High-throughput sequencing technologies and advances in metagenomic analysis of complex DNA mixtures have enabled the recovery of genomic sequences from ancient samples on a budget and timescale not previously achievable. To decipher the Neandertal genome, Green et al. used an approach known as pyrosequencing, which enables the sequencing of hundreds or thousands of DNA molecules at the same time, thus generating smaller pieces of sequences faster and cheaper than classical Sanger sequencing. One of the limitations of this technique is that the length of DNA that can be read in any single sequencing run is very short. For the Neandertal genome, these short sequences were then assembled using a combination of sophisticated alignment algorithms as well as the completed human and chimpanzee genome sequences as guides. This has resulted in ~1.3-fold coverage of the entire Neandertal genome.
Comparison of Neandertal and present-day human genomes can reveal information about genetic changes that have occurred before and after the ancestral population split of modern humans and Neandertals (see the Comparative Genomics section for more). However, low coverage sequencing inevitably leaves a substantial proportion of the genome uncovered. To recover additional information about specific regions of interest, Burbano et al. used a microarray-based approach to sequence ~14,000 protein-coding regions inferred to have changed in the human lineage since the last common ancestor shared with chimpanzees. By generating the sequence of one Neandertal and 50 present-day humans at these positions, the researchers identified 88 amino acid substitutions that have become fixed in humans since our divergence from the Neandertals (see table). Further studies will be needed to investigate the possible functional significance of these genetic changes.