[GenoMax]

For your version (v.1.48?) of picard-tools try:

$ picard-tools CalculateHsMetrics BI=./target_regions.bed TI=./target_regions.bed I=./epilepsy.bam O=./epilepsy.CalculateHSmetrics

If you were to run the command standalone (like below) it will provide you help with all possible options that the command will accept.

$ picard-tools CalculateHsMetrics

The particular version of picard-tools that you installed from Ubuntu repository does not support the reference file option shown by Heisman. This is something you have to get used to and learn. As software develops the developer may add options (or remove some) that were not present in previous versions of the software.

If you want to use the latest version of the software (v. 1.95) you will need to download it from the source here: http://sourceforge.net/projects/picard/files/

[cmccabe]

picard HSmetrics

$ picard-tools CalculateHsMetrics BI=./target_regions.bed TI=./target_regions.bed I=./epilepsy.bam O=./epilepsy.CalculateHSmetrics

ERROR: Option 'INPUT' is required (is the .bam not aligned?) I used the bedToBam in Bedtools 2.17.0. Thanks.

[Heisman]

That implies it cannot find your input file. Could you copy and paste the output of "picard-tools CalculateHsMetrics"? Also do "ls -l" and make sure "epilepsy.bam" is in the directory you are running the command from.

[cmccabe]

picard HSmetrics

dnascopev@ubuntu:~$ cd "picardtools-1.72"
dnascopev@ubuntu:~/picardtools-1.72$ ls -lt
total 21745
-rw-rw-r-- 1 dnascopev dnascopev 16544 Jul 15 16:25 epilepsy.bam
-rw-rw-r-- 1 dnascopev dnascopev 51482 Jul 12 14:35 epilepsy.bed
-rw-r--r-- 1 dnascopev dnascopev 22106574 Jul 14 2012 hg19_fasta.txt

dnascopev@ubuntu:~/picardtools-1.72$ picard-tools CalculateHsMetrics
ERROR: Option 'BAIT_INTERVALS' is required.

 picard-tools CalculateHsMetrics BI=./epilepsy.bed TI=./epilepsy.bed I=./epilepsy.bam O=./epilepsy.CalculateHSmetrics

Thanks.

[Heisman]

Please type "picard-tools CalculateHsMetrics" and copy and paste the output if it gives you like a long list of different options. Perhaps you have to type BAIT_INTERVALS=./epilepsy.bed instead of BI. If you can get a list of options for the version you are using, this will be obvious. You may have to type "picard-tools CalculateHsMetrics -h" or "picard-tools CalculateHsMetrics --help".

[cmccabe]

picard HSmetrics

dnascopev@ubuntu:~/picardtools-1.72$ picard-tools CalculateHsMetrics
ERROR: Option 'BAIT_INTERVALS' is required.

Calculates a set of Hybrid Selection specific metrics from an aligned SAMor BAM file.

Options:

--help
-h Displays options specific to this tool.

--stdhelp
-H Displays options specific to this tool AND options common to all Picard command line
tools.

BAIT_INTERVALS=File
BI=File An interval list file that contains the locations of the baits used. Required.

TARGET_INTERVALS=File
TI=File An interval list file that contains the locations of the targets. Required.

INPUT=File
I=File An aligned SAM or BAM file. Required.

OUTPUT=File
O=File The output file to write the metrics to. Required. Cannot be used in conjuction with
option(s) METRICS_FILE (M)

METRICS_FILE=File
M=File Legacy synonym for OUTPUT, should not be used. Required. Cannot be used in conjuction
with option(s) OUTPUT (O)

 picard-tools CalculateHsMetrics BI=./epilepsy.bed TI=./epilepsy.bed I=./epilepsy.bam O=./epilepsy.CalculateHSmetrics

Thank you.

[Heisman]

Ah, I'm an idiot. The bait interval file needs a special format.

Would you be willing to send me your original bed file and your targets file via dropbox? If you put it in a public folder and make it available to me I can do this for you (or try to).

[cmccabe]

picard HSmetrics

What is your dropbox member ID I should share the folder with? Thanks

[Heisman]

I'm sending you a message with my email. If you can make those files available to me I can check to see if the original bed file you had actually contains aligned reads and I can generate a workable bait interval file for you, as well as run it once to ensure it works.

[cmccabe]

picard HSmetrics

put the bed and the bam in the folder, sorry and thanks.

[Heisman]

So there seems to have been a misunderstanding somewhere; you gave me a bed file and the bam file that was created from the bed file. Two questions:

1. where did the bed file originally come from? Does it represent aligned reads? I don't think it actually does because the regions are many different lengths.

2. Where is the file that represents your target regions? Is it possible that the bed file you provided me actually represents your target regions and that you haven't provided me a file with read information at all?

[cmccabe]

picard HSmetrics

The bed file is an Ion Torrent design file of unaligned targets.

I do not have any read information as I have not ordered these primers rather was looking for a way to in-silco test the off-target reads based on the file. Maybe this is not possible. Thanks.

[Heisman]

Ok, so the bed file is the targets. At least that's clear.

So you would like to get an idea of what percentage of data you can expect to get on target, based on your experimental design, correct? I have never done any type of targeted sequencing with Ion Torrent so I don't know what their typical percent on target metrics are. I would contact them and ask what you can realistically expect.

[cmccabe]

picard HSmetics

Unfourtunaly, the only way right now to calculate that is after the sequencing is complete. I do not want to buy the primer sets (expensive) beforehand and was hoping that there was a way to calculate the predicted -off-targets reads. I guess maybe a BLAT might be a good starting point. Thank you.