[michaellim]

@michaellim: What is your ultimate aim with this RNAseq study? Are you looking to do differential expression or just checking to see what is expressed under some specific condition(s)?

For the immediate issue of not being able to see annotations you can use the gff file from Piet's example and see if that works with IGV. You should compare the pre- and post-trimming FastQC plots to see if there is an improvement in stats. Plots you have posted don't look bad but it is difficult to say if you have adapter contamination unless you try the trimming. No fastq grooming in galaxy should be necessary with MiSeq data. It is already in sanger fastq format.

Once you go away from "model" organisms tools such as galaxy start becoming limiting (as you have already discovered). Depending on your overall goals it may be beneficial to start learning how to do these analyses on command line. If this is a small part of whatever you are trying to do then enlisting the help of a friend/local bioinformatics support folks may be the easiest thing to do so you can get a set of hypotheses to test at the bench and move on.

Hi Genomax,

Thank you for the advise, do you have any recommended handbook/guides/manual/website which teaches some RNA-seq analyses using command lines/softwares?

I would really like to get a bioinformatician who knows about RNA-seq to help, however, no one in my department knows about it. That's why I have been hunting for information online.

It would be easier if all the sequences has been input into Galaxy and IGV, however like what you've said, they only have certain 'model organism' and the rest are not in there. That's why I was thinking of inputting the reference genome (chromosome and plasmid; separately or combined,whichever more appropriate for analyses) myself.

Thank you.

[piet]

But how do I add the plasmid sequences too

usually you will map your reads against a set of reference sequences. All reference sequences are stored in a single FASTA file and this FASTA file is passed to the mapping program (bwa for example). If the genome you want to use as a reference comprises one chromosome and two plasmids than you have to copy the three sequences into a single file. This is called a concatenation and is often done with the Unix command line program 'cat'. For a small genome like E.coli you may also do it with a text editor like notepad.

[michaellim]

usually you will map your reads against a set of reference sequences. All reference sequences are stored in a single FASTA file and this FASTA file is passed to the mapping program (bwa for example). If the genome you want to use as a reference comprises one chromosome and two plasmids than you have to copy the three sequences into a single file. This is called a concatenation and is often done with the Unix command line program 'cat'. For a small genome like E.coli you may also do it with a text editor like notepad.

Hi Piet,

Does that mean I just paste all the chromosome & plasmid sequence together without breaks using one heading, example

">combinedsequences
ccccccccccccccp1p1p1p1p1p1p1p1p2p2p2p2p2p2p2p2p2"

c is chromosome sequence
p1 is plasmid one sequence
p2 is second plasmid's sequence

OR

in the notepad file, there are three headings?
">chromosomeseq
cccccccccccccccccccccccc

>plasmid1
p1p1p1p1p1p1p1p1

>plasmid2
p2p2p2p2p2p2p2p2"

Many thanks!

[Brian Bushnell]

">chromosomeseq
cccccccccccccccccccccccc
>plasmid1
p1p1p1p1p1p1p1p1
>plasmid2
p2p2p2p2p2p2p2p2"

...would be correct.

[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.

[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]

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.

[GenoMax]

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

[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?

[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]

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]

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]

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

[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

[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").