[everestial]

Hi Brian,

Thanks for the message. Should I leave the file as gzipped or extract it to fast before doing any alignment.

Thanks,

[Brian Bushnell]

You can leave them gzipped.

[everestial]

Hi Brian

I have been able to run other example parameters efficiently.
So, I tried to create a index fasta file with the files you mentioned (got if from the JGI website).

I don't seem to be able to prepare a concatenate index file. I put all the mentioned files in one folder in G: and ran the following command
java -Xmx1g -ea -cp G:\bbmap\current\align2.BBmap ref=G:\bbmap\lyrata_genome_index

Folder named lyrata_genome_index contains all three gziped files.
I got the error message:
Error: Could not find or laod main class ref=G:\bbmap\lyrata_genome\index

Again, I tried to prepare the index file for just one file (not including chloroplast and mitochondrial genome).
I get the same error message.

Can you please help.

Also, is there any command to concatenate all these files into one.

Thanks,

[Brian Bushnell]

java -Xmx1g -ea -cp G:\bbmap\current\align2.BBmap ref=G:\bbmap\lyrata_genome_index

is missing a space; should be:

java -Xmx1g -ea -cp G:\bbmap\current\ align2.BBmap ref=G:\bbmap\lyrata_genome_index

To concatenate into one file, in Windows:

type file1 file2 > file3

...taken from http://stackoverflow.com/questions/6...cat-on-windows

[everestial]

Thanks for quick reply.

Concatenating work perfect.
I had used spaced in my previous command and again did the same after I was able concatenate all the scaffolds into one.
But, still getting the error message:
Error: Could not find or load main class align2.BBmap

I check and BBmap.java BBmap.class and several other important BBmap. files do exist inside the required folder (current).

I have been running cmd as administrator in Windows 8.1. HOpefully the problems isn't coming from there.

[Brian Bushnell]

Ah - in this case it's capitalization - should be BBMap, not BBmap. I should have caught that the first time.

[everestial]

Thanks Brian.

[everestial]

Hi Brian,

I was able to index the genome into one file. Looking at the summary it seems that it is chr1, mitochondrial and chlroplast genome. Thats fine for now. I may be able to add sequences from other chromosomes.

For now I tried to map RNAseq reads from one of my sample for the test purpose. I am not worried about splice right now. But, I have been getting errors. I am not sure what the errors mean.
I am posting screen shots.

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,

[GenoMax]

Once you have the index files ready you use them with path=C:\bbmap\refreads directive (if that path is right). Do not use ref=.

[GenoMax]

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.

Here is a post to help understand minimum options you could use: http://seqanswers.com/forums/showpos...68&postcount=3

Read through the thread for several additional pointers that Brian has provided for additional questions.

[everestial]

Seems like there is some problem with java. I am not sure.

First, I deleted the previous index file and created a new one. The cmd prompt window on the left. There is an error message at the bottom saying
Exception in thread.........
But, a ref folder is created in G: and there are fasta index files.

So, I proceed with mapping one of my RNAseq sample
As, the reference had already been set in the previous command I just run align2.BBmap with in=samplename.fasta and out=testF1.fasta
But, still getting the same error.

[everestial]

Here is a post to help understand minimum options you could use: http://seqanswers.com/forums/showpos...68&postcount=3

Read through the thread for several additional pointers that Brian has provided for additional questions.

Well that command for RNAseq reads make a perfect sense. I will follow up with this command and see how my results turn out.

Thanks,

[GenoMax]

Seems like there is some problem with java. I am not sure.

First, I deleted the previous index file and created a new one. The cmd prompt window on the left. There is an error message at the bottom saying
Exception in thread.........
But, a ref folder is created in G: and there are fasta index files.

So, I proceed with mapping one of my RNAseq sample
As, the reference had already been set in the previous command I just run align2.BBmap with in=samplename.fasta and out=testF1.fasta
But, still getting the same error.

You probably want to create a separate folder when you make the index files that way it would be less confusing to use that path in your ref= part.

Can you post the exact commands you are using for:

a) to create the index
b) to do mapping

Screenshots are hard to read.

ref= directive only needs to include path up to a top level directory. In that directory there should be a "ref" directory (and additional sub-directories, assuming your index creation worked right).

[everestial]

Explanation:
I have my F1 reads in F: (due to space concern)
I have my bbmap extracted in G:
I have my concatenated index reads in G:\bbmap named "indexreads.fq"

I run cmd as admin and then move to G: (which I think shouldn't really matter)

To prepare the index file I type
G:\>java -Xmx1g -ea -cp G:\bbmap\current\ align2.BBMap ref=G:\bbmap\indexreads.fq build=1 genScaffoldInfo=true

Message:
Executing align2.BBMap [ref=<path to the indexreads.fq>, build=1, genScaffoldInfo=true]

BBMap version 34.56
Retaining first best site only for ambiguous mappings.
No output file.
Writing reference.
Executing dna.FastaToChromArrays2 [G:\bbmap\indexreads.fq, 1, writeinthread=false, genscaffoldinfo=true, retain, waitforwriting=false, gz=true, maxlen=536670912, writechroms=true, minscaf=1, midpad=300, startpad=8000, stopped=8000, nodisk=false]

Set genScaffoldInfo=true
Writing chunk 1
Set genome to 1

Loaded Reference: 4.224 seconds
Loading index for chunk 1-1, build 1
No index available; generating from reference genome: G:\\ref\index\1\chr1_index_k13_c3_b1.block
Indexing threads started for block 0-1
Exception in thread "Thread-4" java.lang.OutOfMemoryError: Java heap space at align2.IndexMaker4$BlockMaker$CountThread.run<IndexMaker4.java:280>

To map the genome:
G:\>java -Xmx1g -e -cp G:\bbmap\current\ align2.BBMap in=F:\F1_extracted\R1_001.fastq out=F:\mapped_test01.sam maxindel=100000 xstag=firststrand intronlen=10 ambig=random

The program executes but I ge the same exact message at the bottom starting from Loaded Reference (except for time).

[GenoMax]

G:\>java -Xmx1g -ea -cp G:\bbmap\current\ align2.BBMap ref=G:\bbmap\indexreads.fq build=1 genScaffoldInfo=true

Message:
Executing align2.BBMap [ref=<path to the indexreads.fq>, build=1, genScaffoldInfo=true]

BBMap version 34.56
Retaining first best site only for ambiguous mappings.
No output file.
Writing reference.
Executing dna.FastaToChromArrays2 [G:\bbmap\indexreads.fq, 1, writeinthread=false, genscaffoldinfo=true, retain, waitforwriting=false, gz=true, maxlen=536670912, writechroms=true, minscaf=1, midpad=300, startpad=8000, stopped=8000, nodisk=false]

Set genScaffoldInfo=true
Writing chunk 1
Set genome to 1

Loaded Reference: 4.224 seconds
Loading index for chunk 1-1, build 1
No index available; generating from reference genome: G:\\ref\index\1\chr1_index_k13_c3_b1.block
Indexing threads started for block 0-1
Exception in thread "Thread-4" java.lang.OutOfMemoryError: Java heap space at align2.IndexMaker4$BlockMaker$CountThread.run<IndexMaker4.java:280>

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?