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Old 11-18-2013, 12:48 AM   #1
genefan
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Default BSmooth alignment problem

I was aligning short reads obtained from Illumina but always got an error as following:

###############################################
perl bswc_bowtie2_align.pl --out=./outReadslevel/ --sam=./outputSAM/chr ./h.sapiens.bsIndex/chr ./h.sapiens.ref/chr*.fa ./SRA/sra_data.fastq
###############################################
Error: <mate1s/reads> not specified

The alignment pipeline is followed by:
https://github.com/BenLangmead/bsmoo.../master/README
https://github.com/BenLangmead/bsmoo...wtie2_align.pl
# sra_data.fastq is built successfully in the first step..
Does anyone know how to solve this problem?
Thank you in advance!!
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Old 11-18-2013, 01:41 AM   #2
dpryan
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Quote:
Originally Posted by genefan View Post
I was aligning short reads obtained from Illumina but always got an error as following:

###############################################
perl bswc_bowtie2_align.pl --out=./outReadslevel/ --sam=./outputSAM/chr ./h.sapiens.bsIndex/chr ./h.sapiens.ref/chr*.fa ./SRA/sra_data.fastq
###############################################
Error: <mate1s/reads> not specified

The alignment pipeline is followed by:
https://github.com/BenLangmead/bsmoo.../master/README
https://github.com/BenLangmead/bsmoo...wtie2_align.pl
# sra_data.fastq is built successfully in the first step..
Does anyone know how to solve this problem?
Thank you in advance!!
While I've never actually used bsmooth, I can tell you that the syntax isn't quite what you would expect. You might try:

Code:
perl bswc_bowtie2_align.pl --out=outReadslevel/ --sam=outputSAM/chr -- h.sapiens.bsIndex/chr -- h.sapiens.ref/chr*.fa -- -- SRA/sra_data.fastq --
The "--" is important for the parsing. I think you need the "-- --" part near the end as well as it may otherwise assume that the fastq file is actually an option for bowtie2.

Last edited by dpryan; 11-18-2013 at 03:38 AM.
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Old 11-18-2013, 07:51 AM   #3
genefan
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Quote:
Originally Posted by dpryan View Post
While I've never actually used bsmooth, I can tell you that the syntax isn't quite what you would expect. You might try:

Code:
perl bswc_bowtie2_align.pl --out=outReadslevel/ --sam=outputSAM/chr -- h.sapiens.bsIndex/chr -- h.sapiens.ref/chr*.fa -- -- SRA/sra_data.fastq --
The "--" is important for the parsing. I think you need the "-- --" part near the end as well as it may otherwise assume that the fastq file is actually an option for bowtie2.
Thanks a lot, the problem is solved! The "-- --" part near the end is mattered..
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Old 11-18-2013, 07:57 AM   #4
dpryan
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Glad that fixed things. I've seen enough code from Ben Langmead to know that his stuff is rather particular about input syntax.
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Old 11-18-2013, 08:18 AM   #5
genefan
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Quote:
Originally Posted by dpryan View Post
Glad that fixed things. I've seen enough code from Ben Langmead to know that his stuff is rather particular about input syntax.
Yes, this is quite tricky..
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Old 11-18-2013, 01:14 PM   #6
genefan
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Quote:
Originally Posted by dpryan View Post
Glad that fixed things. I've seen enough code from Ben Langmead to know that his stuff is rather particular about input syntax.
One more question:
bowtie2_bs_align.pl [options*] -- <index> -- <refs> -- <bowtie2-args> -- [<mate2s>]

<index>: Name prefix for index files. E.g. if index is in /tmp/idx.*.watson.bt2 and /tmp/idx.*.crick.bt2, specify "/tmp/idx"..

chr1.crick.1.bt2, chr1.crick.2.bt2,chr1.crick.3.bt2, chr1.crick.4.bt2,
chr1.watson.3.bt2 ,chr1.watson.4.bt2, chr1.watson.rev.1.bt2, chr1.watson.rev.2.bt2
chr1.crick.rev.1.bt2 ,chr1.crick.rev.2.bt2 ,chr1.watson.1.bt2 ,chr1.watson.2.bt2
chr2.... and all the other chromosomes..

How should I define the index correctly?
if /tmp/chr is used, it gives "Index with basename does not exist"... when I use /tmp/chr*.bt2, the program works fine, but is this the right solution?
The output is given: chrY.ev.tsv
Is there any way to combine the bt2 files?
Many thanks!!
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Old 11-18-2013, 01:47 PM   #7
dpryan
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How exactly did you create the index files? You should have only a set of index files each strand, not one for each chromosome for each strand.
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Old 11-18-2013, 02:36 PM   #8
genefan
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Quote:
Originally Posted by dpryan View Post
How exactly did you create the index files? You should have only a set of index files each strand, not one for each chromosome for each strand.
I used the following command:
perl bswc_bowtie2_index.pl --name=chr --bowtie2-build=/.../bowtie2-build ./ref/*.fa
https://github.com/BenLangmead/bsmoo...wtie2_index.pl
there are 24 fasta files (chr1.fa, chr2.fa....) in the ref folder..
This will then output the above index files.
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Old 11-18-2013, 04:54 PM   #9
GenoMax
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You can "cat" the *.fa files into a single multi-fasta file and then use that single file as your "reference" for bowtie2-build. That will create one set of index files for the entire genome.
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Old 11-18-2013, 11:07 PM   #10
genefan
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Quote:
Originally Posted by GenoMax View Post
You can "cat" the *.fa files into a single multi-fasta file and then use that single file as your "reference" for bowtie2-build. That will create one set of index files for the entire genome.
Thank you for the suggestion! It should work well in this way..
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