4a. Mutation categories

We defined five categories of mutations to be displayed on the www.wheat-tilling.com website. The first three relate to the minimum number of reads including a mutation that were required by the MAPS pipeline to consider a mutation real. The minimum coverage (MC) to call a mutation was established independently for homozygous (Hom) and heterozygous (Het) mutants using the HomMC and HetMC parameters (See Krasileva et al; Materials and Methods section 3.2.1). We selected as the optimal threshold a minimum coverage of five mutant reads for heterozygous (HetMC5) and three for homozygous mutations (HomMC3). Additional mutations were also identified at lower coverages HetMC4/HomMC3 and HetMC3/HomMC2 but at the expense of potential errors. It is safer to use mutations identified at higher stringencies (HetMC5/HomMC3), but if a desired mutation is identified in the lower categories, it still has a good probability of being real. For a detailed discussion on the use of these categories please see Krasileva et al Main text, Supplemental Online Text section 2.3 and tables S6-S7.

1. HetMC5/HomMC3: Highest stringency level. We estimated the potential error in this category to be below 1% using a series of parameters. Mutations in this category are >99% EMS-type and the % EMS error was calculated at 0.2%.
2. HetMC4/HomMC3: Includes an additional ~350,000 Kronos and ~630,000 Cadenza mutants detected at heterozygous coverage of four. Mutations in this category are ~86% EMS-type in both populations and the % EMS error was calculated at <3%.
3. HetMC3/HomMC2: This includes an additional ~600,000 Kronos and ~1,050,000 Cadenza mutants detected at heterozygous coverage of three and homozygous coverage of two. Mutations in this category are >56% EMS and the %EMS error was calculated at 10%.

Two additional categories were used to classify the mutations above.
4. Multi-Map (MM): This category was used to designate regions which have high sequence similarity across multiple scaffolds and hence reads from regions mapped to multiple scaffolds. These regions corresponds to recently duplicated genes, homoeologues with unusually high sequence similarity or artificially duplicated scaffolds generated during the assembly of the IWGSC reference sequence (see Krasileva et al Materials and Methods Section 3.3).
5. Residual Heterogeneity (RH): This category corresponds to SNPs which were present in multiple individuals and are most likely the result of residual heterogeneity in the initial seed stocks used for the EMS mutagenesis (see Krasileva et al Materials and Methods Section 3.4).

The www.wheat-tilling.com output displays by default only HetMC5/HomMC3 mutations. If users cannot find an adequate mutation within this category they can click “Show lower quality mutations” to display mutations classified as HetMC4/HomMC3, HetMC3/HomMC2, MM and RH (Fig 3e).