Entering edit mode
4.0 years ago
citronxu
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20
Hello everyone,
I'm newcomer to bioinformatics, I've already aligned my RNA-seq reads to the reference and BAM files have been generated as outcome. My question is, if I'd like to do gene functional annotation via, for example, local BLAST, what should I do next to achieve that? obviously BAM file cannot be directly used, is it?
appreciate any help:)
What does that mean? BAM contains reads from the entire experiment. So which genes do you want to include? What is the question you want to answer? Please be precise so we can recommend a suitable strategy.
Hi,Sorry for not make it clear enough. I'm working on Brassica napus and focusing on genes which encode proteins, I'd like to find their homologous counterparts in other well-studied organism, for example, Arabidopsis thaliana. by this way, I can get their possible gene functions.
But for this you do not need RNA-seq data. Rather than this you need sequence alignments between the reference transcriptomes of the two species. I am not much into plants but rapeseed is not so uncommon, is it? Aren't there annotations for this plant that contain exactly what you need? Be sure to extensively browse the web for resources and articles that have already done what you aim to do. I will ping some members that work on plants, they probably know which databases to look up. Probably biomaRt Plants is at least part of the answer.
Thank you for your kind help. I'm doing RNA-seq in order to calculate the gene expression pattern followed by differentially expressed gene discovery between samples, which is able to find out a subset of genes that are influenced by specific treatment. and, ya, meanwhile I'm reading papers and literatures on the internet, yet none has mentioned about it. Like you said, I need the sequence alignments between two species, but right now I'm kinda stuck with how to extract sequences from my BAM file on B.napus or whether there is some software could use BAM file as input to execute the alignment directly.
Are you perhaps working on a de novo version of the brassica genome (other cultivar or such, compared to the reference)?
If not, I suggest to go and have a look at the public resources for brasscia (as mentioned by ATpoint as well). A few examples:
others worth a look: plantDGB, Ensembl plants, PLAZA (those ore more general, == not speciifc Brassica only) but certainly provide useful info for brassica as well. Certainly PLAZA is very useful for what you want to achieve, it is main goal is to (among other things) link genes between species.
More on your specific issue, you do not need your BAM files anymore for the functional part. Since oyu already determined your genes of interest in brassica (through alignment of your reads to the reference genome, expression values, DEG, ... ), now it's a matter of getting the geneIDs of the genes that look interesting to you and go and look for functional info for those.