I am sequencing the ITS1 region in a couple of different plant species. My chromatograms are coming back quite messy (i.e. lots of peaks on top of each other). After giving it some thought, I have come to the conclusion that this is probably due to my individuals being heterozygotes (yes, no)? If yes, how do I deal with this data?
Loci were amplified using a general PCR and sequenced using Sanger sequencing.
The species I am dealing with are the trees from the genus Cordia, family Boraginaceae.
I would agree with Nathan that it could be caused by Indels. However, it could depend on whether you are seeing the double set of peaks throughout the entire sequence. Generally when we see sequence containing base inserts or deletions, the sequence often starts clean extending from the primer. Where the base indel and double sequence begins, the peak heights are half the height of clean peaks. Sequencing in forward and reverse directions should show the heterozygous sequence in the same position. It is also possible that your primers are generating multiple products. To determine if the products are showing indels, you may need to increase the length of the product to before the region where the indel is located. A couple of questions that could be helpful. What is the length of the PCR products? Were the products run on agarose before sequencing and was there a single band? What percent gel did you use?
This sounds like you have heterozygote indels. Have a look at this paper: http://dx.doi.org/10.1371/journal.pcbi.1000113 and see if that helps!
I would agree with Nathan that it could be caused by Indels. However, it could depend on whether you are seeing the double set of peaks throughout the entire sequence. Generally when we see sequence containing base inserts or deletions, the sequence often starts clean extending from the primer. Where the base indel and double sequence begins, the peak heights are half the height of clean peaks. Sequencing in forward and reverse directions should show the heterozygous sequence in the same position. It is also possible that your primers are generating multiple products. To determine if the products are showing indels, you may need to increase the length of the product to before the region where the indel is located. A couple of questions that could be helpful. What is the length of the PCR products? Were the products run on agarose before sequencing and was there a single band? What percent gel did you use?
Doug brings up several good points as well as questions that you should ask yourself. I've encountered chromatograms with multiple peaks, both due to heterozygosity and primer specificity. The messiness of your peaks can have multiple causes. Is a link to a picture/screenshot possible?
The paper Nathan cites looks promising, but the old-school way of dealing with this type of problem is to clone your PCR products. If you have a relatively small set of potential alleles, cloning would be relatively straightforward, if you have lots of individuals, it's probably more time consuming than you want...
Good luck!
It would help to know what organism you are dealing with, and whether or not there are multiple copies of ITS within the genome. Cloning is a good way to deal with heterozygosity and with multiple gene copies. There could be other complications, such as multiple isolates if you're dealing with fungi.
@Julie: see my new edit.
"The species I am dealing with are the trees from the genus Cordia, family Boraginaceae"
Oops, sorry I missed that. I don't know about that genus or family, but I know many plants have multiple ITS sequences in their genome.
I have found both a few heterozygous peaks and a stretch of double, entangled peaks suggesting multiple template perhaps, I was also suggested of pseudogenes.
FYI, I am analysing one species from Boraginaceae, ITS shows 4 bands: 400, 600, 600-700 and 700-800 bp; yet to obtain the sequences. this species we could to identify.
I use Mutation Surveyor software. There is 35 days free trial. https://www.google.fr/search?q=mutation+surveyor+youtube&ie=utf-8&oe=utf-8&gws_rd=cr&ei=evLvVcy8EczvUK3AnOAN
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