Different output when generating step by step airway dataset

rnaseqGene: Different output when generating step by step airway dataset

1

@dec8f401

Last seen 13 hours ago

Spain

I have been following the workflow ‘RNA-seq workflow: gene-level exploratory analysis and differential expression’ and I have a problem:

I tried to generate step by step the airway dataset, downloading the fastq files from www.ebi.ac.uk/ena/browser/view/PRJNA229998?show=reads
Then I download the transcriptome from
ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_29/gencode.v29.transcripts.fa.gz
Then I used Salmon to perform the alignment with the same parameters as the workflow.
Finally, I imported the quants files in R by ‘tximeta’ but the counts did not match. Moreover, when I execute the DESeq2 function, I only get 4 differential expression genes, instead of the roughly 4000 that I should get.

Is there any prefiltering process or any reason for this issue?
I would like to learn these prefiltering and dataset preparation procedures to compare different bioinformatic methods with RNA-Seq.

Thanks in advance and greetings.


tximeta


DESeq2


rnaseqGene

• 60 views

@mikelove

Last seen 8 hours ago

United States

The counts do not match

Is it recognized as GENCODE v29 when you import?

Did you use the same version of Salmon? (I think we used 0.14.1) If not then the counts may not match exactly.

For the DE genes, my top guesses would be: design isn’t correct, or samples aren’t labelled correctly. Make plotCounts of the top gene from the workflow but on your dds.


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