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Patterns of damage in genomic DNA sequences from a Neandertal - PubMed

  • ️Mon Jan 01 2007

Patterns of damage in genomic DNA sequences from a Neandertal

Adrian W Briggs et al. Proc Natl Acad Sci U S A. 2007.

Abstract

High-throughput direct sequencing techniques have recently opened the possibility to sequence genomes from Pleistocene organisms. Here we analyze DNA sequences determined from a Neandertal, a mammoth, and a cave bear. We show that purines are overrepresented at positions adjacent to the breaks in the ancient DNA, suggesting that depurination has contributed to its degradation. We furthermore show that substitutions resulting from miscoding cytosine residues are vastly overrepresented in the DNA sequences and drastically clustered in the ends of the molecules, whereas other substitutions are rare. We present a model where the observed substitution patterns are used to estimate the rate of deamination of cytosine residues in single- and double-stranded portions of the DNA, the length of single-stranded ends, and the frequency of nicks. The results suggest that reliable genome sequences can be obtained from Pleistocene organisms.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

The 454 library preparation process. Double-stranded DNA molecules (i) (yellow) are made blunt-ended by T4 DNA polymerase, 5′-phosphorylated (stars) by T4 polynucleotide kinase (ii) and ligated to one strand of nonphosphorylated double-stranded adaptors A (green) and B (blue) (iii). Ligation products carrying the biotinylated B adaptor are captured on Streptavidin beads (red), and the strand-displacing Bst DNA polymerase is used to extend the nicks between adaptors and template (iv). The DNA strands are then denatured, releasing the A-to-B strands (v), which are isolated and used as templates for emulsion PCR.

Fig. 2.
Fig. 2.

Base composition at ends of Neandertal DNA sequences. The base composition of the human reference sequence is plotted as a function of distance from 5′- and 3′-ends of Neandertal sequences.

Fig. 3.
Fig. 3.

Misincorporation patterns in Neandertal DNA sequences. The frequencies of the 12 possible mismatches are plotted as a function of distance from 5′- and 3′-ends. At each position, the substitution frequency, e.g., C-T, is calculated as the proportion of human reference sequence positions carrying C where the 454 sequence is T. The 10 5′- and 10 3′-most nucleotides were removed from the 3′- and 5′-graphs, respectively.

Fig. 4.
Fig. 4.

Miscoding lesions and the 454 process. During preparation of templates for 454 sequencing, the ends of DNA fragments are first repaired by T4 DNA polymerase (A), and in a later step linkers are filled in by Bst DNA polymerase (B). During blunt-end repair by T4 DNA polymerase (A), miscoding lesions (black circles) on 3′-overhanging ends are removed, whereas miscoding lesions on 5′-overhangs result in complementary misincorporations (white circles) in the resultant 454 sequences. Similarly, extension by the strand-displacing Bst DNA polymerase (B) causes miscoding lesions in the template DNA downstream of nicks or gaps to result in complementary misincorporations in the sequences generated.

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