An efficient pipeline for ancient DNA mapping and recovery of endogenous ancient DNA from whole‐genome sequencing data

Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next‐generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from raw sequencing data. This is often difficult due to degradation of ancient DNA and high levels of contamination, especially homologous contamination that has extremely similar genetic background with that of the real ancient DNA. In this study, we collected whole‐genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from homologous contaminations, we attempted to recover reads based on ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing homologous contaminations to very low proportions. Our goal in this research was to develop useful recommendations for ancient DNA mapping and for separation of endogenous DNA to facilitate future studies of ancient DNA.     

Combined hybridization capture and shotgun sequencing for ancient DNA analysis of extinct wild and domestic dromedary camel

The performance of hybridization capture combined with next‐generation sequencing (NGS) has seen limited investigation with samples from hot and arid regions until now. We applied hybridization capture and shotgun sequencing to recover DNA sequences from bone specimens of ancient‐domestic dromedary (Camelus dromedarius) and its extinct ancestor, the wild dromedary from Jordan, Syria, Turkey and the Arabian Peninsula, respectively. Our results show that hybridization capture increased the percentage of mitochondrial DNA (mtDNA) recovery by an average 187‐fold and in some cases yielded virtually complete mitochondrial (mt) genomes at multifold coverage in a single capture experiment. Furthermore, we tested the effect of hybridization temperature and time by using a touchdown approach on a limited number of samples. We observed no significant difference in the number of unique dromedary mtDNA reads retrieved with the standard capture compared to the touchdown method. In total, we obtained 14 partial mitochondrial genomes from ancient‐domestic dromedaries with 17–95% length coverage and 1.27–47.1‐fold read depths for the covered regions. Using whole‐genome shotgun sequencing, we successfully recovered endogenous dromedary nuclear DNA (nuDNA) from domestic and wild dromedary specimens with 1–1.06‐fold read depths for covered regions. Our results highlight that despite recent methodological advances, obtaining ancient DNA (aDNA) from specimens recovered from hot, arid environments is still problematic. Hybridization protocols require specific optimization, and samples at the limit of DNA preservation need multiple replications of DNA extraction and hybridization capture as has been shown previously for Middle Pleistocene specimens.     

Performance of amplicon and shotgun sequencing for accurate biomass estimation in invertebrate community samples

New applications of DNA and RNA sequencing are expanding the field of biodiversity discovery and ecological monitoring, yet questions remain regarding precision and efficiency. Due to primer bias, the ability of metabarcoding to accurately depict biomass of different taxa from bulk communities remains unclear, while PCR‐free whole mitochondrial genome (mitogenome) sequencing may provide a more reliable alternative. Here, we used a set of documented mock communities comprising 13 species of freshwater macroinvertebrates of estimated individual biomass, to compare the detection efficiency of COI metabarcoding (three different amplicons) and shotgun mitogenome sequencing. Additionally, we used individual COI barcoding and de novo mitochondrial genome sequencing, to provide reference sequences for OTU assignment and metagenome mapping (mitogenome skimming), respectively. We found that, even though both methods occasionally failed to recover very low abundance species, metabarcoding was less consistent, by failing to recover some species with higher abundances, probably due to primer bias. Shotgun sequencing results provided highly significant correlations between read number and biomass in all but one species. Conversely, the read–biomass relationships obtained from metabarcoding varied across amplicons. Specifically, we found significant relationships for eight of 13 (amplicons B1FR‐450 bp, FF130R‐130 bp) or four of 13 (amplicon FFFR, 658 bp) species. Combining the results of all three COI amplicons (multiamplicon approach) improved the read–biomass correlations for some of the species. Overall, mitogenomic sequencing yielded more informative predictions of biomass content from bulk macroinvertebrate communities than metabarcoding. However, for large‐scale ecological studies, metabarcoding currently remains the most commonly used approach for diversity assessment.     

High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA

Analyses of degraded DNA are typically hampered by contamination, especially when employing universal primers such as commonly used in environmental DNA studies. In addition to false-positive results, the amplification of contaminant DNA may cause false-negative results because of competition, or bias, during the PCR. In this study, we test the utility of human-specific blocking primers in mammal diversity analyses of ancient permafrost samples from Siberia. Using quantitative PCR (qPCR) on human and mammoth DNA, we first optimized the design and concentration of blocking primer in the PCR. Subsequently, 454 pyrosequencing of ancient permafrost samples amplified with and without the addition of blocking primer revealed that DNA sequences from a diversity of mammalian representatives of the Beringian megafauna were retrieved only when the blocking primer was added to the PCR. Notably, we observe the first retrieval of woolly rhinoceros (Coelodonta antiquitatis) DNA from ancient permafrost cores. In contrast, reactions without blocking primer resulted in complete dominance by human DNA sequences. These results demonstrate that in ancient environmental analyses, the PCR can be biased towards the amplification of contaminant sequences to such an extent that retrieval of the endogenous DNA is severely restricted. The application of blocking primers is a promising tool to avoid this bias and can greatly enhance the quantity and the diversity of the endogenous DNA sequences that are amplified.     

Fine-mapping of shotgun template-libraries; an efficient strategy for the systematic sequencing of genomic DNA.

To test the effectiveness of ordering shotgun DNA-templates prior to sequence analysis, the 450 kb left arm of yeast chromosome XII was randomly subcloned into a phagemid vector. Clones were ordered by hybridisation to an average map density of one new insert every 125 bp and are currently used for sequencing the chromosomal fragment. An 11.5 kb overlap between the template map and a DNA fragment that had been sequenced earlier allowed an independent evaluation of the strategy's effectiveness. To this end, clones were selected from the map and tag-sequenced from either end, thus comparing the map position with the actual location within the 11.5 kb. Of 65 selected clones, taken mostly at random from a total of 423, 58 mapped on average about a quarter of a clone length around their predicted position, with the other seven being between 0.6 and 1.5 clone length off. 75-86 sequencing reactions on clones selected from the map would have been sufficient for completely sequencing both strands of the 11.5 kb fragment. The results demonstrate the efficacy of such template sorting, considerably assisting sequencing at relatively little cost on the mapping level.     

DNA sequencing: present limitations and prospects for the future

As the human genome program gets under way, we examine the progress made since the first human genome meeting in Santa Cruz in 1985. The lessons of the last 5 years demonstrate that progress has been much slower than anticipated. The new technology being developed in 1985 was fluorescent sequencing and multiplexing. These techniques are now established, but they still have to produce a substantial sequence to rival those determined by conventional technology. Inspection of the EMBL and GenBank databases shows few large sequences have been determined and that there is a large discrepancy between what is theoretically possible and what has been achieved so far.     

Method for discovering novel DNA viruses in blood using viral particle selection and shotgun sequencing

Rapid identification of viruses is needed to monitor the blood supply for emerging threats. Here we present a method that meets these criteria and allows for the shotgun sequencing of novel, uncultured DNA viruses directly from human blood. This method employs selection based on the physical properties of viruses combined with sequence-independent amplification and cloning. We show that both single- and double-stranded DNA viruses can be recovered from blood samples using this approach. In addition, we report the discovery of novel anellovirus sequences in the blood of healthy donors. PCR primers designed to amplify these novel anellovirus sequences were then used to verify the presence of these viruses in the general donor population.     

Rapid screening of complex DNA samples by single-molecule amplification and sequencing

Microbial cloning makes Sanger sequencing of complex DNA samples possible but is labor intensive. We present a simple, rapid and robust method that enables laboratories without special equipment to perform single-molecule amplicon sequencing, although in a low-throughput manner, from sub-picogram quantities of DNA. The method can also be used for quick quality control of next-generation sequencing libraries, as was demonstrated for a metagenomic sample.     

Molecular genetic analysis of the polymorphic apolipoprotein E in modern and ancient human DNA samples

In this report, methodical bases for the molecular genetic analysis of the three common apolipoprotein E alleles APOE*2, APOE*3 and APOE*4 in DNA isolated from ancient human skeletal remains are described. Considering that ancient DNA target regions for amplification are generally quite small, the detection method is based on short amplification products in the range from 71 bp to 75 bp. The applicability of the modified method for APOE genotyping was examined in modern human DNA samples.     

Comparing whole‐genome shotgun sequencing and DNA metabarcoding approaches for species identification and quantification of pollen species mixtures

Molecular identification of mixed‐species pollen samples has a range of applications in various fields of research. To date, such molecular identification has primarily been carried out via amplicon sequencing, but whole‐genome shotgun (WGS) sequencing of pollen DNA has potential advantages, including (1) more genetic information per sample and (2) the potential for better quantitative matching. In this study, we tested the performance of WGS sequencing methodology and publicly available reference sequences in identifying species and quantifying their relative abundance in pollen mock communities. Using mock communities previously analyzed with DNA metabarcoding, we sequenced approximately 200Mbp for each sample using Illumina HiSeq and MiSeq. Taxonomic identifications were based on the Kraken k‐mer identification method with reference libraries constructed from full‐genome and short read archive data from the NCBI database. We found WGS to be a reliable method for taxonomic identification of pollen with near 100% identification of species in mixtures but generating higher rates of false positives (reads not identified to the correct taxon at the required taxonomic level) relative to rbcL and ITS2 amplicon sequencing. For quantification of relative species abundance, WGS data provided a stronger correlation between pollen grain proportion and sequence read proportion, but diverged more from a 1:1 relationship, likely due to the higher rate of false positives. Currently, a limitation of WGS‐based pollen identification is the lack of representation of plant diversity in publicly available genome databases. As databases improve and costs drop, we expect that eventually genomics methods will become the methods of choice for species identification and quantification of mixed‐species pollen samples.     

Comparing the performance of three ancient DNA extraction methods for high‐throughput sequencing

The DNA molecules that can be extracted from archaeological and palaeontological remains are often degraded and massively contaminated with environmental microbial material. This reduces the efficacy of shotgun approaches for sequencing ancient genomes, despite the decreasing sequencing costs of high‐throughput sequencing (HTS). Improving the recovery of endogenous molecules from the DNA extraction and purification steps could, thus, help advance the characterization of ancient genomes. Here, we apply the three most commonly used DNA extraction methods to five ancient bone samples spanning a ~30 thousand year temporal range and originating from a diversity of environments, from South America to Alaska. We show that methods based on the purification of DNA fragments using silica columns are more advantageous than in solution methods and increase not only the total amount of DNA molecules retrieved but also the relative importance of endogenous DNA fragments and their molecular diversity. Therefore, these methods provide a cost‐effective solution for downstream applications, including DNA sequencing on HTS platforms.     

Genomic-scale capture and sequencing of endogenous DNA from feces

Genomic-level analyses of DNA from non-invasive sources would facilitate powerful conservation and evolutionary studies in natural populations of endangered and otherwise elusive species. However, the typical low quantity and poor quality of DNA that is extracted from non-invasive samples have generally precluded such work. Here we apply a modified DNA capture protocol that, when used in combination with massively-parallel sequencing technology, facilitates efficient and highly-accurate resequencing of megabases of specified nuclear genomic regions from fecal DNA samples. We validated our approach by comparing genetic variants identified from corresponding fecal and blood DNA samples of six western chimpanzees (Pan troglodytes verus) across more than 1.5 megabases of chromosome 21, chromosome X, and the complete mitochondrial genome. Our results suggest that it is now feasible to conduct genomic studies in natural populations for which constraints on invasive sampling have otherwise long been a barrier. The data we collected also provided an opportunity to examine western chimpanzee genetic diversity at unprecedented scale. Despite high mitochondrial genome diversity (π = 0.585%), western chimpanzees have a low ratio (0.42) of X chromosomal (π = 0.034%) to autosomal (chromosome 21 π = 0.081%) sequence diversity, a pattern that may reflect an unusual demographic history of this subspecies.     

Characterization of masson pine (Pinus massoniana Lamb.) microsatellite DNA by 454 genome shotgun sequencing

Microsatellites (simple sequence repeats, SSRs) are important genetic markers in tree breeding and conservation. Here we utilized high-throughput 454 sequencing technology to mine microsatellites from masson pine (MP) genomic DNA. First, we analyzed the characteristics of SSRs in all nonredundant MP reads (genome survey sequences, GSSs) and compared them with loblolly pine (LP) GSSs and BACs (bacterial artificial chromosome clone sequences), and three other nonconiferous species GSSs. Second, a set of MP GSS–SSR primer pairs were designed. There were extremely low overall GSS–SSR densities (28 SSR/Mb) in MP when compared with LP (48 SSR/Mb) and the other species. AT, AAT, AAAT, and AAAAAT were the richest motifs in di-, tri-, tetra-, and hexanucleotides, respectively. Two hundred forty GSS–SSR primer pairs were designed in total, and 20 novel polymorphic markers were identified using three populations (two natural and one clonal seed orchard) as evaluating samples. These markers should be useful for future MP population genetics studies.     

DNA methylation in mouse A-repeats in DNA methyltransferase-knockout ES cells and in normal cells determined by bisulfite genomic sequencing

Mouse ES cells with a null mutation of the known DNA methyltransferase retain some residual DNA methylation and can methylate foreign sequences de novo. We have used bisulfite genomic sequencing to examine the sequence specificity and distributions of methylation of a hypermethylated CG island sequence, mouse A-repeats. There were 13 CG dinucleotides in the region examined, 12 of which were methylated to variable extents in all DNAs. We found that: (1) there is considerable residual DNA methylation in ES cells lacking the known DNA methyltransferase (29% of normal methylation in the complete knockout ES DNA); (2) this other activity methylates at exactly the same CG sites as the major methyltransferase; and (3) differences in the distribution of methylated sites between A-repeats in these DNAs are consistent with this other activity methylating in a random de novo fashion. Also, the lack of any methylation in non-CG sites argues that, in other studies where non-CG methylation sites have been found by bisulfite sequencing, detection of such sites of non-CG methylation is not an inherent artifact in this methodology.     

中国古代人骨体质人类学的研究进展与展望

近10年来,中国古代人骨遗存的体质人类学研究迅猛发展,研究内容的广度和深度已完全超越了上世纪以人种类型分析为主的情况。这些研究涉及到古代人群的演化、古代疾病与健康、文化习俗相关的骨骼异常、骨骼功能适应、古人口学等多个领域,并积累了一批重要的古代人群体质人类学数据。这些研究工作丰富了我们对东亚地区全新世人群演化历史的认识,体现了多学科的研究理念和研究方法的创新。本文对我国近10年来全新世古代人骨材料的体质人类学研究进展进行了回顾和展望。     

Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing.

This paper describes a computer method for handling gel reading data produced by the shotgun method of DNA sequencing. The method greatly reduces the time the sequencer needs to spend checking and editing his data and yet it produces a consensus sequence for which the accuracy of determination of every base can be clearly shown. The program can take a batch of new gel readings, screen them against vector sequences removing any that match, and then compare and align all the sequences to produce a final consensus. No information is lost in this process as alignments are achieved by making only insertions and because all the individual gel readings are added to a database from which they can be retrieved and displayed lined up one above the other. This allows the user to check on the alignments achieved by the program and if necessary change them. As each gel reading is added to the database the consensus is automatically updated accordingly and used for the next comparisons. This is a much faster process than comparing each new gel against every individual gel in the database.     

Proposed nomenclature for human butyrylcholinesterase genetic variants identified by DNA sequencing

1. New information identifying nucleotide alterations of human butyrylcholinesterase allows the use of more specific nomenclature for the variants commonly known as atypical, fluoride, silent, and K variant. 2. In addition to suggesting a system of trivial names and abbreviations, we provide a list of formal names that follow the guidelines of the Committee for Human Gene Nomenclature. 3. It is suggested that formal names be included in publications whenever possible.     

Functional analysis of the apple fruit microbiome based on shotgun metagenomic sequencing of conventional and organic orchard samples

Fruits harbour abundant and diverse microbial communities that protect them from post-harvest pathogens. Identification of functional traits associated with a given microbiota can provide a better understanding of their potential influence. Here, we focused on the epiphytic microbiome of apple fruit. We suggest that shotgun metagenomic data can indicate specific functions carried out by different groups and provide information on their potential impact. Samples were collected from the surface of ‘Golden Delicious’ apples from four orchards that differ in their geographic location and management practice. Approximately 1 million metagenes were predicted based on a high-quality assembly. Functional profiling of the microbiome of fruits from orchards differing in their management practice revealed a functional shift in the microbiota. The organic orchard microbiome was enriched in pathways involved in plant defence activities; the conventional orchard microbiome was enriched in pathways related to the synthesis of antibiotics. The functional significance of the variations was explored using microbial network modelling algorithms to reveal the metabolic role of specific phylogenetic groups. The analysis identified several associations supported by other published studies. For example, the analysis revealed the nutritional dependencies of the Capnodiales group, including the Alternaria pathogen, on aromatic compounds.     

Efficient identification of point mutations by automated DNA sequencing of artificial heterozygote samples

DNA sequencing templates of individual point mutants of thelacI target gene were amplified by polymerase chain reaction (PCR). By mixing the PCR fragments from two individual mutants in a defined ratio, samples of artificial heterozygous composition were prepared. These samples were then submitted to automated DNA sequencing. The simultaneous, visual comparison of the mixed mutant traces using a graphics program efficiently revealed all heterozygous positions. Based on the individual intensities of the heterozygous base signals the identified point mutations could be assigned to the corresponding mutants. This efficient approach doubles the sample throughput for both the sequencing reactions and the gel electrophoresis using an automated DNA sequencing system.