Differences per site in alignments of ancient DNA fragments. All nucleotide differences (top) and transversion differences (bottom) in different alignments to reference genomes of increasing divergence. Each read is required to have one uniquely best Mega BLAST alignment to the reference genome (estimate shown as the black line). The semiglobal alignment forces the full sequence to align to the genomic region identified by the local alignment (estimate shown as red line). These full alignments are further filtered for having a positive alignment score (blue line). The green crosses show the differences between human and the reference species in the ENCODE multiple sequence alignments. The divergence times on the x-axis are from  and , except for human for which we choose an arbitrary divergence time of 1 million years to Neandertal.