8. What to do if you have an incomplete gene model

As mentioned above, the predicted consequence of a mutation is based on the IWGSC CSS gene model. Therefore, if the gene model is incomplete, missing or incorrect then the predicted consequence of a mutation may not be adequate. Users can manually annotate their gene model based on the IWGSC or the UCW_Kronos_U and TGAC_Cadenza_U scaffolds and then used this to determine the effect of the mutations on their alternative or improved gene model. We explain this process below and in the wheat-training website.

###Use of CODDLE and PARSESNP to identify mutations CODDLE and PARSESNP are two programs which can be used to predict the consequence of mutations according to the improved gene model.

1. Obtain the scaffold name of the IWGSC CSS, Kronos_U or Cadenza_U scaffold that the sequence is on.
2. Use the scaffold name as the search query on www.wheat-tilling.com to download the excel file with all the mutations in this scaffold.
3. Use this to create a variants text file: this should consist of two columns. The first column should contain all the mutation positions following the format REF base, scaffold position, MUT base. The second column should contain the name of the mutant line containing this mutation. See Fig 4 for an example. This should be saved as a '.txt' file.
4. Hint: to generate the information for column 1 you can concatenate the 'ref', 'pos' and 'mt' columns from the exported csv file. Paste the formula “=concatenate(H2,E2,I2)” into the excel output to produce this format for the first mutation in row 2.

1. Download the genomic sequence of the whole IWGSC CSS scaffold by clicking on the name of the scaffold in the HTML output table
2. Make a coding sequence for your gene using fragments of the genomic sequence. It is very important that there are no SNPs between the genomic sequence and coding sequence.
3. Go to the CODDLE website
4. Copy and paste your genomic sequence into the ‘Submit genomic sequence’ box (Fig 5a)
5. Copy and paste your CDS into the ‘Submit cDNA sequence’ box (Fig 5b)
6. Click ‘Begin Processing’ (Fig 5c)
7. You will first be taken to a page with predicted conserved blocks in your protein, click the ‘Proceed with PARSESNP’ button

1. On the PARSESNP page upload your variants text file (point 3 above; Fig 6a), set the ‘No. of variants to enter by hand’ (Fig 6b) to 0 and click ‘PARSE-SNPs in Your Gene’ (Fig 6c)
2. On the next page (your variants file displayed in a table on the webpage) click ‘submit’.
3. The result will be a table with information including the predicted effect of all the mutations in your variants file on your gene of interest.
4. From here you can proceed with selecting your mutant line as normal and then ordering seeds through SeedStor.