Dear Noorul, This is an interesting question and I have few suggestions for this. However, I hope that experts will help you in advance to solve this problem. Please check the quality of the sequences before BLAST them. After aligning the sequences, it is necessary to remove the ends off sequences where there are too many wrong bases. This will help you to get a good alignment. If you don't do so, you will get different results for the same species. Further, when you align the forward and reverse sequences, consider the length of sequences. When the sequencing length is very long, then there may be no overlap between forward and reverse sequences. As a result, there may be too many N at the end of one sequence and you won't be able to get a good align. If you use such aligned sequences for the BLAST search, you will get different results. However, if you have already considered these factors , you will have to wait for the ideas of experts. Sometimes, concentration of the template DNA may have a impact for your PCR inhibition as you have used same PCR conditions always. Hope that following link also useful for you.

http://www.biosyn.com/TEW/Ten-Things-That-Can-Kill-Your-PCR.aspx

Dear Noorul,

DNA sequence alignment was done properly first and than make to blast. The sequence trim slowly front and back evenly. For example given in the NCBI website go to check and clear your doubts.

Sometimes the PCR procedure was not same as all species. The PCR was done and check to amplify different annealing 50 °C or 55°C or 60°C. What's the exact result found to be were they 50 or 55, and also 60 take to gel run the thick bands shown which annealing denote and make further DNA gel extraction and DNA Elution.

This is my small thing idea, Further some experts to refer for good and excellent ideas.

Thank you.

Hi Noorul,

I am not an expert in this, but I have been doing some PCR and sequencing for my PhD to develop a phylogeny. If I understand your question correctly you have sent several samples from the same species (different individuals?) to an offsite lab to have the PCR and sequencing performed and you get variable results back. In my experience there are a number of things that could cause the inconsistent BLAST results. There could be contamination in your DNA extraction or in the original sample. Your samples might have gotten mixed up. The original ID could be incorrect (it happens - even to experts). If you already asked the lab to re-run the sample and it gave the same results, it seems unlikely to be an issue with the PCR conditions. You might try another DNA extraction. If the results match the first sample you sent off for sequencing (the one you got the expected BLAST result) it was probably contamination in the previous extraction. If you have a sample from another individual of the same species you could try extracting DNA from that one. If those results match your first one, then it seems like your second sample (The one with the unexpected results) probably is from another species. I can't think of any way you can really check from your end without sending away more samples.

The weak gel bands could indicate that there was not much PCR product. The primers may not be a good match for that particular species or the PCR conditions might not be optimised for that species. You could ask the lab to run a temperature gradient for your DNA sample if this hasn't already been done. Have the primers you are using been used for this species before? If not you might consider designing a species specific primer.

All the best with it!

Thank you all for your help. I think I should re-extract and try to get high DNA quality.

No problem Noorul.

When you finish the extraction can you see a DNA pellet with the naked eye? If not you could try decreasing the amount of buffer you use for resuspension. I generally use 20 uL TE if I can't see a pellet.

Dear Noorul,

Maybe it's too late. Did you solve your problem? If not I can give some ideas.

When you say "it gave me completely different species result from the blast. " (for the batch 2), taxonomic identification was closed to shark or ray species ? Or it was very distant taxa ?

In case (1) it should be not a contamination. Alternatively (not a contamination) maybe you amplified pseudogenes. Your barcode was a coding or not coding gene? If it was coding gene (eg COI), ou have to check the translate of your DNA sequence into protein sequence. If you find a lot of stop codon, it may be a pseudognes.

In case 2 It certainly due to contamination, it would expected that you taxa should be distant. You likely sampled the correct shark species, but depending of your sampling protocol, you sampled also DNA from others species (prey, parasites, free DNA....). If primer set worked better on the non target species, you can have fatal DNA template competition.

Furthermore, I think you are not the only one with this problem. I just found COI sequences identified as a shark species ( Carcharhinus limbatus ) but that in fact belonged to a fish species:

Check Genbank Identifier: FJ237542

Cheers

Can you please answer how did you solve the same problem