[dpryan]

It's difficult to diagnose your problem when you use multiple pipes. Just run the enumerate command and save that to a file, then try the subsequent commands one at a time and then post which didn't work (probably sort-bed or bedops) and some of the input you're giving it.

[krafiq]

I just tried building a bowtie index using the genome.fa file (without splitting it) and it gave me 4 .ebwt files. And then I used the script:

./enumerateUniquelyMappableSpace.pl 50 Genome | sort-bed - | bedops -m - > Genome.50.mappable_only.bed

but it gave the following error:

Failed to read 50
Warning: Could not find any reads in "-"
# reads processed: 0
# reads with at least one reported alignment: 0 (0.00%)
# reads that failed to align: 0 (0.00%)
No alignments

[dpryan]

EricHaugen: What's the "bowtie_index_prefix" in the 2nd option you gave me above?

It's the prefix for the output index files, so it can be anything you want. Normal examples would be "hg19", "mm9" and "mm10", for human and two mouse genome versions. When bowtie is later invoked to do alignments, this same prefix is given to it so it knows what to align things against.

[dpryan]

dpryan: I'm sorry-could you please clarify a bit as to what exactly I should do?
Also, is there a way to get the source code for bowtie?

The source code for bowtie is available on its website here or here.

Regarding the remainder, enumerateUniquelyMappableSpace is just a perl script that executes a few other commands, some of which won't work for you because of how the script is structured. I already deleted the Hotspot code (I don't use it) so I can't immediately give you exact changes, but the gist is that you can just edit the code to have bowtie-build index genome.fa rather than a too-long list of scaffold.fa files. There may be a few other lines that will throw errors for similar reasons and you can likely use the same strategy. This all assumes that you know enough to edit a bit of code, of course.

[krafiq]

EricHaugen: What's the "bowtie_index_prefix" in the 2nd option you gave me above?

[krafiq]

dpryan: I'm sorry-could you please clarify a bit as to what exactly I should do?
Also, is there a way to get the source code for bowtie?

[dpryan]

Well, if you read through the perl scripts, it'll become pretty apparent that they only designed hotspot around human/mouse/etc. genomes (rather than your situation with scaffolds), so you're probably going to have to just edit the script. It's just trying to run bowtie-build, which will effectively concatentate everything together anyway (it looks like they normally use individual chromosome files so things can more easily be split to later run on a cluster). The script is pretty simple, so go ahead and change it to suite your usage needs.

[krafiq]

dpryan: I had to split the genome.fa file to get the individual files in the first place to use the hotspot software. should i still cat them? won't that bring it back to genome.fa?

[dpryan]

Try concatenating the files together first (you'll have to do it in a couple batches, since the command will be too long for "cat" too) and just use the multifasta file.

[krafiq]

My enumerateUniquelyMappableSpace script is calling this line:
bowtie-build $chromosomeFiles $genome

The chromosome files variable in this case is a list of 30,000 file names. So when I run the script, it gives me the following error:

Argument list too long

is there a way around this?

[sivasubramani]

In HHblits package, there is a script which does the way you want.

HHblits_src/scripts/splitfasta.pl

[EricHaugen]

It looks like "bowtie-build" isn't in your PATH, so the shell couldn't find it.

Try adding a line near the top of "enumerateUniquelyMappableSpace" like:

export PATH=$PATH:/location/of/this/script/folder

Then it should be able to find bowtie-build, and the Perl script it calls later will be able to find your bowtie executable there also.

[krafiq]

Thanks all!!

EricHaugen: I'm trying option 1 for now. I'm trying to run the script again with bowtie and bowtie-build in the same folder as the script. But it's giving me the following error:
./enumerateUniquelyMappableSpace: line 30: bowtie-build: command not found

And then it goes on to give the following error multiple times:
Failed to find bowtie index file Genome.1.ebwt

Does anyone know why and what I should change?

Thanks!

[fengqi]

Did you try
'samtools faidx genome.fasta chrX > chrX.fasta'

[EricHaugen]

Two options:

1. Change "chr" to "scaffold" in the enumerateUniquelyMappableSpace bash wrapper script, to list the individual fasta files.

2. Just run the whole genome fasta file, after building a bowtie index, with:

enumerateUniquelyMappableSpace.pl read_length bowtie_index_prefix genome.fa | sort-bed - | bedops -m - > genome.read_length.mappable_only.bed

If "sort-bed" runs out of memory here, the BEDOPS suite includes a "bbms" script that can be used in place of sort-bed.