Normalizing Read Coverage

[IonTom]

Dear All,

I would like to compare read depth between two sets of samples
for targeted amplicon regions. Now i would like to normalize all
samples to make their read depth comparable. Which method would
would you recommend. There are lots of good methods for RNA-Seq but which of these ca also be used for DNA-SEQ. And what to do about the GC bias when calculating fold changes ?


Best wishes and thank for your help.

[rnaeye]

bedtools coverage tool may help.

http://bedtools.readthedocs.org/en/latest/content/tools/coverage.html

[criscruz]

Hi everyone;

I'm working with Ion PGM to generate whole genome sequences of some RNA viruses. Then I want to do a phylognetic tree with the consensus sequences of each virus I could identify. So the question is, how many coverage or reads per base I need in order to make a good consensus sequences to my phylogenetic analysis.
I don't want to see or analyze the variant or quasespecies. So I need just the minimal necessary

thanks for your time
my best

Cris

[GenoMax]

Do you have reference sequences available? Otherwise you will need to account for the platform specific errors inherent to Ion.

Is this a rapidly mutating viral species or a mixed population? Without getting some additional information about the project and/or the quality/amount of the data it would be difficult to give you a useful answer.

[criscruz]

Hi GenoMax;

thanks for your comment. Well, I hope identify Dengue and other flavivirus. I have reference sequences available. Falvivirus have ~11kb

best

[GenoMax]

Hi GenoMax;

thanks for your comment. Well, I hope identify Dengue and other flavivirus. I have reference sequences available. Falvivirus have ~11kb

best

Have you done any QC/test alignments? What aligner did you try?

[criscruz]

Hi,
I'm not a bioinformatic. I'm involving in wet lab but I'm working with new one in this field. He gave me a graphic where I could see a not uniform distribution of reads along the genome. So the logical question I did was what is the minimum coverage per base to trust in the consensus sequence.
he told me he performed a QC before alignment and the assembling was denovo using newbler

[GenoMax]

Why is your colleague doing an assembly when you say that reference genome(s) are available?

If you don't feel the coverage is deep enough to trust the consensus you may have to do a) more sequencing b) go with the best call that you can make from what you have.