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No ancient DNA damage in Actinobacteria from the Neanderthal bone.

Zaremba-Niedźwiedzka K, Andersson SG - PLoS ONE (2013)

Bottom Line: However, phylogenetic analyses did not identify any sediment clones that were closely related to the bone-derived sequences.We analysed the patterns of nucleotide differences in the individual sequence reads compared to the assembled consensus sequences of the rRNA gene sequences.Such studies can help identify targeted measures to increase the relative amount of endogenous DNA in the sample.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Evolution, Cell and Molecular Biology, Science for Life Laboratory, Biomedical Centre, Uppsala University, Uppsala, Sweden.

ABSTRACT

Background: The Neanderthal genome was recently sequenced using DNA extracted from a 38,000-year-old fossil. At the start of the project, the fraction of mammalian and bacterial DNA in the sample was estimated to be <6% and 9%, respectively. Treatment with restriction enzymes prior to sequencing increased the relative proportion of mammalian DNA to 15%, but the large majority of sequences remain uncharacterized.

Principal findings: Our taxonomic profiling of 3.95 Gb of Neanderthal DNA isolated from the Vindija Neanderthal Vi33.16 fossil showed that 90% of about 50,000 rRNA gene sequence reads were of bacterial origin, of which Actinobacteria accounted for more than 75%. Actinobacteria also represented more than 80% of the PCR-amplified 16S rRNA gene sequences from a cave sediment sample taken from the same G layer as the Neanderthal bone. However, phylogenetic analyses did not identify any sediment clones that were closely related to the bone-derived sequences. We analysed the patterns of nucleotide differences in the individual sequence reads compared to the assembled consensus sequences of the rRNA gene sequences. The typical ancient nucleotide substitution pattern with a majority of C to T changes indicative of DNA damage was observed for the Neanderthal rRNA gene sequences, but not for the Streptomyces-like rRNA gene sequences.

Conclusions/significance: Our analyses suggest that the Actinobacteria, and especially members of the Streptomycetales, contribute the majority of sequences in the DNA extracted from the Neanderthal fossil Vi33.16. The bacterial DNA showed no signs of damage, and we hypothesize that it was derived from bacteria that have been enriched inside the bone. The bioinformatic approach used here paves the way for future studies of microbial compositions and patterns of DNA damage in bacteria from archaeological bones. Such studies can help identify targeted measures to increase the relative amount of endogenous DNA in the sample.

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Phylogeny of rRNA gene sequences.The longest rRNA consensus sequence assembled from the Neanderthal data and assigned to Streptmycetales is shown in red (SSU_Streptomycetales C11). The rRNA gene sequences amplified from the cave sediment are shown in green colour. Streptomyces coelicolor, used as a reference in the alignment is shown in blue. The PCR-amplified sequences from the cave sediment are shown in green. “ACT primers clone A5_G07” refer to amplifications with the actinobacterial-specific primers, while the four sequences obtained from the universal primers 27f and 1492r are referred to as “universal primers clone u2_C02, 05, 08 and 09″. The phylogeny was inferred using the maximum likelihood method. Numbers refer to bootstrap support values higher than 75%.
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pone-0062799-g003: Phylogeny of rRNA gene sequences.The longest rRNA consensus sequence assembled from the Neanderthal data and assigned to Streptmycetales is shown in red (SSU_Streptomycetales C11). The rRNA gene sequences amplified from the cave sediment are shown in green colour. Streptomyces coelicolor, used as a reference in the alignment is shown in blue. The PCR-amplified sequences from the cave sediment are shown in green. “ACT primers clone A5_G07” refer to amplifications with the actinobacterial-specific primers, while the four sequences obtained from the universal primers 27f and 1492r are referred to as “universal primers clone u2_C02, 05, 08 and 09″. The phylogeny was inferred using the maximum likelihood method. Numbers refer to bootstrap support values higher than 75%.

Mentions: The maximum likelihood phylogeny showed that the consensus C11 sequence clustered with Streptomyces vitaminophilus with 100% bootstrap support (Figure 3). The same placement was also observed for five shorter consensus sequences obtained if short and long reads were assembled separately (data not shown). The sequence identity of the consensus C11 sequence and S. vitaminophilus SSU rRNA gene was 97% over 1361 bp, and contained three indels of 13–15 bp. A comparison with the rRNA gene sequences of S. griseus and S. coelicolor indicated that the indels correspond to one insertion and two deletions in S. vitaminophilus (see positions 378, 644 and 1322 in Figure S3). Additionally, the consensus C11 and the S. vitaminophilus rRNA gene sequences differ by 29 substitutions, 1 bp indel and 9 homopolymer differences distributed across the genes (Figure S3). The read coverage of the consensus sequence was very high, with a mean of 261 and a minimum of 158 reads in the sequence aligned to S. vitaminophilus. The mean Phred quality score was 73, which corresponds to less than one error in 10,000 bp, and the minimum quality was 27 at one position. However, it should be recalled that these scores do not reflect the possibility of polymorphisms at any position, just the dominance of one particular base over all others.


No ancient DNA damage in Actinobacteria from the Neanderthal bone.

Zaremba-Niedźwiedzka K, Andersson SG - PLoS ONE (2013)

Phylogeny of rRNA gene sequences.The longest rRNA consensus sequence assembled from the Neanderthal data and assigned to Streptmycetales is shown in red (SSU_Streptomycetales C11). The rRNA gene sequences amplified from the cave sediment are shown in green colour. Streptomyces coelicolor, used as a reference in the alignment is shown in blue. The PCR-amplified sequences from the cave sediment are shown in green. “ACT primers clone A5_G07” refer to amplifications with the actinobacterial-specific primers, while the four sequences obtained from the universal primers 27f and 1492r are referred to as “universal primers clone u2_C02, 05, 08 and 09″. The phylogeny was inferred using the maximum likelihood method. Numbers refer to bootstrap support values higher than 75%.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643900&req=5

pone-0062799-g003: Phylogeny of rRNA gene sequences.The longest rRNA consensus sequence assembled from the Neanderthal data and assigned to Streptmycetales is shown in red (SSU_Streptomycetales C11). The rRNA gene sequences amplified from the cave sediment are shown in green colour. Streptomyces coelicolor, used as a reference in the alignment is shown in blue. The PCR-amplified sequences from the cave sediment are shown in green. “ACT primers clone A5_G07” refer to amplifications with the actinobacterial-specific primers, while the four sequences obtained from the universal primers 27f and 1492r are referred to as “universal primers clone u2_C02, 05, 08 and 09″. The phylogeny was inferred using the maximum likelihood method. Numbers refer to bootstrap support values higher than 75%.
Mentions: The maximum likelihood phylogeny showed that the consensus C11 sequence clustered with Streptomyces vitaminophilus with 100% bootstrap support (Figure 3). The same placement was also observed for five shorter consensus sequences obtained if short and long reads were assembled separately (data not shown). The sequence identity of the consensus C11 sequence and S. vitaminophilus SSU rRNA gene was 97% over 1361 bp, and contained three indels of 13–15 bp. A comparison with the rRNA gene sequences of S. griseus and S. coelicolor indicated that the indels correspond to one insertion and two deletions in S. vitaminophilus (see positions 378, 644 and 1322 in Figure S3). Additionally, the consensus C11 and the S. vitaminophilus rRNA gene sequences differ by 29 substitutions, 1 bp indel and 9 homopolymer differences distributed across the genes (Figure S3). The read coverage of the consensus sequence was very high, with a mean of 261 and a minimum of 158 reads in the sequence aligned to S. vitaminophilus. The mean Phred quality score was 73, which corresponds to less than one error in 10,000 bp, and the minimum quality was 27 at one position. However, it should be recalled that these scores do not reflect the possibility of polymorphisms at any position, just the dominance of one particular base over all others.

Bottom Line: However, phylogenetic analyses did not identify any sediment clones that were closely related to the bone-derived sequences.We analysed the patterns of nucleotide differences in the individual sequence reads compared to the assembled consensus sequences of the rRNA gene sequences.Such studies can help identify targeted measures to increase the relative amount of endogenous DNA in the sample.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Evolution, Cell and Molecular Biology, Science for Life Laboratory, Biomedical Centre, Uppsala University, Uppsala, Sweden.

ABSTRACT

Background: The Neanderthal genome was recently sequenced using DNA extracted from a 38,000-year-old fossil. At the start of the project, the fraction of mammalian and bacterial DNA in the sample was estimated to be <6% and 9%, respectively. Treatment with restriction enzymes prior to sequencing increased the relative proportion of mammalian DNA to 15%, but the large majority of sequences remain uncharacterized.

Principal findings: Our taxonomic profiling of 3.95 Gb of Neanderthal DNA isolated from the Vindija Neanderthal Vi33.16 fossil showed that 90% of about 50,000 rRNA gene sequence reads were of bacterial origin, of which Actinobacteria accounted for more than 75%. Actinobacteria also represented more than 80% of the PCR-amplified 16S rRNA gene sequences from a cave sediment sample taken from the same G layer as the Neanderthal bone. However, phylogenetic analyses did not identify any sediment clones that were closely related to the bone-derived sequences. We analysed the patterns of nucleotide differences in the individual sequence reads compared to the assembled consensus sequences of the rRNA gene sequences. The typical ancient nucleotide substitution pattern with a majority of C to T changes indicative of DNA damage was observed for the Neanderthal rRNA gene sequences, but not for the Streptomyces-like rRNA gene sequences.

Conclusions/significance: Our analyses suggest that the Actinobacteria, and especially members of the Streptomycetales, contribute the majority of sequences in the DNA extracted from the Neanderthal fossil Vi33.16. The bacterial DNA showed no signs of damage, and we hypothesize that it was derived from bacteria that have been enriched inside the bone. The bioinformatic approach used here paves the way for future studies of microbial compositions and patterns of DNA damage in bacteria from archaeological bones. Such studies can help identify targeted measures to increase the relative amount of endogenous DNA in the sample.

Show MeSH
Related in: MedlinePlus