BLAST- no similarity found!

[Sr_08]

I am working on a bacterial strain which has not yet been sequenced so far.
I designed the primers using the sequence information of the closest related species. My PCR also worked! But, when I tried to Blast it returned a message saying no significant similarity found . I id try changing the thresold condn and unchecking low complexity/ masking....

Dont how to figure this out?
I nputs/advice would be apprciated.
thanks

[Anastasiya]

Try to decrease word size.

[Sr_08]

I did try.....................

[danfive]

Hi have you looked at your sequence data.
I've cleaned up data due to mysterious interference (small yet detectable big dye peaks). You detect and mask this contamination or whatever it is because your data has large peaks and these are half the size or less.

Good luck.

[Warthaug]

Which blast program are you using? Some of them allow you to control the stringency of your search. If yours allows for this, reduce the stringency to see if you cannot find lesser-related species.

As Dan said, make sure you've checked you sequences before hand for obvious signs of sequencing errors. You may also want to consider removing the sequence from the end of your sequencing reactions - this area tends to be more error-prone. If you have your original 4-color trace you can usually see where noise begins to encroach on your signal.

Bryan

[Sr_08]

Thank you everyone for helping me figure out!


I am using NCBI BLAST program; and I reduced the thresold value to 1 and uncheck the low complexity

As Bryan said will try to remove some sequences at the start and at the end.
Will keep you updated. thnx

[queryist]

Choose nr/nt from the databases.