Question: RNA_seq Data analysis Help
gravatar for deamonkinge
11 weeks ago by
deamonkinge10 wrote:

Hi there, I am new to RNA_seq and to Bioinformatics and I would greatly appreciate if someone could help me out a little with the analysis of some of my RNA_seq data.

So I am analyzing RNA_seq data from Bacteria using Galaxy - RNA was isolated from wild type bacteria and from a mutant bacteria. RNA was sequenced using Illumina NextSeq 500 system.

So the first thing I did when given the RNA_seq data was to perform a quality check on the RNA_seq data which was in the form of FastQ files. I performed the quality check using ''FastQC'', after a quality check I found that the reads were of very good quality so I proceeded with mapping of the reads back onto the bacterial reference genome. For this I used ''Bowtie2'' - I allowed Bowtie to perform soft clipping during the mapping step.

I then obtained a BAM file from the mapping stage - I used the ''BamCoverage'' Tool to change the Bam files into BigWig files. I then used ''IGV'' to visualize the mapping of my reads onto the reference genome. You can see this in the picture I attached to this question - In the picture you can see ''Wild Type'' forward and reverse files and also ''Mutant'' forward and reverse files. At the bottom of the picture you can see the Gene / Genes that the reads are mapping to. You can see in the picture that there are many reads from the Mutant forward file mapping to the gene ''NWMN_RS14115'' which is a gene that is on the forward strand.

My question is : Why are there Big blocks of reads mapping in and around the ends of the gene and less reads mapping to the middle of the gene? Shouldn't all the reads be falling evenly within the confines of the gene? Why are the reads mapping in such uneven batches - especially at the ends of the gene?

Any help would be greatly appreciated! Thanks

ADD COMMENTlink modified 11 weeks ago by Hussain Ather700 • written 11 weeks ago by deamonkinge10
gravatar for Hussain Ather
11 weeks ago by
Hussain Ather700
National Institutes of Health, Bethesda, MD
Hussain Ather700 wrote:

It's possible there's a 5' or 3' end bias. A ratio of 5' or 3' bias can be found using Picard. 3' bias can be due to polyA selection.

ADD COMMENTlink written 11 weeks ago by Hussain Ather700
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