Need help obtaining the sequence of the wild-type/mutant alleles?
Say you only know through sequencing a small window around the mutation, for example:
GAGTATGAAGCCCTCCACGAAGATGAGTTG
GAGTATGAAGCCCTC--CGAAGATGAGTTG
We can find the genomic position of the mutation with BLAST.
Under BLAST Genomes, type Danio rerio > Search.
Enter the wild-type sequence from above > BLAST.
The top result should be the correct match. Check that Query cover and Percentage Identity (Per. Ident) are both 100%. Click on the result.
You will obtain the start and stop positions of the small sequence, in our case: chr20:35900655–35900684.
The mutation is the deletion of CA in the middle. Count from the start and you should find that the deletion is chr20:35900670–71. Record it somewhere for the future.
Now go to the UCSC Genome Browser for Danio rerio reference genome danRer11:
Search for the position of the mutation: chr20:35900670.
Check that you are at the right place, for example that you see your gene of interest and that this nucleotide/the surrounding ones are those you expect from sequencing (you can zoom out for this).
In Position, you should have your position of interest, in our case: chr20:35,900,670-35,900,670.
Add 100 extra bases upstream (5') and 100 extra downstream (3').
Click extended case/color options > change Letters per line to a big number e.g. 999.
You now have a 200-bp genomic window centered on the position of your mutation (i.e. ~ 100 bp on either side). This is your wild-type sequence, in our case:
AAAAATAGGTGGATGGAAATGTAGCTACAGATAACAGGTACTGAGATGTGTTGTTGTCTCTGCAGTTGAGGTGGTGGTGGAGTATGAGTATGAAGCCCTCCACGAAGATGAGTTGACCCTCAGGCTTGGAGACATCATCAAAAACGTACGACGCATCGAAGAGGAGGGATGGATGGAAGGAGACCTCAACGGCAAACGAGG
To create the mutant sequence, simply replace the deleted CA by --. You should obtain:
AAAAATAGGTGGATGGAAATGTAGCTACAGATAACAGGTACTGAGATGTGTTGTTGTCTCTGCAGTTGAGGTGGTGGTGGAGTATGAGTATGAAGCCCTC--CGAAGATGAGTTGACCCTCAGGCTTGGAGACATCATCAAAAACGTACGACGCATCGAAGAGGAGGGATGGATGGAAGGAGACCTCAACGGCAAACGAGG