Quantify gene expression with kallisto

[nazerafauzia]

Hi Team,

I want to quantify the gene expression, but my experiment requires a different transcriptome that is not available in kallisto. In the A-to-Z tutorial Analysis #2 mentioned that I can index any FASTA file through RNAlysis, by entering the “FASTQ” menu -> “RNA sequencing quantification” -> “Create kallisto index…”. But I don't understand what kind of file I have to load. Is it the FASTA file of the full genome sequencing?

Thanks.
Nazera

[GuyTeichman]

Hi Nazera,
The file you need to load in order to create a kallisto index is a FASTA of your organism's transcriptome.
Meaning, each sequence in the FASTA file is a transcript. If a gene has multiple transcripts (e.g. alternative splicing), then each of those transcripts should be its own sequence in the FASTA file.

Other aligners (such as HISAT2) can work with a reference genome instead of a reference transcriptome, but kallisto was created to work specifically with transcriptomes.

If you have any other questions, feel free to reply or open another thread.

Good luck,
Guy.

[nazerafauzia]

Hi Guy,
Thank you for your reply.
I've tried to load my FASTA file (.fa), but it mentioned that RNAlysis encountered the following error, which I don't understand.
Do you know how to solve this issue?

Thanks.
Nazera

Traceback (most recent call last):

File "C:\Users\nazer\AppData\Local\Programs\Python\Python310\lib\site-packages\rnalysis\gui\gui.py", line 3425, in finish_generic_job
raise worker_output[0]

File "C:\Users\nazer\AppData\Local\Programs\Python\Python310\lib\site-packages\rnalysis\gui\gui_widgets.py", line 268, in run
result = self.partial()

File "C:\Users\nazer\AppData\Local\Programs\Python\Python310\lib\site-packages\rnalysis\fastq.py", line 70, in kallisto_create_index
io.run_subprocess(call)

File "C:\Users\nazer\AppData\Local\Programs\Python\Python310\lib\site-packages\rnalysis\utils\io.py", line 1294, in run_subprocess
with subprocess.Popen(args, stdout=stdout, stderr=stderr) as process:

File "C:\Users\nazer\AppData\Local\Programs\Python\Python310\lib\subprocess.py", line 971, in init
self._execute_child(args, executable, preexec_fn, close_fds,

File "C:\Users\nazer\AppData\Local\Programs\Python\Python310\lib\subprocess.py", line 1440, in _execute_child
hp, ht, pid, tid = _winapi.CreateProcess(executable, args,

FileNotFoundError: [WinError 2] The system cannot find the file specified

[GuyTeichman]

Hi Nazera,
My guess is that the path you specified to your kallisto installation directory is incorrect. Could you please send me a screen shot of your "create kallisto index" window just before you click "start"?

Guy

[nazerafauzia]

Hi Guy,
Here is the screenshot

Nazera

[GuyTeichman]

Hi,
If I'm not mistaken, the actual installation folder should be a folder named 'kallisto'. It is probably inside the directory you extracted your kallisto files into (meaning, inside the directory 'kallisto_windows-v0.46.1').
So, you should be pointing RNAlysis to ".../kallisto_windows-v0.46.1/kallisto".
Please try that and let me know if it works.

Best,
Guy.

[nazerafauzia]

Hi,
It works.
Thank you so much!

Nazera