This protocol describes a method for converting short single-stranded and double-stranded DNA into libraries compatible with high-throughput sequencing using Illumina technology. This method has ...primarily been developed to improve sequence retrieval from ancient DNA, but it is also applicable to the sequencing of short or degraded DNA from other sources, and it can also be used for sequencing oligonucleotides. Single-stranded library preparation is performed by ligating a biotinylated adapter oligonucleotide to the 3' ends of heat-denatured DNA. The resulting strands are then immobilized on streptavidin-coated beads and copied with a polymerase. A second adapter is attached by blunt-end ligation, and library preparation is completed by PCR amplification. We estimate that intact DNA strands are recovered in the library with ∼50% efficiency. Libraries can be generated from up to 12 DNA or oligonucleotide samples in parallel within 2 d.
It has been shown that highly fragmented DNA is most efficiently converted into DNA libraries for sequencing if both strands of the DNA fragments are processed independently. We present an updated ...protocol for library preparation from single-stranded DNA, which is based on the splinted ligation of an adapter oligonucleotide to the 3' ends of single DNA strands, the synthesis of a complementary strand using a DNA polymerase and the addition of a 5' adapter via blunt-end ligation. The efficiency of library preparation is determined individually for each sample using a spike-in oligonucleotide. The whole workflow, including library preparation, quantification and amplification, requires two work days for up to 16 libraries. Alternatively, we provide documentation and electronic protocols enabling automated library preparation of 96 samples in parallel on a Bravo NGS Workstation (Agilent Technologies). After library preparation, molecules with uninformative short inserts (shorter than ~30-35 base pairs) can be removed by polyacrylamide gel electrophoresis if desired.
Contamination by present-day human and microbial DNA is one of the major hindrances for large-scale genomic studies using ancient biological material. We describe a new molecular method, U selection, ...which exploits one of the most distinctive features of ancient DNA--the presence of deoxyuracils--for selective enrichment of endogenous DNA against a complex background of contamination during DNA library preparation. By applying the method to Neanderthal DNA extracts that are heavily contaminated with present-day human DNA, we show that the fraction of useful sequence information increases ∼ 10-fold and that the resulting sequences are more efficiently depleted of human contamination than when using purely computational approaches. Furthermore, we show that U selection can lead to a four- to fivefold increase in the proportion of endogenous DNA sequences relative to those of microbial contaminants in some samples. U selection may thus help to lower the costs for ancient genome sequencing of nonhuman samples also.
Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in ...molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp.
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Although great progress has been made in improving methods for generating DNA sequences from ancient biological samples, many, if not most, samples are still not amenable for analyses due to ...overwhelming contamination with microbial or modern human DNA. Here we explore different DNA decontamination procedures for ancient bones and teeth for use prior to DNA library preparation and high-throughput sequencing. Two procedures showed promising results: (
) the release of surface-bound DNA by phosphate buffer and (
) the removal of DNA contamination by sodium hypochlorite treatment. Exposure to phosphate removes on average 64% of the microbial DNA from bone powder but only 37% of the endogenous DNA (from the organism under study), increasing the percentage of informative sequences by a factor of two on average. An average 4.6-fold increase, in one case reaching 24-fold, is achieved by sodium hypochlorite treatment, albeit at the expense of destroying 63% of the endogenous DNA preserved in the bone. While both pretreatment methods described here greatly reduce the cost of genome sequencing from ancient material due to efficient depletion of microbial DNA, we find that the removal of human DNA contamination remains a challenging problem.
DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, ...library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3΄ biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude.
Denisovans, a sister group of Neandertals, have been described on the basis of a nuclear genome sequence from a finger phalanx (Denisova 3) found in Denisova Cave in the Altai Mountains. The only ...other Denisovan specimen described to date is a molar (Denisova 4) found at the same site. This tooth carries a mtDNA sequence similar to that ofDenisova 3. Here we present nuclear DNA sequences fromDenisova 4and a morphological description, as well as mitochondrial and nuclear DNA sequence data, from another molar (Denisova 8) found in Denisova Cave in 2010. This new molar is similar toDenisova 4in being very large and lacking traits typical of Neandertals and modern humans. Nuclear DNA sequences from the two molars form a clade withDenisova 3. The mtDNA ofDenisova 8is more diverged and has accumulated fewer substitutions than the mtDNAs of the other two specimens, suggesting Denisovans were present in the region over an extended period. The nuclear DNA sequence diversity among the three Denisovans is comparable to that among six Neandertals, but lower than that among present-day humans.
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Of all obligate intracellular bacteria, Wolbachia is probably the most common. In general, Wolbachia are either widespread, opportunistic reproductive parasites of arthropods or essential mutualists ...in a single group of filarial nematodes, including many species of medical significance. To date, a robust phylogenetic backbone of Wolbachia is lacking and consequently, many Wolbachia-related phenomena cannot be discussed in a broader evolutionary context. Here we present the first comprehensive phylogenomic analysis of Wolbachia supergroup relationships based on new whole-genome-shotgun data. Our results suggest that Wolbachia has switched between its two major host groups at least twice. The ability of some arthropod-infecting Wolbachia to universally infect and to adapt to a broad range of hosts quickly is restricted to a single monophyletic lineage (containing supergroups A and B). Thus, the currently observable pandemic has likely a single evolutionary origin and is unique within the radiation of Wolbachia strains.
We present a DNA library preparation method that has allowed us to reconstruct a high-coverage (30×) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome ...allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of "missing evolution" in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans.
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Genetic triggers for sex determination are frequently co-inherited with other linked genes that may also influence one or more sex-specific phenotypes. To better understand how sex-limited regions ...evolve and function, we studied a small W chromosome-specific region of the frog Xenopus laevis that contains only three genes (dm-w, scan-w, ccdc69-w) and that drives female differentiation. Using gene editing, we found that the sex-determining function of this region requires dm-w but that scan-w and ccdc69-w are not essential for viability, female development, or fertility. Analysis of mesonephros+gonad transcriptomes during sexual differentiation illustrates masculinization of the dm-w knockout transcriptome, and identifies mostly non-overlapping sets of differentially expressed genes in separate knockout lines for each of these three W-specific gene compared to wildtype sisters. Capture sequencing of almost all Xenopus species and PCR surveys indicate that the female-determining function of dm-w is present in only a subset of species that carry this gene. These findings map out a dynamic evolutionary history of a newly evolved W chromosome-specific genomic region, whose components have distinctive functions that frequently degraded during Xenopus diversification, and evidence the evolutionary consequences of recombination suppression.
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