Improving ancient DNA genome assembly.
Ontology highlight
ABSTRACT:
SUBMITTER: Seitz A
PROVIDER: S-EPMC5384568 | biostudies-literature | 2017
REPOSITORIES: biostudies-literature
Publications
Most reconstruction methods for genomes of ancient origin that are used today require a closely related reference. In order to identify genomic rearrangements or the deletion of whole genes, <i>de novo</i> assembly has to be used. However, because of inherent problems with ancient DNA, its <i>de novo</i> assembly is highly complicated. In order to tackle the diversity in the length of the input reads, we propose a two-layer approach, where multiple assemblies are generated in the first layer, wh ...[more]
Similar Datasets
Project description:Comparing the performance of three ancient DNA extraction methods for high-throughput sequencing
| PRJEB10532 | ENA
Project description:BACKGROUND: Next-Generation Sequencing has revolutionized our approach to ancient DNA (aDNA) research, by providing complete genomic sequences of ancient individuals and extinct species. However, the recovery of genetic material from long-dead organisms is still complicated by a number of issues, including post-mortem DNA damage and high levels of environmental contamination. Together with error profiles specific to the type of sequencing platforms used, these specificities could limit our ability to map sequencing reads against modern reference genomes and therefore limit our ability to identify endogenous ancient reads, reducing the efficiency of shotgun sequencing aDNA. RESULTS: In this study, we compare different computational methods for improving the accuracy and sensitivity of aDNA sequence identification, based on shotgun sequencing reads recovered from Pleistocene horse extracts using Illumina GAIIx and Helicos Heliscope platforms. We show that the performance of the Burrows Wheeler Aligner (BWA), that has been developed for mapping of undamaged sequencing reads using platforms with low rates of indel-types of sequencing errors, can be employed at acceptable run-times by modifying default parameters in a platform-specific manner. We also examine if trimming likely damaged positions at read ends can increase the recovery of genuine aDNA fragments and if accurate identification of human contamination can be achieved using a strategy previously suggested based on best hit filtering. We show that combining our different mapping and filtering approaches can increase the number of high-quality endogenous hits recovered by up to 33%. CONCLUSIONS: We have shown that Illumina and Helicos sequences recovered from aDNA extracts could not be aligned to modern reference genomes with the same efficiency unless mapping parameters are optimized for the specific types of errors generated by these platforms and by post-mortem DNA damage. Our findings have important implications for future aDNA research, as we define mapping guidelines that improve our ability to identify genuine aDNA sequences, which in turn could improve the genotyping accuracy of ancient specimens. Our framework provides a significant improvement to the standard procedures used for characterizing ancient genomes, which is challenged by contamination and often low amounts of DNA material.
| S-EPMC3468387 | biostudies-literature
Project description:Poor DNA preservation is the most limiting factor in ancient genomic research. In the majority of ancient bones and teeth, endogenous DNA molecules represent a minor fraction of the whole DNA extract, rendering shot-gun sequencing inefficient for obtaining genomic data. Based on ancient human bone samples from temperate and tropical environments, we show that an EDTA-based enzymatic 'pre-digestion' of powdered bone increases the proportion of endogenous DNA several fold. By performing the pre-digestion step between 30?min and 6?hours on five bones, we observe an asymptotic increase in endogenous DNA content, with a 2.7-fold average increase reached at 1?hour. We repeat the experiment using a brief pre-digestion (15 or 30?mins) on 21 ancient bones and teeth from a variety of archaeological contexts and observe an improvement in 16 of these. We here advocate the implementation of a brief pre-digestion step as a standard procedure in ancient DNA extractions. Finally, we demonstrate on 14 ancient teeth that by targeting the outer layer of the roots we obtain up to 14 times more endogenous DNA than when using the inner dentine. Our presented methods are likely to increase the proportion of ancient samples that are suitable for genome-scale characterization.
| S-EPMC4472031 | biostudies-literature
Project description:BackgroundSecond-generation sequencing technologies have revolutionized our ability to recover genetic information from the past, allowing the characterization of the first complete genomes from past individuals and extinct species. Recently, third generation Helicos sequencing platforms, which perform true Single-Molecule DNA Sequencing (tSMS), have shown great potential for sequencing DNA molecules from Pleistocene fossils. Here, we aim at improving even further the performance of tSMS for ancient DNA by testing two novel tSMS template preparation methods for Pleistocene bone fossils, namely oligonucleotide spiking and treatment with DNA phosphatase.ResultsWe found that a significantly larger fraction of the horse genome could be covered following oligonucleotide spiking however not reproducibly and at the cost of extra post-sequencing filtering procedures and skewed %GC content. In contrast, we showed that treating ancient DNA extracts with DNA phosphatase improved the amount of endogenous sequence information recovered per sequencing channel by up to 3.3-fold, while still providing molecular signatures of endogenous ancient DNA damage, including cytosine deamination and fragmentation by depurination. Additionally, we confirmed the existence of molecular preservation niches in large bone crystals from which DNA could be preferentially extracted.ConclusionsWe propose DNA phosphatase treatment as a mechanism to increase sequence coverage of ancient genomes when using Helicos tSMS as a sequencing platform. Together with mild denaturation temperatures that favor access to endogenous ancient templates over modern DNA contaminants, this simple preparation procedure can improve overall Helicos tSMS performance when damaged DNA templates are targeted.
| S-EPMC3430569 | biostudies-literature
Project description:We report a major improvement to the assembly of published short read sequencing data from an ancient variola virus (VARV) genome by the removal of contig-capping sequencing tags and manual searches for gap-spanning reads. The new assembly, together with camelpox and taterapox genomes, permitted new dates to be calculated for the last common ancestor of all VARV genomes. The analysis of recently sequenced VARV-like cowpox virus genomes showed that single nucleotide polymorphisms (SNPs) and amino acid changes in the vaccinia virus (VACV)-Cop-O1L ortholog, predicted to be associated with VARV host specificity and virulence, were introduced into the lineage before the divergence of these viruses. A comparison of the ancient and modern VARV genome sequences also revealed a measurable drift towards adenine + thymine (A + T) richness.
| S-EPMC5618019 | biostudies-literature
Project description:DNA de novo assembly can be used to reconstruct longer stretches of DNA (contigs), including genes and even genomes, from short DNA sequencing reads. Applying this technique to metagenomic data derived from archaeological remains, such as paleofeces and dental calculus, we can investigate past microbiome functional diversity that may be absent or underrepresented in the modern microbiome gene catalogue. However, compared to modern samples, ancient samples are often burdened with environmental contamination, resulting in metagenomic datasets that represent mixtures of ancient and modern DNA. The ability to rapidly and reliably establish the authenticity and integrity of ancient samples is essential for ancient DNA studies, and the ability to distinguish between ancient and modern sequences is particularly important for ancient microbiome studies. Characteristic patterns of ancient DNA damage, namely DNA fragmentation and cytosine deamination (observed as C-to-T transitions) are typically used to authenticate ancient samples and sequences, but existing tools for inspecting and filtering aDNA damage either compute it at the read level, which leads to high data loss and lower quality when used in combination with de novo assembly, or require manual inspection, which is impractical for ancient assemblies that typically contain tens to hundreds of thousands of contigs. To address these challenges, we designed PyDamage, a robust, automated approach for aDNA damage estimation and authentication of de novo assembled aDNA. PyDamage uses a likelihood ratio based approach to discriminate between truly ancient contigs and contigs originating from modern contamination. We test PyDamage on both on simulated aDNA data and archaeological paleofeces, and we demonstrate its ability to reliably and automatically identify contigs bearing DNA damage characteristic of aDNA. Coupled with aDNA de novo assembly, Pydamage opens up new doors to explore functional diversity in ancient metagenomic datasets.
| S-EPMC8323603 | biostudies-literature
Project description:Abstract 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. Our method for ancient DNA mapping and filtering contamination can decrease the contamination to very low level. We believe that the work we present can facilitate future studies of ancient DNA.
| S-EPMC7790629 | biostudies-literature
Project description:Genomes sequenced using short-read, next-generation sequencing technologies can have many errors and may be fragmented into thousands of small contigs. These incomplete and fragmented assemblies lead to errors in gene identification, such that single genes spread across multiple contigs are annotated as separate gene models. Such biases can confound inferences about the number and identity of genes within species, as well as gene gain and loss between species.We present AGOUTI (Annotated Genome Optimization Using Transcriptome Information), a tool that uses RNA sequencing data to simultaneously combine contigs into scaffolds and fragmented gene models into single models. We show that AGOUTI improves both the contiguity of genome assemblies and the accuracy of gene annotation, providing updated versions of each as output. Running AGOUTI on both simulated and real datasets, we show that it is highly accurate and that it achieves greater accuracy and contiguity when compared with other existing methods.AGOUTI is a powerful and effective scaffolder and, unlike most scaffolders, is expected to be more effective in larger genomes because of the commensurate increase in intron length. AGOUTI is able to scaffold thousands of contigs while simultaneously reducing the number of gene models by hundreds or thousands. The software is available free of charge under the MIT license.
| S-EPMC4952227 | biostudies-literature
Project description:BackgroundThe ostrich (Struthio camelus) is the tallest and heaviest living bird. Ostrich meat is considered a healthy red meat, with an annual worldwide production ranging from 12,000 to 15,000 tons. As part of the avian phylogenomics project, we sequenced the ostrich genome for phylogenetic and comparative genomics analyses. The initial Illumina-based assembly of this genome had a scaffold N50 of 3.59 Mb and a total size of 1.23 Gb. Since longer scaffolds are critical for many genomic analyses, particularly for chromosome-level comparative analysis, we generated optical mapping (OM) data to obtain an improved assembly. The OM technique is a non-PCR-based method to generate genome-wide restriction enzyme maps, which improves the quality of de novo genome assembly.FindingsIn order to generate OM data, we digested the ostrich genome with KpnI, which yielded 1.99 million DNA molecules (>250 kb) and covered the genome at least 500×. The pattern of molecules was subsequently assembled to align with the Illumina-based assembly to achieve sequence extension. This resulted in an OM assembly with a scaffold N50 of 17.71 Mb, which is 5 times as large as that of the initial assembly. The number of scaffolds covering 90% of the genome was reduced from 414 to 75, which means an average of ~3 super-scaffolds for each chromosome. Upon integrating the OM data with previously published FISH (fluorescence in situ hybridization) markers, we recovered the full PAR (pseudoatosomal region) on the ostrich Z chromosome with 4 super-scaffolds, as well as most of the degenerated regions.ConclusionsThe OM data significantly improved the assembled scaffolds of the ostrich genome and facilitated chromosome evolution studies in birds. Similar strategies can be applied to other genome sequencing projects to obtain better assemblies.
| S-EPMC4427950 | biostudies-literature
Project description:BackgroundHorseshoe crabs are marine arthropods with a fossil record extending back approximately 450 million years. They exhibit remarkable morphological stability over their long evolutionary history, retaining a number of ancestral arthropod traits, and are often cited as examples of "living fossils." As arthropods, they belong to the Ecdysozoa, an ancient super-phylum whose sequenced genomes (including insects and nematodes) have thus far shown more divergence from the ancestral pattern of eumetazoan genome organization than cnidarians, deuterostomes and lophotrochozoans. However, much of ecdysozoan diversity remains unrepresented in comparative genomic analyses.ResultsHere we apply a new strategy of combined de novo assembly and genetic mapping to examine the chromosome-scale genome organization of the Atlantic horseshoe crab, Limulus polyphemus. We constructed a genetic linkage map of this 2.7 Gbp genome by sequencing the nuclear DNA of 34 wild-collected, full-sibling embryos and their parents at a mean redundancy of 1.1x per sample. The map includes 84,307 sequence markers grouped into 1,876 distinct genetic intervals and 5,775 candidate conserved protein coding genes.ConclusionsComparison with other metazoan genomes shows that the L. polyphemus genome preserves ancestral bilaterian linkage groups, and that a common ancestor of modern horseshoe crabs underwent one or more ancient whole genome duplications 300 million years ago, followed by extensive chromosome fusion. These results provide a counter-example to the often noted correlation between whole genome duplication and evolutionary radiations. The new, low-cost genetic mapping method for obtaining a chromosome-scale view of non-model organism genomes that we demonstrate here does not require laboratory culture, and is potentially applicable to a broad range of other species.
| S-EPMC4066314 | biostudies-literature