Hello,
I hope someone can help me
I am currently working with a genome and heterologous hybridization microarrays data from agilent. The probes for the microarray were design previously to the NGS sequencing from my genome.
So they used of a closely related Genome (species B ) to design the 60-mer probes.
Meanwhile, the genome of specie A (my genome) was sequenced.
Since the previous analysis of the microarray data gave small changes in the expression of the genome and also a lot of background so I inquired if the probes design were appropriated and if possible by removing some inappropriate probes the result would be more correct.
I used blastn from stand alone blast to verify if the probes matched my genome. However, most of the probes weren’t a perfect match. Most of them have several mismatches, some gaps and rarely the full length.
From what I see can I say that gene 300 will hybridize with only Ras_56701_D or with Ras_56701_D , Ras_56377_D , Ras_46891_R_m or more or none. Well if 43 nucleotides with 8 mismatches would hybridize so would 42 with 8 mismatches.
Do I need to see were in the probe the mismatches and the gaps happen? Could a gap in a certain place be less “damaging” to the hybridization than several mismatches?
What is the acceptable threshold? There is some better way to score these alignments?
Thanks.
Regard,
Sam.
Hi Sam, this is a really interesting question. It's important to note that molecular binding, (what you're measuring), will not be a linear function of the count of mismatches. Series of CGCG will bind more easily/tightly than entropic 4-mers. So it might not be a good idea to try to look at the data in such a way. Instead focus on the probe-level intensities and their annotation files. The microarray will be defined with assay ID#s and you can look at their statistics. Was this a sample-sample comparison experimental design? I can't tell what you're trying to score threshold.
Hi karl, Thank for your reply. The condition A (sample A-my genome) it’s the same in several microarrays, the Sample B is the same species (A) but in a different condition. When I talk of a threshold I was thinking of cutoff value. I will try to look at my microarray data (raw data) and see if I can make sense of it. I hoped that I could remove some of the probes by the blastn result before analyzing the microarray data so the probes that are not appropriated would be remove. Thanks.