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Old 05-22-2015, 10:19 AM   #1
millrick10
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Default Using bowtie2 with min and max fragment fragment

I am pretty new to bioinformatics, and I was hoping that I could get some help with a bowtie2 script that I am putting together.

I am aligning ChIP-seq paired-end reads (2 samples: Input control and ChIP sample) to a reference and I want to include a minimum fragment size of 200bp and maximum fragment size of 600bp. A pseudocode version of my script is below.

/(path)/bowtie2 I 200 X 600 -x /(path)/genomeIndex -1 (chip/IN).fastq -2 (chip/IN).fastq -S alignment.sam

When I run this code, I get the error:

"Extra parameter(s) specified: "I", "200", "X", "600"
Note that if <mates> files are specified using -1/-2, a <singles> file cannot
also be specified. Please run bowtie separately for mates and singles.
Error: Encountered internal Bowtie 2 exception (#1)"

and:

"(ERR): bowtie2-align exited with value 1"

I am not specifying any singles files, only mate pair files, so I am thoroughly confused. Any suggestions?
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Old 05-22-2015, 10:29 AM   #2
westerman
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I think you have double dashes for '-I' and '-X'. They should be single dashes or the long name with double dash; e.g., '--minins' and '--maxins'. Take care of that problem first and then let us know if there are problems with '-1' and '-2'.
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Old 05-22-2015, 10:34 AM   #3
millrick10
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@westerman

Nope, they are single dashes in "-I" and "-X." For good measure, I also swapped "--minins" for "-I" and "--maxins" for "-X" and it still gave me the same error.

Thanks for the idea though.
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Old 05-22-2015, 10:38 AM   #4
westerman
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Could you paste in the exact error you get when using '--minins' and '--maxins'


Also the results of 'bowtie2 --version'

Last edited by westerman; 05-22-2015 at 10:40 AM.
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Old 05-22-2015, 10:44 AM   #5
millrick10
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@westerman

Sure thing, it is below.

My code:
/(path)/bowtie2 --minins 200 --maxins 600 -x /(path)/genomeIndex -1 (reads1).fastq -2 (reads2).fastq -S (alignment).sam

The error:
Extra parameter(s) specified: "--minins", "200", "--maxins", "600"
Note that if <mates> files are specified using -1/-2, a <singles> file cannot
also be specified. Please run bowtie separately for mates and singles.
Error: Encountered internal Bowtie 2 exception (#1)
Command: /(path)/bowtie2-align-s --wrapper basic-0 -x /(path)/bowtie2-2.2.3/indexes/(genomeIndex) -S (alignment).sam -1 (reads1).fastq -2 (reads2).fastq --minins 200 --maxins 600
(ERR): bowtie2-align exited with value 1

Also, I have to apologize, in the code that I originally posted, I did have double dashes with I and X. I fixed it though, and it did not get rid of the error.
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Old 05-22-2015, 10:49 AM   #6
millrick10
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@westerman

Results of "bowtie2 --version":
"64-bit
Built on pc-201305311418.ebalto.jhmi.edu
Thu May 29 12:48:36 EDT 2014
Compiler: gcc version 4.2.1 (Based on Apple Inc. build 5658) (LLVM build 2336.11.00)
Options: -O3 -m64 -msse2 -funroll-loops -g3 -DPOPCNT_CAPABILITY
Sizeof {int, long, long long, void*, size_t, off_t}: {4, 8, 8, 8, 8, 8}"
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Old 05-22-2015, 11:09 AM   #7
westerman
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This is strange. Not the 'Note that ...' part. That message (as per the source code) will be output if extra parameters are found. The strange part is why bowtie2 thinks you have extra parameters. What happens if you take out the minins and maxins parameters?

BTW, the source code says "... TODO: Argument parsing is very, very flawed. ..." which, having looked at it, is a statement I agree with.

Last edited by westerman; 05-22-2015 at 11:17 AM. Reason: syntax
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Old 05-22-2015, 11:25 AM   #8
GenoMax
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What OS is this on @millrick10?

I am not getting an error with your exact options/order they are in.

Last edited by GenoMax; 05-22-2015 at 11:28 AM.
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Old 05-22-2015, 11:32 AM   #9
westerman
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@GenoMax and @millrick10

It does look like a Mac system which may cause problems.

BTW, my bowtie --version (on Linux) looks like:

/group/bioinfo/apps/apps/bowtie2-2.2.5/bowtie2-align-s version 2.2.5
64-bit
Built on localhost.localdomain
Thu Mar 5 18:34:52 EST 2015
Compiler: gcc version 4.1.2 20080704 (Red Hat 4.1.2-54)
Options: -O3 -m64 -msse2 -funroll-loops -g3 -DPOPCNT_CAPABILITY
Sizeof {int, long, long long, void*, size_t, off_t}: {4, 8, 8, 8, 8, 8}
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Old 05-22-2015, 11:43 AM   #10
GenoMax
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Tested on a Mac with latest bowtie (v.2.2.5) and the lambda test data that comes with bowtie. No errors.

@millrick10: Are you using the latest Mac binary?
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Old 05-22-2015, 02:47 PM   #11
millrick10
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So if I run the code below, it works. I had to take out my options (I want to use "--minins 200" and "--maxins 600").

/(path)/bowtie2 -x /(path)/genomeIndex -1 /(path)/(reads1).fastq -2 /(path)/(reads2).fastq -S (alignment).sam

If I run it with my desired options (code below):

/(path)/bowtie2 --minins 200 --maxins 600 -x /(path)/genomeIndex -1 /(path)/(reads1).fastq -2 /(path)/(reads2).fastq -S (alignment).sam

I get the error:

Extra parameter(s) specified: "—-maxins", "600"
Note that if <mates> files are specified using -1/-2, a <singles> file cannot
also be specified. Please run bowtie separately for mates and singles.
Error: Encountered internal Bowtie 2 exception (#1)
Command: /(path)/bowtie2-align-s --wrapper basic-0 --minins 200 -x /(path)/genomeIndex -S (alignment).sam -1 (reads1).fastq -2 (reads2).fastq --maxins 600
(ERR): bowtie2-align exited with value 1

@GenoMax, I am using OS X version 10.9.5.

I also tried updating my bowtie2 from 2.2.3 to 2.2.5 and that has not helped.
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Old 05-22-2015, 05:16 PM   #12
GenoMax
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That's odd. It must be the shell script that you are using that is causing the error.

Did you get the pre-compiled binary or compiled from source yourself?
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Old 05-27-2015, 06:03 PM   #13
wetSEQer
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What if you disable the -I and -X option, just run a basic bowtie2 run and see what happens?
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