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  • anoopkmr
    replied
    This tools helps me create bed files from a gene list:
    https://genome-euro.ucsc.edu/cgi-bin/hgTables

    Choose bed format while downloading.

      Leave a comment:

    • Gopo
      replied
      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

        Leave a comment:

      • Gopo
        replied
        [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]

          Leave a comment:

        • Gopo
          replied
          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

            Leave a comment:

          • Gopo
            replied
            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

              Leave a comment:

            • mhmtgenc
              replied
              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?

                Leave a comment:

              • Gopo
                replied
                Try CollectHSmetrics - part of Picard Tools
                For details see:
                Tool documentation
                https://broadinstitute.github.io/picard/command-line-overview.html

                  Leave a comment:

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

                  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
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