Bowtie2: Error accepting mate files

[Collidor]

I'm trying to align numerous fastq files to the hg19 reference with bowtie2. I have a shell script that should read in the fastq files (paired end) and align them to their reference as per the script laid out in bowtie's instruction manual.

#cd /home/psychgene/Documents/bowtie
bowtie2 -x hg19 -q -1 $/media/psychgene/My\ Passport/RNA-Seq\ FASTQ/ERR188028_1.fastq -2 $/media/psychgene/My\ Passport/RNA-Seq\ FASTQ/ERR188028_2.fastq -S ERR188028.sam

executing this script returns this error:

Warning: Same mate file "$/media/psychgene/My" appears as argument to both -1 and -2
Extra parameter(s) specified: "Passport/RNA-Seq", "FASTQ/ERR188028_1.fastq", "Passport/RNA-Seq", "FASTQ/ERR188028_2.fastq"
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: /usr/bin/bowtie2-align --wrapper basic-0 -q -x hg19 -S ERR188028.sam -1 $/media/psychgene/My -2 $/media/psychgene/My Passport/RNA-Seq FASTQ/ERR188028_1.fastq Passport/RNA-Seq FASTQ/ERR188028_2.fastq 
bowtie2-align exited with value 1
psychgene@psychgene-VirtualBox:~/Documents/bowtie$ sh FASTQ.sh
Warning: Same mate file "$/media/psychgene/My" appears as argument to both -1 and -2
Extra parameter(s) specified: "Passport/RNA-Seq", "FASTQ/ERR188028_1.fastq", "Passport/RNA-Seq", "FASTQ/ERR188028_2.fastq"
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: /usr/bin/bowtie2-align --wrapper basic-0 -x hg19 -q -S ERR188028.sam -1 $/media/psychgene/My -2 $/media/psychgene/My Passport/RNA-Seq FASTQ/ERR188028_1.fastq Passport/RNA-Seq FASTQ/ERR188028_2.fastq 
bowtie2-align exited with value 1

I've even tried to remove the -2 file and run as a -U alignment, which returns a similar error message:

Warning: Same mate file "$/media/psychgene/My" appears as argument to both -1 and -2
Extra parameter(s) specified: "Passport/RNA-Seq", "FASTQ/ERR188028_1.fastq", "Passport/RNA-Seq", "FASTQ/ERR188028_2.fastq"
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: /usr/bin/bowtie2-align --wrapper basic-0 -x hg19 -q -S ERR188028.sam -1 $/media/psychgene/My -2 $/media/psychgene/My Passport/RNA-Seq FASTQ/ERR188028_1.fastq Passport/RNA-Seq FASTQ/ERR188028_2.fastq 
bowtie2-align exited with value 1
psychgene@psychgene-VirtualBox:~/Documents/bowtie$ sh FASTQ.sh
Extra parameter(s) specified: "Passport/RNA-Seq", "FASTQ/ERR188028_1.fastq"
Error: Encountered internal Bowtie 2 exception (#1)
Command: /usr/bin/bowtie2-align --wrapper basic-0 -x hg19 -q -S ERR188028.sam -U $/media/psychgene/My Passport/RNA-Seq FASTQ/ERR188028_1.fastq 
bowtie2-align exited with value 1

Any technical support is appreciated!

Thanks

[Brian Bushnell]

It looks like escaping the spaces is not working. I suggest you rename the input files to not have any spaces. Though maybe it would work if you surrounded the file names by quotes.

[Collidor]

Unforunately neither seemed to resolve this issue.

Do i need to specify each index file of hg19? There are 6 files that I downloaded: hg19.1.bt2 through hg19.4.bt2, hg19.rev1.bt2, and hg19.rev2.bt2. The manual only stated

lambda_phage

as reference, even though lambda_phage corresponds to multiple index files.

[Brian Bushnell]

Oh, let me rephrase - it's not the file, but the directory. Did you try renaming this:

/media/psychgene/My\ Passport/RNA-Seq\ FASTQ/ERR188028_1.fastq

to this:

/media/psychgene/My_Passport/RNA-Seq_FASTQ/ERR188028_1.fastq

...so that there are no spaces in the path?

[Collidor]

Thanks Brian, I think that may have done something positive, but I'm now encountering a new error. Have any thoughts?:

Warning: Could not open read file "$/media/psychgene/My_Passport/RNA-Seq_FASTQ/ERR188028_1.fastq" for reading; skipping...
Error: No input read files were valid
bowtie2-align exited with value 1

I've tried different arrangments of adding -q prefix and renaming extension to .fq.

[Brian Bushnell]

It may not have permissions set correctly... or, it may not exist. Please post the output of this command:

ls -l $/media/psychgene/My_Passport/RNA-Seq_FASTQ/ERR188028_1.fastq

Also, it could have something to do with the dollar sign, which probably should not be there; it's for expanding variables.

[Collidor]

Great catch! The $ appears to have been the culprit. bowtie has accepted the reads and is processing them.
Thanks for your insight!