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Table 5 Benefits and limits of different genotyping techniques for EPICs (for direct sequencing, see Results-Discussion, last section).

From: An efficient method to find potentially universal population genetic markers, applied to metazoans

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'

  1. 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.