count matrices for genes and transcripts for all samples

Question

[fatimarasool135]

Hi According to this protocol

http://ccb.jhu.edu/software/stringtie/index.shtml?t=manual

I have generated the gtf files for ballgown but now i want to use the Deseq. for the Deseq tool i have to generate the text file of gene and transcripts files. I have run the python script for one samples as python prepDE.py ./ballgown/sample1/sample1.gtf

I gave me 2 output files. I run this command for one sample. My number of samples is 3. Can i have to run the same command for other 2 samples?

[ATpoint]

I would use tximport to aggregate the abundances to the gene level and then feed this into DESeq2: https://bioconductor.org/packages/release/bioc/vignettes/tximport/inst/doc/tximport.html#stringtie

[fatimarasool135]

Hi AT point, I have run this command for all samples python prepDE.py ./ballgown/sample1/sample1.gtf python prepDE.py ./ballgown/sample2/sample2.gtf python prepDE.py ./ballgown/sample3/sample3.gtf

Two output files are generated in each run. Then i renamed files according to sample in command. and now i have 6 files. for next step , I have to run this command for all 3 samples separately? Load gene(/transcript) count matrix and labels

countData <- as.matrix(read.csv("gene_count_matrix.csv", row.names="gene_id"))

I do not know how to work on Deseq2 . Can you please guide me in detail.

Answer 1

[Haci]

You do not have to run prepDE as many times as the number of your samples. The -i option (i meaning input) enables you to provide a list of samples, generating a single count file with genes/transcripts as rows and samples as columns, exactly what Deseq2 uses as input. Here is a

[fatimarasool135]

Hi Haci, I have use this method. which is written in manual of stringtie. I did not use the i option with list of samples because in this method i got error of incorrect path so i use the other method. that is list below...I have run this command for 3 samples . now i have 6 files for gene and transcripts. These six files used in DEseq?

`./ballgown/sample1/sample1.gtf

./ballgown/sample2/sample2.gtf

./ballgown/sample3/sample3.gtf`

I am new to Deseq and R laguage Can you please gave me script of Deseq , I have ead the manual but cant understand it how to start it. I have just install it in my system

[Haci]

Here is the Deseq2 tutorial: https://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html

The tutorial is very comprehensive and covers a lot scenarios for how to go from counts to the Deseq2 object. One way is the tximport, as suggested by ATPoint above. If you repeatedly encounter problems with prepDE you might consider using tximport instead.

Speaking of encountering problems with prepDE, please pay attention to the error messages, for example an error of "incorrect path" tells you to provide the "correct" path of the gtf files in the list of samples file. You can go to the directory where the gtf files sit and type "pwd" to print the full path of the directory. You should then append something like "/fileX.gtf" for each file.