[GenoMax]

Put your reference sequence in a new directory and then make the index.

G:\>java -Xmx4g -ea -cp G:\bbmap\current\ align2.BBMap ref=G:\MAKE_NEW\indexreads.fq build=1 genScaffoldInfo=true

[everestial]

You appear to be running out of memory. Increase -Xmx1g to -Xmx4g in the first command. Until this completes without errors do not run the second command.

How big is your reference?

The reference is 200 mb file.

[everestial]

Put your reference sequence in a new directory and then make the index.

G:\>java -Xmx4g -ea -cp G:\bbmap\current\ align2.BBMap ref=G:\MAKE_NEW\indexreads.fq build=1 genScaffoldInfo=true

My ram is 4gb so I am going to use 3gb instead and if it fails I will run with 4gb. I will let you know what happens.

Thanks,

[Brian Bushnell]

Also, to allow splice junction (which I would expect for RNAseq reads) what should I do. I have been reading the readme file. I understand the concept but for some reason whole coding process still doesn't make very good sense to me.

I was able to do fastqc quality check on iplant. Is there any parameter in BBMap equivalent to this quality check app.

Thanks,

The default settings of BBMap work fine for plant introns. As for quality... the bhist, qhist, qahist, aqhist, mhist, and ehist flags will allow you to print histograms indicating read quality. You can plot them in Excel or whatever; but BBMap does not directly provide graphical output like Fastqc.

As for your error messages, as GenoMax noted, you are running out of memory. You can determine how to set the -Xmx flag by running AssemblyStats like this:

java -Xmx1g -ea -cp G:\bbmap\current\ jgi.AssemblyStats2 in=reference.fa k=13

At the bottom, it will tell you how much memory is needed.

Also, neither BBMap nor AssemblyStats support a reference in fastq format (.fq), it has to be in fasta (.fa) format. I'm not entirely sure what "indexreads.fq" is, but the "ref=" argument needs to point to the fasta file of the genome.

Edit:

A 200Mbp genome should run fine with 2g, but not with 1g.

[everestial]

My ram is 4gb so I am going to use 3gb instead and if it fails I will run with 4gb. I will let you know what happens.

Thanks,

on Xmx3g and 4g
I get the message that the space could not be reserved.
Then I se it to Xmx1500m
still same message.

[GenoMax]

Are you using 32-bit windows on this machine (and 32-bit java)?

[everestial]

The default settings of BBMap work fine for plant introns. As for quality... the bhist, qhist, qahist, aqhist, mhist, and ehist flags will allow you to print histograms indicating read quality. You can plot them in Excel or whatever; but BBMap does not directly provide graphical output like Fastqc.

As for your error messages, as GenoMax noted, you are running out of memory. You can determine how to set the -Xmx flag by running AssemblyStats like this:

java -Xmx1g -ea -cp G:\bbmap\current\ jgi.AssemblyStats2 in=reference.fa k=13

At the bottom, it will tell you how much memory is needed.

Also, neither BBMap nor AssemblyStats support a reference in fastq format (.fq), it has to be in fasta (.fa) format. I'm not entirely sure what "indexreads.fq" is, but the "ref=" argument needs to point to the fasta file of the genome.

Edit:

A 200Mbp genome should run fine with 2g, but not with 1g.

I am going to run the command incorporating all you advise. Hope it will work.
Thanks,

[everestial]

I am going to run the command incorporating all you advise. Hope it will work.
Thanks,

Alrite, overall it seems like a memory issue. I corrected the files to fa (fasta) format by new concatenation. RAM of my computer is 4gb but I cannot allocate above 1400mb of the available memorey. I get the erorr message: could not reserve enough space for 1536000KB object heap.

Asseblystats isn't helping either.

[Brian Bushnell]

Sounds like you have a 32-bit version of Windows and/or Java, which may be enough in this case, we'll see. What was the output of AssemblyStats?

[everestial]

Alrite, overall it seems like a memory issue. I corrected the files to fa (fasta) format by new concatenation. RAM of my computer is 4gb but I cannot allocate above 1400mb of the available memorey. I get the erorr message: could not reserve enough space for 1536000KB object heap.

Asseblystats isn't helping either.

Well, AssemblyStats worked now:
BBMap minimum memory estimate at k=13: -Xmx2770m <at least 3080 MB physical RAM>

My RAM is 4gb. What would you suggest in this case? Is there a way to boost up the memory usage?

[Brian Bushnell]

Do you know whether your OS is 32-bit or not? Right-click on Computer (should be on the desktop) and you should get a description of the operating system, indicating whether it is 32-bit or 64-bit. At least, it works that way in Windows 7.

[everestial]

Sounds like you have a 32-bit version of Windows and/or Java, which may be enough in this case, we'll see. What was the output of AssemblyStats?

My windows is 8.1 64 bit. And, I install java 64 bit too (recent release). AssemblyStats finally worked.
The memory required is 3080 mb. My computer is 4 gb.

[Brian Bushnell]

You can decrease the amount of memory needed with the flags "usemodulo" and "k=12". What is the largest amount of memory Java will let you use?

[everestial]

I check and my computer is 64 bit.

[Brian Bushnell]

OK, try this:

java -Xmx1400m -ea -cp G:\bbmap\current\ align2.BBMap ref=G:\bbmap\genome.fasta k=12 usemodulo

...and see if that works.