please some one help me step by step I was really exhausted

Question

[jivarajivaraj]

I want to work with Ribo-seq data by bowtie2

I did step by step

I downloaded bowtie2-2.2.4 source zip, extracted it in drive c, edit make file by notepad++

Then I downloaded TDM gcc 4.8.1-3 64 bit (my lap top is 64 bit), installed it in drive c

Now what can I do for bowtie installing? I mean rest of steps to bowtie2 be prepared

I got confused I went to advanced setting, environment variable and add ;c:\TDM gcc\bin to the user path but when I type cd c:\bowtie2-2.2 in mingw command prompt, it says it is not recognised as external or internal command, operable program or batch file and says the system cant find the path specified

Please tell me what can I do may b I can work with bowtie2?

[jivarajivaraj]

Hello Adrian

After copying eg1.sam file from C:\bowtie2-2.2.4\bowtie2-2.2.4 to c:\samtools then I typed

cd c:\samtools
samtools.exe view -Sb eg1.sam > eg1.bam

it said:

[samopen] SAM header is present: 1 sequences.

Is it the result or error?

Thank you to the moon

[Adrian Pelin]

This is the result.

[jivarajivaraj]

Thank you

Please don't leave me alone with bioinformatics, I cant find anyone here in my country to help me as useful as you and other guys in biostar helped

Sorry can I ask from where I can learn about commands? for example how you know to convert sam to bam u should type samtools.exe view -sb eg1.sam > eg1.bam and another command like this?

[Adrian Pelin]

I would STRONGLY, and I repeat STRONGLY, suggest that you switch to a linux type environment. Why? Because you will get to a point where you will not be able to use Windows to do analysis. People have suggested you do that here C: hello i know i must accept that im a stupid. Now, about samtools, you need to learn to read documentation. Documentation is documents written by authors of the program explaining how to use it. Read everything here.

[jivarajivaraj]

Hello Adrian

Sorry may I know please how I can do these above mentioned steps on another genome for example homo sapience?

Which I should download and where place it to run your mentioned commands?

[jivarajivaraj]

Sorry Adrian

I downloaded brassica reference genome and a file of reads and according to command I built index and watched index in bowtie file, now I should visualize created sam by IGV but I dont know how I should. I downloaded some files from IGV page (IGVzip and IGVtools) and BamView but I don't know how they work

My supervisor exactly asked me: "select the needed program from the BEDTOOLS toolkit and try to produce an output that is suited as an input file for the Genome browser or IGV visualising tools"

I mean how I can view the generated output by IGV while even I don't know how to launch IGV itself

May you please tell me the steps as already...

Really I'm shy to ask every things but I got confused by reading IGV instruction

Thank you

Answer 1

[Adrian Pelin]

From the many questions you posted already, I see you have tried different things, when people seem to suggest you do something, you just open another question.

Let's try this again.

Step 1) Download this package: http://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.2.4/bowtie2-2.2.4-mingw-win64.zip/download

Step 2) Unzip it. Place it in a folder you can navigate to, something simple, such as C:\bowtie2_win64

Step 3) Assuming you have windows 7 (you should say what operating system you are using), open "cmd.exe", this is command prompt, you can open it by pressing WindowsKey+R and typing cmd.exe + enter

Step 4) In command prompt (black windows), navigate to where you have unzipped your bowtie, in Step2 I told you to put it in C:\bowtie2_win64, so write in command prompt cd C:\bowtie2_win64.

Now you need to test whether the precompiled windows binary works for you, so:

Step 5) Index example fasta file, type bowtie2-build-s.exe example/reference/lambda_virus.fa lambda_virus

Step 6) If all goes well, next you need to align the sample reads provided bowtie2-align-s.exe -x lambda_virus -U example/reads/reads_1.fq -S eg1.sam

If all went well, you should see:

10000 reads; of these:
  10000 (100.00%) were unpaired; of these:
    596 (5.96%) aligned 0 times
    9404 (94.04%) aligned exactly 1 time
    0 (0.00%) aligned >1 times
94.04% overall alignment rate

And if all went well, you are ready to try it on your own data.

[jivarajivaraj]

thank u very much.

[jivarajivaraj]

hello

again me...!

I did these steps:

in command prompt I typed bowtie2-build-s.exe example/reference/lambda_virus.fa lambda_virus

then bowtie2-align-s.exe -x lambda_virus -U example/reads/reads_1.fq -S eg1.sam

and at last after trying many times!!! I got this:

10000 reads; of these:
  10000 (100.00%) were unpaired; of these:
    596 (5.96%) aligned 0 times
    9404 (94.04%) aligned exactly 1 time
    0 (0.00%) aligned >1 times
94.04% overall alignment rate

whether is the result in SAM format??? if not how i can convert it to SAM format, then how i can convert obtained SAM to BAM? (windows7-64bit)

thanks a lot

[Adrian Pelin]

You need samtools, windows version is available here.

After unpacking it, just issue the command:

samtools.exe view -Sb eg1.sam > eg1.bam

Read more about samtools.

[jivarajivaraj]

thank u...

[jivarajivaraj]

I downloaded http://bow.codeplex.com/downloads/get/379402

unpacked in c drive

opened cmd

and taped: cd c:\samtools

then I typed: samtools.exe view -Sb eg1.sam > eg1.bam

it says:

[main_samview] fail to open eg1.sam for reading

what should I do now?

[Adrian Pelin]

you should place eg1.sam in c:\samtools\. The file isn't there, so that's what the error message is telling you, failed to open for reading.

[jivarajivaraj]

Hello Adrian

merry xmas and happy new year to you

Answer 2

[Daniel]

As Adrian has said above, you should really use a linux environment to do this kind of analysis. It would also make installing software easier.

Bio-Linux is a linux environment and has bioinformatics software already installed so you can download it to a usb, put it in your computer and start doing bioinformatics. It won't change your computer, unless you decide you want to install it permanently.

You can get it here: http://environmentalomics.org/bio-linux/

There is also full documentation on using the linux environment and running bioinformatics programs (there's not an RNA-seq section, but the genome assembly section would probably be useful).

[jivarajivaraj]

hey thanks,

this post is belong to months ago, now i am a semi professional in bioinformatics...but from heart i should say that Adrian helped me in such a way that saved me from a too bad condition...

[Daniel]

I'm sorry, I didn't realise it was so old. It appeared at the top of my feed for some reason! Glad to know you're doing alright.

[jivarajivaraj]

not at all... like you friend, for your comment!!!