Background:

Seeds of an advanced durum breeding line, UAD0951096_F2:5, were mutagenized with 0.7% EMS and 500 mutant plants grown through one further generation to seed. In the next generation (M2) DNA from 99 of these plants and the unmutagenized control UAD0951096_F2:5 was sequenced following reduction by exome capture using the 106.9 Mb Roche NimbleGen SeqCap Wheat Exome Design.

To improve variant calling, a novel consolidated Durum Exome Capture Reference (DECaR) was built from available durum whole genome assemblies: reads from the unmutagenized control sample were mapped into two publically available durum wheat whole genome assemblies of Svevo and Kronos EI v1 , and then combined with assembled unmapped control reads. Putative mutations were identified by comparing the DECaR reference with each mutant plant’s sequence. A read depth is reported for each putative mutation to indicate the comparative level of certainty for the mutation call.

How to:

  1. BLAST your FASTA-formatted sequence of interest in the internal BLAST portal on the Blast page.
  2. The most similar sequences in the available sequenced exome will be displayed within their contigs. Scroll down and expand the BLAST Search Results panel to see the base pair resolution alignment of your sequence of interest with the reference.
  3. Scroll back up and choose your favourite hit by clicking on the button to the left marked Select.
  4. You will be taken to the Search page where you will find all the putative mutations in your sequence of interest called within the sequenced plants of the population. Note: if you see no results, no mutations were called in these 99 plants for your sequence of interest.
  5. Results in the Search page can be sorted by #, Mutant ID, Contig, Position, Depth and Chromosome (Chr).
  6. Column headings have short descriptions in pop-ups when you mouse over the heading. See the table below for longer descriptions.
  7. Copy or note your favourite mutant id, contig number and mutation position (plus any other details you like). You can use these to search directly in the Search page next time, without having to repeat the BLAST.
  8. Clicking on the Flanking Sequence link will expand the sequence fragment with at least 50 bp on either side of the putative mutation. The mutation will be shown in square brackets, e.g. a G in the control that is an A in your selected mutant plant will be shown as [G/A].
  9. Each mutation called in the entire dataset has a unique number ‘#’ that you will also see in the name of the FASTA-formatted flanking sequence, e.g. >#123.
  10. Select and copy the sequence as text to your own preferred file.

Tilling table headers:

The output table on the Search page contains one row for each mutation found.

Column name Description
# Each mutation called in the entire dataset has a unique mutation number ‘#’, a.k.a. internal ID, linking together the individual mutant plant with a mutation position on a DECaR contig.
Mutant ID Identifier of the individual plant harbouring a mutation of interest.
Contig Name of contig using internal DECaR reference nomenclature.
Position Mutation position in bases relative to the start of the respective DECaR contig.
Mutation type The induced mutation type – normally either a substitution of one base for another, or a deletion of one or more bases.
Control Base call at the position in the non-mutagenized control.
Mutant Base call at the position in the EMS-mutagenized individual.
Depth Number of reads at the mutation position (read number of each putative mutated base following removal of poor quality/duplicated sequences - mutant allele coverage).
Zygosity Predicted zygosity of the mutation in the M2 sequenced mutant plant.
Chr Chromosome of the sequence containing the mutation.
Flanking Sequence Sequence fragment of at least 50 bp on either side of the mutation and up to 200 bp.