This tools helps me create bed files from a gene list:
Choose bed format while downloading.
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You will have to make the BED file yourself. Here is a guide:
Best,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
GopoLeave a comment:
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[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
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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
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So, you will have to generate the BED file yourself based on the coordinates of the genes or exons that you are interested in.
# note that you might have to add readgroups to the file ALL-samples.bam (whatever your BAM file is named) withCode:# 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
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=LENIENTLeave a comment:
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Try CollectHSmetrics - part of Picard Tools
For details see:
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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
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Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...04-04-2024, 04:25 PM
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