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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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