[GenoMax]

Bedtools 2.17 is compilied on Ubuntu and the command:

bin/bamToBed -i Amplicons.bed -g genomes/human.hg19.genome > Amplicons.bam does run but there is an error extracting the gz file and it is 0 bytes. Thanks.

What "gz" file? There isn't one in the command you are executing. Can you post the exact error?

[cmccabe]

Bed2Bam

There is an amplicons.bab folder in the bedtools-2.17 directory that says gz and when I extract it An error occured extrating the files. Thank you.

[GenoMax]

Can you post a listing of files in this folder?

$ ls -lt

[Heisman]

Is your "Amplicons.bed" file actually gzip compressed? Otherwise this doesn't make sense. Your command line doesn't include "amplicons.bab" so I don't see why that is relevant for the command you typed. If you type "less Amplicons.bed", can you read the file, or does it tell you it may be binary format or something like that?

[cmccabe]

bed2Bam

total 141
-rw-rw-r-- 1 dnascopev dnascopev 16544 Jul 12 12:16 amplicons.bam
-rw-rw-r-- 1 dnascopev dnascopev 51482 Jul 12 12:06 epilepsy.bed
drwxrwxr-x 2 dnascopev dnascopev 1024 Jul 12 11:48 bin
drwxrwxr-x 2 dnascopev dnascopev 3072 Jul 12 11:48 obj
drwxr-xr-x 2 dnascopev dnascopev 1024 Jul 11 13:38 data
drwxr-xr-x 4 dnascopev dnascopev 1024 Jul 11 13:38 docs
drwxr-xr-x 2 dnascopev dnascopev 1024 Jul 11 13:38 genomes
drwxr-xr-x 2 dnascopev dnascopev 1024 Jul 11 13:38 scripts
drwxr-xr-x 40 dnascopev dnascopev 1024 Jul 11 13:38 src
-rw-r--r-- 1 dnascopev dnascopev 17988 Nov 3 2012 LICENSE
-rw-r--r-- 1 dnascopev dnascopev 6693 Nov 3 2012 Makefile
-rw-r--r-- 1 dnascopev dnascopev 1505 Nov 3 2012 README.rst
-rw-r--r-- 1 dnascopev dnascopev 32293 Nov 3 2012 RELEASE_HISTORY
drwxr-xr-x 20 dnascopev dnascopev 1024 Nov 3 2012 test

Thanks.

[Heisman]

You don't have a file called "Amplicons.bed" in that folder. Where is that file? You're trying to run the command using that file but you aren't specifying the correct path to the file.

[GenoMax]

Looks like this must have already worked (looking at the date stamps).

epilepsy.bed == Amplicons.bed

resulted in the "Amplicons.bam" file.

[cmccabe]

bed2Bam

total 141
-rw-rw-r-- 1 dnascopev dnascopev 16544 Jul 12 12:16 amplicons.bam
-rw-rw-r-- 1 dnascopev dnascopev 51482 Jul 12 12:06 epilepsy.bed
drwxrwxr-x 2 dnascopev dnascopev 1024 Jul 12 11:48 bin
drwxrwxr-x 2 dnascopev dnascopev 3072 Jul 12 11:48 obj
drwxr-xr-x 2 dnascopev dnascopev 1024 Jul 11 13:38 data
drwxr-xr-x 4 dnascopev dnascopev 1024 Jul 11 13:38 docs
drwxr-xr-x 2 dnascopev dnascopev 1024 Jul 11 13:38 genomes
drwxr-xr-x 2 dnascopev dnascopev 1024 Jul 11 13:38 scripts
drwxr-xr-x 40 dnascopev dnascopev 1024 Jul 11 13:38 src
-rw-r--r-- 1 dnascopev dnascopev 17988 Nov 3 2012 LICENSE
-rw-r--r-- 1 dnascopev dnascopev 6693 Nov 3 2012 Makefile
-rw-r--r-- 1 dnascopev dnascopev 1505 Nov 3 2012 README.rst
-rw-r--r-- 1 dnascopev dnascopev 32293 Nov 3 2012 RELEASE_HISTORY
drwxr-xr-x 20 dnascopev dnascopev 1024 Nov 3 2012 test

I changed to amplicons.bed and a GZ folder with the BAM results that is 0 bytes and I get an error occurred while extracting the files. The file type in unknown so maybe that is part of it, but is 0 bytes really right?Thanks.

[Heisman]

Oh, are you trying to extract the .bam file with "gunzip" or an equivalent?

Do you have samtools installed and compiled? If so, try to do "samtools view amplicons.bam" and you'll see what it actually looks like.

[cmccabe]

bed2Bam

Tool: bedtools bedtobam (aka bedToBam)
Version: v2.17.0
Summary: Converts feature records to BAM format.

Usage: bedtools bedtobam [OPTIONS] -i <bed/gff/vcf> -g <genome>

Options:
-mapq Set the mappinq quality for the BAM records.
(INT) Default: 255

-bed12 The BED file is in BED12 format. The BAM CIGAR
string will reflect BED "blocks".

-ubam Write uncompressed BAM output. Default writes compressed BAM.

Notes:
(1) BED files must be at least BED4 to create BAM (needs name field).

Looks like the bed is the problem, when I did dos2unix I got the above.

The bed:
chr1 40539720 40539896 AMPL4669271522 . PPT1
chr1 40542435 40542658 AMPL5154908878 . PPT1
chr1 40544200 40544360 AMPL3675665035 . PPT1

Thanks.

[cmccabe]

bed2Bam

dos2unix epilepsy.bed says skipping epilepsy.bed, not a regular file. Thanks.

[Heisman]

I'm confused. Above you show an "amplicons.bam" file of size 16544 bytes; definitely not 0. Why do you believe something is wrong?

[Heisman]

If you do "head -3 epilepsy.bed | od -c" and paste the output it should be clear if the file is fine.

[cmccabe]

bed2Bam

I have samtools installed, how do I compile it?

Eventhough the epilepsy.bam is in a gz folder picard will reconize it?

I am try to calculate the off-target reads and going to use HS metrics. Thank you for all your help and patience.

[Heisman]

I remember what we're trying to do; I think we'll get there eventually.

A tip: with most programs in this field, once you download and untar the directory, there is usually a README or INSTALL file that will tell you what to do. If you go to the samtools directory and type "cat INSTALL", you'll see what to do. Try to install it, and then try the "pathToSamtools/samtools view -h amplicons.bam and see if it looks ok to you.