Rna-seq for differential expression analysis
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9.1 years ago

Hi,

I am a beginner in bioinformatics and I would like to learn the basic pipeline for quantifying expression of rna sequences from transcriptomic data. I am using a bacterial transcriptome for model. Here is what I have done so far:

I got the transcriptomic data off NCBI, the file was in SRA format, so I used fastq-dump in the sra toolkit to convert sra file into two fastq files (since the data was paired end).

I used bowtie2 to align the fastq files to a reference genome for the same bacteria I found off NCBI (the fasta file ends in .ffn)

I converted the sam to bam, sorted, and indexed bam file using sam tools.

I know I need to use something like bedtools to quantify the bam file, but I am not sure how exactly to go about this. Also, can someone make sure the reference genome I am using for this pipeline is correct? The link to it is ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/Clostridium_difficile_630_uid57679/ and the file is NC_009089.ffn. Any help is appreciated!

Update: I was able to get the read counts using bedtools! Now I have a file in this format:

gi|126697566|ref|NC_009089.1|:1-1320    0    1320    7904

Basically it tells me the location(start 0, end 1320) and the count (7904).

How do I take this data and figure out which exact genes they relate to? I want to get the read counts for another strain of same bacteria and see which genes are differentially expressed between the two.

Thank you

differential-expression transcriptome • 3.1k views
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Maybe you can try using HTSeq? That will give you the per gene expression count. However, you will have to find the GTF file for your bacteria to use it

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Thank you for the reply, I have found the gff file for the bacteria. I will try using HTSeq now!

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Could you get the read counts for your data? If yes, can you explain your pipe? Thanks in advance.

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