I obtain around 10 million reads for each sample in my Chip-Seq assay . But after the Bioinformatics clean the sample, map and remove the duplicates we only have around 10% of the reads that we can use, close to 1 million reads. Do you think this number of reads is enough to be comfortable about the future results?
We start with around 10ng of DNA and we think that our protein of interest is going to be linked to a reduced number of genes... maybe this is the reason why we obtain a short number of reads? The bioinformatics are not Chip seq experts but they are not very comfortable with the number of reads. Any suggestions?
Hola Laura,
as long as you have the input (no-chip) control then remember that you can quantitatively and also qualitatively determine the enrichment of your target locus/loci."Ideally" the more reads the better but sometime you have to make the most of what you have and get as much interpretable/meaningful data as you can. It will always depend what your final scope is.
Also, if your bionformatitians are not true expert in analysing this kind of data then I'd suggest to talk to bioinformaticians that do have experience in chip-seq in order to seek for advice. Remember these guys (bionformatitians) are fancy statisticians+ and they are very important at both the planing stage and at the analysis stage of your NGS experiment. By consulting with bioinformaticians at the end of your NGS approach they will be only able to tell you what your experiment died of. Always, always and always, when possible, talk to them before hand, that is when you plan your experiment, this will guarantee you get the best chance with NGS experiments.
Good luck!
Hi Laura,
Optimum number of ChIP-seq reads depends on your genome size and the number of binding sites you expect for your protein. So if its a bacterial genome 1 million would be OK to go for it. See this link for more info:
http://www.betacell.org/documents/administered/about/guidelines/ENCODE_BCBC_ChIP-Seq_Standards_V01_20110503.pdf
However, you are losing 90% of the reads after QC and Mapping. That is not a good sign. It would mean that most of the reads you are getting are PCR duplicates and may be highly nonspecific.
Using more amount of DNA for library prep will reduce the PCR duplicates and abundance of reads would reflect ChIPed reads more closely.
Hola Laura,
as long as you have the input (no-chip) control then remember that you can quantitatively and also qualitatively determine the enrichment of your target locus/loci."Ideally" the more reads the better but sometime you have to make the most of what you have and get as much interpretable/meaningful data as you can. It will always depend what your final scope is.
Also, if your bionformatitians are not true expert in analysing this kind of data then I'd suggest to talk to bioinformaticians that do have experience in chip-seq in order to seek for advice. Remember these guys (bionformatitians) are fancy statisticians+ and they are very important at both the planing stage and at the analysis stage of your NGS experiment. By consulting with bioinformaticians at the end of your NGS approach they will be only able to tell you what your experiment died of. Always, always and always, when possible, talk to them before hand, that is when you plan your experiment, this will guarantee you get the best chance with NGS experiments.
Good luck!
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