I have a Drosophila mutant, wherein I suspected the presence of an 8.5 kb insertion in the genome, by PCR. End sequencing of the same revealed an AT-rich sequence. To confirm its presence and in the search for other mutations in the genome, we performed WGS. The reads were clean and we detected point mutations, but the insertion never popped up !!! Is it omitted while library preps?
It depends on a couple of things, how were the libraries prepared? Whether there was PCR step involved or these were PCR free libraries? In case of PCR step, you can expect the same bias as you see in amplification of AT rich region during PCR. In case of PCR-free libraries, you can expect there won't be any bias.
The next question is whether the coverage was enough to to see this AT rich region, whether it was paired-end or single read sequencing.
Hi,
Maybe your insertion is due to a transposable element (8.5kb is a good TE size), TEs are generally AT-rich. Repeated sequences are difficult to assemble with NGS sequencing. Maybe you can take a look at that.
Hi Jonathan, actually, the mutant arose as a product of P element mutagenesis. However, it is the P element excised line, which is devoid of the P element. The end seq of the putative insertion showed homology to gypsy. But I really do not know, how a gypsy can crop in it !!!!
Also, when I try to amplify the insertion, the PCRs fail. I am just at a loss.
You mention that you suspected 8.5 Kb insertion, was it on the basis of PCR? What Polymerase did you use to amplify such large fragment?
Categorically, now you mention that your PCRs are not working. So, how do you know the insertion is there? And can you do a Southern blot on the whole genome to figure out if the insertion is a real insertion? When you had the 8.5 Kb PCR fragment, did you sequence it?
Simran, I used Platinum High Fidelity Taq Pol from ABI. It is a really good polymerase with TdT activity. The primers amplify a region of 300 bp approx in the WT, but the mutant shows up as almost 8.5 kb. The full gene, along with the insertion should be around 17 kb, which I have got just once.
I have been trying to get a Southern done, but my Southerns fail. I have till now, tried with DIG-probes, but now am planning to use radiolabeled probes.
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