Table 5.
Benefits and limits of different genotyping techniques for EPICs (for direct sequencing, see Results-Discussion, last section).
| Genotyping technique | 454 sequencing | Cloning-sequencing | Melting curve genotyping | SSCP | Intron length polymorphism |
|---|---|---|---|---|---|
| Information (models of allele evolution) | Diploid Seq. (various) | Diploid Seq. (various) | Genotype (IAM)i | Genotype (IAM)i | Genotype (IAM)i |
| Fragment size | Seq <400 bp* | No limit ** | Best <350 bp | Best <350 bp | No limit |
| Allele number | No limit | No limit | Very limited. If high, impairs genotyping | Some alleles may not be distinguished | Some alleles may not be distinguished |
| Throughput | high | low | high | Medium | medium |
| PCR | Classical | Classical | Real Time | Classical | Classical |
| Cloning | No | Yes | No | No | No |
| Pooling loci | After careful quantification | Possible# but problematical | No | Possible | Common |
| Electrophoresis | No | For sequencing | No | Yes | Yes |
| Sequencing | 1 run = Numerous individuals × numerous loci | Several reactions required per individual | No | No | No |
| Number human action steps μ | L + 1 + 1 | L + NL + 10NL | L | L + L/3 μ' | L + L/3 μ' |
Seq.: sequence. i: IAM: Infinite Allele Model (evolutionary distance is considered identical between all allele pairs). *: Larger amplicons can be included in the run, but only the first 400 bp will be sequenced; however, to favor equal representation among loci, amplicons of relatively uniform lengths (500-900) are preferred. Sequencing can be oriented if desired (start with one of the PCR primers only). This size threshold may increase in future. **: but cloning efficiency may depend on size (and size must be compatible with PCR). #: not recommended, since loci may be very unequally inserted by cloning vectors (often one missing). μ : For each technique, the number of steps requiring human action is expressed as a function of the numbers of loci (L) and of genotyped individuals (N) and deduced from the five preceeding rows of the table. We do not consider the possibility of multiplex PCR (i.e. several loci in a single PCR reaction) though this reduces the number of steps). μ': Pooling three loci together for electrophoresis is generally easy (whether or not an automatic sequencer is used); more loci can be pooled when allelic distributions do not overlap.