We believe that random primers more likely produce various cDNA fragments, which are combined using PCR to generate chimeric DNA products.
Although the presence of divergent SFVs could explain such discrepancy, low viral load in the blood samples is more likely, because our PCR primers have been shown to amplify a large variety of African SFVs but also several rather divergent Asian SFVs (16 – 18, 22 ).
The difference in sequence depth within the position-tags is therefore more likely an effect of primer qualities in combination with the fact that different tag sequences could affect the primer annealing by forming secondary structures.
As methylation seems to be more inhomogeneous at the 3'-end of the small promoter fragment it is even more likely for the U-primer to find a suitable template.
Preferential amplification often occurs in multiplex PCR as a result of different efficiencies of different primer pairs and from creation of primer dimers, which are more likely to occur as the number of primers increases [ 48].
The proportion of EST-derived RGHs was much higher than that of genome-derived RGHs, which suggests that EST-derived primers were more likely to be functional.
This analysis revealed that classification was more likely to be impacted by primer bias than by read length.
For any particular genomic region, primer pairs are more likely to amplify a single amplicon from a unique region or amplify many amplicons from a multicopy region, rather than amplify two products from separate regions.
As described previously, our results indicate that primer pairs are more likely to fail in instances in which the exon-exon junction they span has a small intron as compared to primer pairs spanning exon-exon junctions of large intron size or primers pairs that do not span exon-exon junctions.
Five SSR primer pairs detected more than one polymorphic locus, which could be due to translocation events in the genomic regions involving loci detected by primer pairs, or more likely, could be due to the presence of conserved SSRs occurring in multi-gene families or duplicated genomic regions.
