Question

Bowtie2 command does not produce output file

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3.3 years ago

a.hayat20 • 0

Hi all,

I am new to Bioinformatics and have been stuck with aligning my data for 3 weeks! I feel like I have tried everything. I would like to align my ATAC-seq data using Bowtie2. Now I have previously done that using the command:

bowtie2 -x "name of index" -1 fast.reads -2 fast.reads -S name.sam

However, now when I run this command it updates/modifies the bowtie2 executable file for some reason!

The full command that I use is exactly as below:

cd bowtie2-2.3.4-mingw-x86_64/example/reference
bowtie2-2.3.4-mingw-x86_64\bowtie2 -x GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index \
    -1 bowtie2-2.3.4-mingw-x86_64/example/index/out.1.fastq \
    -2 /bowtie2-2.3.4-mingw-x86_64/example/index/out.2.fastq \
    -S 1.sam

Thanks so much for your help!

RNA-Seq ChIP-Seq alignment Assembly genome • 2.9k views

ADD COMMENT • link 3.3 years ago by a.hayat20 • 0

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first path looks like linux:

bowtie2-2.3.4-mingw-x86_64/example/reference

second path looks like windows ??

bowtie2-2.3.4-mingw-x86_64\bowtie2

ADD REPLY • link 3.3 years ago by Pierre Lindenbaum 161k

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Hello, you said no output file. Does your run get any error? Can you post some running log? The reference of bowtie2, can you check the name? For example my bowtie2 index file is "GRCh38.1.bt2, GRCh38.2.bt2, GRCh38.3.bt2..." so my -x is -x GRCh38 that remove file extention from file name. Your -x is -x GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index so the file name should be something like "GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index.1.bt2"

ADD REPLY • link 3.3 years ago by MatthewP ★ 1.4k

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Hi Matthew P and seidel - Thanks so much for your help!

I just downloaded Bowtie2 again and when I run the by dragging the file to the terminal (bowtie2 --help), it gives me this error.

/Downloads/bowtie2-2.4-2.2-macos-x86_64/bowtie2 
dyld: lazy symbol binding failed: Symbol not found: ____chkstk_darwin
  Referenced from: /Downloads/bowtie2-2.4-2.2-macos-x86_64/bowtie2-align-s (which was built for Mac OS X 10.15)
  Expected in: /usr/lib/libSystem.B.dylib

dyld: Symbol not found: ____chkstk_darwin
  Referenced from: /Downloads/bowtie2-2.4-2.2-macos-x86_64/bowtie2-align-s (which was built for Mac OS X 10.15)
  Expected in: /usr/lib/libSystem.B.dylib

(ERR): Description of arguments failed!
Exiting now ...

I just dragged the unix executable file (bowtie2) with the help option and the above error came up..

Not sure what is going on.. It seems to be a new thing each time! Thank you!

ADD REPLY • link updated 3.3 years ago by i.sudbery 19k • written 3.3 years ago by a.hayat20 • 0

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Which version of macOS are you using?

You may be best off using conda to install/use bioinformatics software. You can get started here (just first part for conda) : Creating workflows with snakemake and conda

ADD REPLY • link 3.3 years ago by GenoMax 142k

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Hi GenoMax,

Thanks so much! I installed conda and then installed Bowtie2 (it was so easy!) and I was able to call it. I am a bit confused about how to run the bowtie2 alignment command now. I know that the bowtie2 is the "environment", is the human reference genome also in the environment? How should I go on about aligning my data now?

Thanks!!!

ADD REPLY • link 3.3 years ago by a.hayat20 • 0

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conda activate bowtie2 (if that is the name of your conda env)
bowtie2 -x GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index \
    -1 /path_to/out.1.fastq \
    -2 /path_to/out.2.fastq \
    -S 1.sam

ADD REPLY • link 3.3 years ago by GenoMax 142k

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Oh, I think its working!! My Sam file is being created! I used the code Seidel shared with me: bowtie2 -x bowtie2-2.3.4-mingw-x86_64/example/reference/GCA_000001405.15_GRCh38_no_alt_analysis_set foo1.fastq

I ran this after installing conda.

THANK YOU SO MUCH! You are all life savers!!!!

ADD REPLY • link 3.3 years ago by a.hayat20 • 0

score 2 · Answer 1 · 2021-01-27

Some things for you to try. First off, get comfortable running toy examples.

Step1: type bowtie2. Do you get any output? Do you get A:

cws@pi ~$ bowtie2
No index, query, or output file specified!
Bowtie 2 version 2.3.4.1 by Ben Langmead (langmea@cs.jhu.edu, www.cs.jhu.edu/~langmea)
Usage:
    bowtie2 [options]* -x <bt2-idx> {-1 <m1> -2 <m2> | -U <r> | --interleaved <i>} [-S <sam>] 
[...]

or B:

cws@pi ~$ bowtie2
bash: bowtie2: command not found

If you get A, you can run bowtie2. If you get B, you'll have to find the location of bowtie2 and type the full path to the program. You might have to experiment to get this to work. Based on the above, you might be able to type: bowtie2-2.3.4-mingw-x86_64/bowtie2, but this is something you'll have to experiment with and solve on your own. But it's the easiest and first experiment to perform. (You should be able to type bowtie2 from anywhere, but if you can't, typing the full path is the solution to that problem).

Step 2: confirm that you can specify an index and perform an alignment. Again, think toy examples, and get comfortable with what it takes to run them. Grab a few reads to use as a toy:

# we grab 25 reads (remember 4 lines per read) into a local file:
head -100 bowtie2-2.3.4-mingw-x86_64/example/index/out.1.fastq > foo1.fastq

Then locate your index. Based on the above, it is probably in this directory: bowtie2-2.3.4-mingw-x86_64/example/reference/

so examine the contents:

ls -al bowtie2-2.3.4-mingw-x86_64/example/reference/

and then specify the basename of the files ending in .bt2

With your index, and a few reads, see if you can put the pieces together in a minimal way:

bowtie2 -x bowtie2-2.3.4-mingw-x86_64/example/reference/GCA_000001405.15_GRCh38_no_alt_analysis_set foo1.fastq

If you get SAM output, you're good to go to build the real command. If not, experiment with these steps until they work. If something doesn't work, think of the simplest test you can perform to test an assumption.