[dinesh21.ksd]

tnx for the reply. i try ur suggested website http://support.illumina.com/sequenci...e/igenome.html. but i cant download in our campus internet because of some security problem.

i try to ask in my university and get a permission to download that file .

once again thank u

[GenoMax]

guys, now i am working on earthworm RNA sequencing. i need to study the gene expression of earthworm RNA seq.

for gene expression analysis i use tophat and cufflinks.

(ref: Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks)

i am already install all the software (tophat, cufflinks, bowtie, CummeRbund). for running these software i need reference genome.

in that above ref. paper reference genome download via their website. but i am not able to download the reference genome. they ask username and password.

so kindly help me to download the reference genome.

then also help me to choose the reference genome which one is suitable for earthworm.

Dinesh: First a suggestion. Start a new post for a question that is not directly related to a thread even if the title of the thread appears to be similar to what you are asking.

You are the earthworm expert (relatively speaking) so you need to include additional information about which earthworm species are you specifically looking at/for.

Here is an example earthworm genome that may or may not work for you.

http://badger.bio.ed.ac.uk/earthworm/home/download

If the genome you are looking for is not available publicly (e.g. Amynthas) then you may need to assemble a transcriptome yourself using RNAseq data.

[mastal]

guys, now i am working on earthworm RNA sequencing. i need to study the gene expression of earthworm RNA seq.

for gene expression analysis i use tophat and cufflinks.

(ref: Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks)

i am already install all the software (tophat, cufflinks, bowtie, CummeRbund). for running these software i need reference genome.

in that above ref. paper reference genome download via their website. but i am not able to download the reference genome. they ask username and password.

so kindly help me to download the reference genome.

then also help me to choose the reference genome which one is suitable for earthworm.

I find I can download the pre-built indexes without being asked for a username or password, from either the Bowtie, Bowtie2, or Illumina iGenomes web pages, links given below.

http://bowtie-bio.sourceforge.net/index.shtml

http://bowtie-bio.sourceforge.net/bowtie2/index.shtml

http://support.illumina.com/sequencing/sequencing_software/igenome.html

You can always build your own index.

I think your problem is going to be that the only worm reference genome on these web sites is C. elegans, which is probably not closely enough related to
whichever earthworm species you are working with.

If you can't find a closely-related reference genome, you can always try de novo transcriptome assembly.

[dinesh21.ksd]

guys, now i am working on earthworm RNA sequencing. i need to study the gene expression of earthworm RNA seq.

for gene expression analysis i use tophat and cufflinks.

(ref: Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks)

i am already install all the software (tophat, cufflinks, bowtie, CummeRbund). for running these software i need reference genome.

in that above ref. paper reference genome download via their website. but i am not able to download the reference genome. they ask username and password.

so kindly help me to download the reference genome.

then also help me to choose the reference genome which one is suitable for earthworm.

[Brian Bushnell]

Hi Brian,

By the way, so for the mapping to work, does that mean, all the FASTQ files and the reference genome (fasta file) must be in the "current" folder or anywhere in the computer?

Thank you

No, that does not matter. If they are in the current folder you can use the file name alone (e.g. "reads.fq") and if they are in a different folder you can use the absolute path (e.g. "C:\genetics\reads.fq").

[michaellim]

Oh, that's completely my mistake. Sorry! Command should be:

java -ea -Xmx1g -cp C:\bbmap\current\ align2.BBMap ref=ecoli.fasta in=reads.fq out=mapped.sam

However, you need to replace "ecoli.fasta" and "reads.fq" with the actual filenames and paths to your reference file and reads file(s).

Hi Brian,

By the way, so for the mapping to work, does that mean, all the FASTQ files and the reference genome (fasta file) must be in the "current" folder or anywhere in the computer?

Thank you

[michaellim]

Oh, that's completely my mistake. Sorry! Command should be:

java -ea -Xmx1g -cp C:\bbmap\current\ align2.BBMap ref=ecoli.fasta in=reads.fq out=mapped.sam

However, you need to replace "ecoli.fasta" and "reads.fq" with the actual filenames and paths to your reference file and reads file(s).

Hi Brian,

Something happened this time, but still something missing? Oh ok, got it. thank you... Silly me.. hahaha

Thank you

[Brian Bushnell]

Oh, that's completely my mistake. Sorry! Command should be:

java -ea -Xmx1g -cp C:\bbmap\current\ align2.BBMap ref=ecoli.fasta in=reads.fq out=mapped.sam

However, you need to replace "ecoli.fasta" and "reads.fq" with the actual filenames and paths to your reference file and reads file(s).

[michaellim]

If you cut-and-pasted this:
"could not find or load main class align2.bbmap"

...then the problem is capitalization; Java is case-sensitive (should be BBMap). If not, can you copy and paste the full command line and full error message?

Hi Brian,

Here's the picture for you to have a look.

Thank you.

[Brian Bushnell]

If you cut-and-pasted this:
"could not find or load main class align2.bbmap"

...then the problem is capitalization; Java is case-sensitive (should be BBMap). If not, can you copy and paste the full command line and full error message?

[michaellim]

Is there an extra space in "C:\bbmap\ align2.BBMap" after bbmap\? Don't have a PC handy to test.

Yup, there is a space. am I missing something?

[GenoMax]

Is there an extra space in "C:\bbmap\ align2.BBMap" after bbmap\? Don't have a PC handy to test.

[michaellim]

Michael,

It is written in Java, so the executable is actually java.exe, which needs to already be on the computer. If not you can get it here.

As for installing BBMap, you need to unzip it (producing a .tar file), then untar it (producing a directory "bbmap"). After that it depends on your operating system, but in any case you need a command prompt (which you get in Windows by generally going to start->run->"cmd" (then push enter).

For Linux and OSX running is very easy, just execute a shellscript (such as "bbmap.sh") by typing the name then hitting enter, which will give you instructions. For Windows it's a little more involved and depends on the specific program, but for BBMap, the command would be something like this, if you unzipped it in C:

java -ea -Xmx1g -cp C:\bbmap\ align2.BBMap ref=ecoli.fasta in=reads.fq out=mapped.sam

If you still have any trouble, please let me know your operating system, where you unzipped the program, and where the reference and read files are located so I can give more specific instructions.

Hi Brian,

Yup in C:, copied the entire command but it states "could not find or load main class align2.bbmap"

any idea what should I do? Maybe you can devise an executable icon file like those ClustalW alignment which we can just click and run for commandline dummies like me! haha..

Thank you.

[Brian Bushnell]

Michael,

It is written in Java, so the executable is actually java.exe, which needs to already be on the computer. If not you can get it here.

As for installing BBMap, you need to unzip it (producing a .tar file), then untar it (producing a directory "bbmap"). After that it depends on your operating system, but in any case you need a command prompt (which you get in Windows by generally going to start->run->"cmd" (then push enter).

For Linux and OSX running is very easy, just execute a shellscript (such as "bbmap.sh") by typing the name then hitting enter, which will give you instructions. For Windows it's a little more involved and depends on the specific program, but for BBMap, the command would be something like this, if you unzipped it in C:

java -ea -Xmx1g -cp C:\bbmap\ align2.BBMap ref=ecoli.fasta in=reads.fq out=mapped.sam

If you still have any trouble, please let me know your operating system, where you unzipped the program, and where the reference and read files are located so I can give more specific instructions.

[michaellim]

">chromosomeseq
cccccccccccccccccccccccc
>plasmid1
p1p1p1p1p1p1p1p1
>plasmid2
p2p2p2p2p2p2p2p2"

...would be correct.

Thanks Brian,

By the way, I've downloaded the BBMap, unzipped it, but I must be not doing it correctly, I cannot seem to find an executable file to click to open the software. Can you please advise?

Thank you.