rnaseqGene: Different output when generating step by step airway dataset
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.
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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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