I was just wondering what is more important when sequencing DNA PCR products, is it the quality of the sequence (by that I mean the clear peaks) or is it the BLAST results? My problem is, my sequences are poor but when I BLAST them I get reliable results e.g. E-value=0.0; Max. Identity=95%.
Will it be "scientifically" reasonable using such sequences for further analyses?
From experience, I only trust the sequencing data. If your sequences are poor, I would advise you to clean your DNA amplicon before sending it to sequencing. There are some great kits available for that. Usually they are called PCR clean-up kits and are offered by several companies. On the other hand your DNA concentration might also be too low and you need more rounds of PCR. I would not use BLAST alone to check your sequences. If you do use it, you must make sure that you have a complete coverage of your sequence and your identity should be at 100%. After all what you really want is to ensure that you have no mutations in your sequence and BLAST can be misleading in some cases. Good luck!
Garbage in- garbage out. You need to start with reliable sequence data otherwise you're getting false hits. Often, the poor peaks can result from template that's not properly purified.
To avoid such problem always do PCR with high fidelity DNA polymerase such as phusion HF polymerase.This polymerase does not add any mispairing nucleotide during PCR amplification. And if you send such pcr amplified product for sequencing then you can trust whatever the nucleotide sequence u get. It will be better if you do sequencing from both side of the strand if size of the gene is higher than 700bp. You can align of sequence of both side and make a full sequnce.
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