Special Feature: The Neandertal Genome

The Samples

To sequence the Neandertal genome, Green et al. began by analyzing 21 Neandertal bones from Vindija Cave in Croatia. The team removed just 50 to 100 mg of bone powder from each bone and screened each sample for the presence of Neandertal mitochondrial DNA (mtDNA) by PCR. Three of the bones, which were determined to represent three different female individuals, were ultimately chosen for sequencing analysis.

The first bone dates to about 38,000 years ago and has been used for earlier genome sequencing efforts, including determination of a complete mitochondrial DNA sequence. The second bone is undated but was found in an older stratigraphic layer than the first bone. The third bone has been dated to about 44,500 years ago.

The Samples

Ancient DNA Challenges

Extracting and analyzing ancient DNA is fraught with challenges. Genetic material degrades into small fragments over time, with errors often introduced into the aging sequence. Moreover, 95 to 99% of fossil DNA can be sequences from microbes that have infiltrated the decaying bone. Ancient human remains pose additional challenges in that well-preserved samples are rare and have typically been handled by curators and researchers, thus raising the possibility that they have been contaminated by modern human DNA. Because Neandertals and humans are so closely related, distinguishing between their DNA can be nearly impossible.

Several precautions were taken in Green et al. to minimize contamination during the Neandertal genome project including DNA extraction procedures performed in a "clean room" with sterile equipment; the addition of unique sequence tags to ancient DNA molecules in the clean room, making it possible to identify contamination from other DNA sources; and the use of enzymes that preferentially cut microbial DNA to increase the relative proportion of Neandertal DNA in the sequencing libraries.

Sequencing Approach

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.

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Examples of genes in which amino acid substitutions have become fixed in humans since our divergence from Neandertals.

Gene Symbol Gene Name Molecular Function
ABCC12 ATP-binding cassette, subfamily C (CFTR/MRP), member 12 ATP-binding cassette (ABC) transporter
CASC5 cancer susceptibility candidate 5 --
KCNH8 potassium voltage-gated channel, subfamily H (eag-related), member 8 Voltage-gated potassium channel
LYST lysosomal trafficking regulator Select regulatory molecule
MCHR2 melanin-concentrating hormone receptor 2 G-protein coupled receptor
OR5K4 olfactory receptor, family 5, subfamily K, member 4 --
PNLIP pancreatic lipase Lipase
SPAG17 sperm associated antigen 17 --
STAB1 stabilin 1 Extracellular matrix structural protein
ZNHIT2 zinc finger, HIT type 2 Zinc finger transcription factor
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