how to identify gene expression and genes responsible for virulence

Question

[bhavya.v]

Hi, I have clinical urine samples from which i need to identify the gene expression changes in the pathogen after treatment. (i know the bacteria present in urine through urine culture test)

Q1. Should I do 16s DNA sequencing to identify the strain of the bacteria? How do I know the genes present in the DNA? Can I do annotation of the data and identify the genes to see which genes are virulent or responsible for biofilm formation?

Q2. I will be doing Rna-Seq to identify the expression of the genes. Do i need to provide the reference genome as the one sequenced above?

Do I need to do DNA sequencing and rna expression profiling to make my data look validated? What exactly is the right route to go to conclude my result.

Totally confused, I am new to this and this is my first bioinformatics clubbed project.

[Kristoffer Vitting-Seerup]

Could you tell us if the bacteria is a known or novel one? Meaning does it have a reference genome? A gene annotation database? You can try searching NCBI's database but I'd also recommend googling and looking for papers.

[bhavya.v]

I am working on Urinary tract infection causing bacteria from clinical patient samples. I know which bacteria caused infection just by name as i did bacterial urine culture to check the count of bacteria (eg. I know if it is e.coli or streptococcus, etc) but I dont know its sequence. It has reference genome at NCBI most definitely. But, do i need to sequence the isolated bacteria and use its genome as reference for identification of gene-expression from rna-seq

[bhavya.v]

I dont know the strain of the bacteria, all I know from urine culture report is that the bacteria present in urine sample is E.coli with >100000cfu/ml count, and the resistant-sensitivity pattern against antibiotics before and after treatment.

So, I want to see if the reason for change in the sensitivity against antibiotics can be checked through DNA sequencing by identifying SNP and genes responsible for the change

Answer 1

[Buffo]

Q1. Yes, you can identify bacteria by 16S amplification and sequencing. Q2. Yes, you can perform differential expression analysis before treatment/after treatment (do not forget replicates, at least 3). Genome reference is recommended but if it does not exist you can perform a de novo transcriptome assembly.

Do I need to do DNA sequencing?

Just for the 16S, if the bacterial genome does not exist and you have money you can sequence it and assembly :)

Answer 2

[Kristoffer Vitting-Seerup]

If your bacteria of interest already have an annotated genome (and you are sure exactly which bacteria and strain etc it is) you dont need to sequence DNA - you can just use RNASeq directly and map it against the known genome and already annotated genes.