverage-metrics.txt
              Best,
              Gopo
              Last edited by Gopo; 08-08-2018, 10:12 PM. Reason: wrong syntax for displaying code

              Comment

              • anoopkmr
                Junior Member
                • Aug 2018
                • 4
                #8
                This tools helps me create bed files from a gene list:
                https://genome-euro.ucsc.edu/cgi-bin/hgTables


                Choose bed format while downloading.

                Comment

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