Time and Cost
It took four researchers approximately six months of dedicated lab time each to complete the initial effort of sequencing and genotyping six different nDNA loci (three per species). In the end however, only four of these six loci were sequenced completely due to complications. By comparison, all lab work, including primer development and optimization, for the entire 454 sequencing phase of the project was completed by one researcher in approximately six months of lab time (Table 5). There was some overlap of primer development between project phases and these time differences do not take into account the differences in sequence processing time. Regardless, our best estimate is that 454 sequencing as outlined here is three to four times more time efficient (in terms of cost and manpower) than traditional Sanger-based methods for sequencing multiple nDNA markers.
 | Table 5 Approximate lab time needed to complete each sequencing objective. |
The major costs of the targeted 454 method is the full plate of sequencing which includes library quality testing, quantification, and emPCR, and the cost for 400 primers (2 labeled primers each for 20 individuals across 2 species and 5 loci). This puts the total cost at $24,560 to sequence approximately 16 populations or 3200 individual nDNA loci. At $4.00 per individual sequence, the cost to Sanger sequence 3200 loci in the forward and reverse direction is approximately $25,725, already above the 454 price point. Additionally, if any cloning becomes necessary this cost savings quickly increases (Table 6). Overall, in species with moderate to high levels of genetic diversity and heterozygosity, 454 sequencing will be a much more cost effective way to sequence multiple nDNA intron loci.
 | Table 6 Costs of sequencing 3200 loci. |
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