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Old 12-07-2017, 04:41 AM   #1
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Default RNA_seq Data analysis Help - Trouble viewing files in IGV.

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
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Old 01-11-2018, 03:22 PM   #2
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This locus looks like it has pretty low read coverage based on the very blocky looking pileup. I'll say that a lot erratic coverage results from low read depth at a locus. Also if this is your first dip into RNA-Seq be ready to come across many things that seem strange or aren't what you expected when it comes to the raw level alignments like you're looking at.
/* Shawn Driscoll, Gene Expression Laboratory, Pfaff
Salk Institute for Biological Studies, La Jolla, CA, USA */
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