Effect of the enzyme and PCR conditions on the quality of high-throughput DNA sequencing results.

Library preparation protocols for high-throughput DNA sequencing (HTS) include amplification steps in which errors can build up. In order to have confidence in the sequencing data, it is important to understand the effects of different Taq polymerases and PCR amplification protocols on the DNA molecules sequenced. We compared thirteen enzymes in three different marker systems: simple, single copy nuclear gene and complex multi-gene family. We also tested a modified PCR protocol, which has been suggested to reduce errors associated with amplification steps. We find that enzyme choice has a large impact on the proportion of correct sequences recovered. The most complex marker systems yielded fewer correct reads, and the proportion of correct reads was greatly affected by the enzyme used. Modified cycling conditions did reduce the number of incorrect sequences obtained in some cases, but enzyme had a much greater impact on the number of correct reads. Thus, the coverage required for the safe identification of genotypes using one of the low quality enzymes could be seven times larger than with more efficient enzymes in a biallelic system with equal amplification of the two alleles. Consequently, enzyme selection for downstream HTS has important consequences, especially in complex genetic systems.

Y-chromosome analysis in Retuertas horses.

Several studies based on a variety of genetic markers have attempted to establish the origins of horse domestication. Thus far a discrepancy between the results of mitochondrial DNA analysis, which show high levels of diversity, and results from the Y-chromosome, with almost no genetic variability, has been identified. Most previous work on the horse Y-chromosome has focused on widespread, popular breeds or local Asian breeds. It is possible that these breeds represent a reduced set of the genetic variation present in the species. Additional genetic variation may be present in local breeds and ancient feral populations, such as the Retuertas horse in Spain. In this study we analyzed the Y-chromosome of the Retuertas horse, a feral horse population on the Iberian Peninsula that is at least several hundred years old, and whose genetic diversity and morphology suggests that it has been reproductively isolated for a long time. Data from the Retuertas horse was compared to another 11 breeds from the region (Portugal, Spain and France) or likely of Iberian origin, and then to data from 15 more breeds from around the globe. We sequenced 31 introns, Zinc finger Y-chromosomal protein (ZFY) and anonymous Y-linked fragments and genotyped 6 microsatellite loci found on the Y-chromosome. We found no sequence variation among all individuals and all breeds studied. However, fifteen differences were discovered between our data set and reference sequences in GenBank. We show that these likely represent errors within the deposited sequences, and suggest that they should not be used as comparative data for future projects.