Regarding Depth coverage

Question

[manojkumarbioinfo]

Hi. all

I'm new to field of NGS. and I am trying to calculate the Dept Coverage for my alignment.

For this I use picard tool for calculating

I have used CalculateHsMetrics for calculating my depth but I cant get my output

It show the following message

bioinformatics@BIF-Server:~/Documents/Mano/test$ java -jar /home/bioinformatics/Documents/Mano/Tools/picard-tools-1.141/picard.jar CalculateHsMetrics I=realignment_reads.bam R=/home/bioinformatics/Documents/Mano/Reference/hg19/hg19.fasta TI=RB1.bed BI=RB1.bed VALIDATION_STRINGENCY=SILENT O=result
[Thu Dec 31 18:17:09 IST 2015] picard.analysis.directed.CalculateHsMetrics BAIT_INTERVALS=[RB1.bed] TARGET_INTERVALS=[RB1.bed] INPUT=realignment_reads.bam OUTPUT=result VALIDATION_STRINGENCY=SILENT REFERENCE_SEQUENCE=/home/bioinformatics/Documents/Mano/Reference/hg19/hg19.fasta    METRIC_ACCUMULATION_LEVEL=[ALL_READS] VERBOSITY=INFO QUIET=false COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false GA4GH_CLIENT_SECRETS=client_secrets.json
[Thu Dec 31 18:17:09 IST 2015] Executing as bioinformatics@BIF-Server on Linux 3.19.0-41-generic amd64; OpenJDK 64-Bit Server VM 1.7.0_91-b02; Picard version: 1.141(8ece590411350163e7689e9e77aab8efcb622170_1447695087) IntelDeflater
[Thu Dec 31 18:17:09 IST 2015] picard.analysis.directed.CalculateHsMetrics done. Elapsed time: 0.00 minutes.
Runtime.totalMemory()=2058354688
To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp
Exception in thread "main" java.lang.IllegalStateException: Interval list file must contain header. 
    at htsjdk.samtools.util.IntervalList.fromReader(IntervalList.java:436)
    at htsjdk.samtools.util.IntervalList.fromFile(IntervalList.java:381)
    at htsjdk.samtools.util.IntervalList.fromFiles(IntervalList.java:410)
    at picard.analysis.directed.CollectTargetedMetrics.doWork(CollectTargetedMetrics.java:85)
    at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:209)
    at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:95)
    at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:105)

Answer 1

[Pierre Lindenbaum]

picard cannot read bed. You need to convert your bed using http://broadinstitute.github.io/picard/command-line-overview.html#BedToIntervalList

[manojkumarbioinfo]

@pirerre I tried to convert my bed file to interval list but I did not understood what is SEQUENCE_DICTIONARY (File)

[Pierre Lindenbaum]

A file ref.dict created with CreateSequenceDictionary in https://broadinstitute.github.io/picard/command-line-overview.html

[manojkumarbioinfo]

Thanks @Pierre I got my results.