Table 1. Comparison of mapping-by-sequencing methodologies.
| Outcross | Backcross | ||
|---|---|---|---|
| Bulked (HA mapping) | Serial (EMS density) | Bulked (VDM) | |
| Principle | Mapping interval inferred through segregation of known HA SNPs in pooled F2 recombinants | Mapping interval inferred through increased density of EMS-induced variants after serial backcrosses | Mapping interval inferred through segregation of de novo discovered SNPs in pooled F2 recombinants |
| Cross with | Hawaiian (or other polymorphic strain) | Background nonmutagenized strain (or other available strain) | Background nonmutagenized strain (or other available strain) |
| Step by step | Cross, pool 20–50 F2 homozygous recombinants, WGSa the pool | Backcross 3–6 times, WGSa the backcrossed strain | Cross, pool 20–50 F2 homozygous recombinants, WGSa the pool |
| Variants followed | Variants from mapping strain (HA variants) | Variants from the mutagenized strain (i.e., EMS induced) | Variants from the mutagenized strain (EMS induced only or EMS + background strain variants) |
| Other strains to sequence | Background strain (for variant identification subtraction)b | Mapping strain (for mapping and variant identification subtractions)b | Mapping strain (for mapping and variant identification subtractions)b |
| Mapping plots | HA variant allele frequency | Density of EMS-induced SNPs per physical bin | Mutant variant allele frequency |
| Main advantages | Highest map resolution (>100,000 SNPs) | Mutant strain is already backcrossed after mapping protocol | High mapping resolution |
| Can be used to map the absence of a mutation | Basic genetic tests can be performed during backcrosses | Can be used in all mutation categories | |
| Fast (requires only one cross) | Convenient with complex screening strains | Can be used for mapping the absence of a mutation | |
| Convenient with difficult phenotypes | Fast (requires only one cross) | ||
| Appropriate for species where polymorphic strain unavailable | Basic genetic tests can be performed during backcross | ||
| Appropriate for species where polymorphic strain unavailable | |||
| NOT appropriate for | Phenotypes that might be affected by Hawaiian background | Spontaneous mutations, mutant strains generated without EMS or high density mutations | |
| Complicated background strains (background mutations e.g., modifier screens) | Mapping the absence of a mutation | ||
a
Followed by standard bioinformatics analysis: Mapping and alignment to reference genome, variant calling and annotation (see Bioinformatics and Pipelines). This can be done with Cloudmap, home-made pipelines, or as part of sequencing service.
b
Background (or mapping strain) variants can also be obtained by sequencing other mutants from the screen.