The use of archived tags in retrospective genetic analysis of fish

Collections of historical tissue samples from fish (e.g. scales and otoliths) stored in museums and fisheries institutions are precious sources of DNA for conducting retrospective genetic analysis. However, in some cases, only external tags used for documentation of spatial dynamics of fish populations have been preserved. Here, we test the usefulness of fish tags as a source of DNA for genetic analysis. We extract DNA from historical tags from cod collected in Greenlandic waters between 1950 and 1968. We show that the quantity and quality of DNA recovered from tags is comparable to DNA from archived otoliths from the same individuals. Surprisingly, levels of cross‐contamination do not seem to be significantly higher in DNA from external (tag) than internal (otolith) sources. Our study therefore demonstrates that historical tags can be a highly valuable source of DNA for retrospective genetic analysis of fish.     

Multiplex preamplification PCR and microsatellite validation enables accurate single nucleotide polymorphism genotyping of historical fish scales

Incorporating historical tissues into the study of ecological, conservation and management questions can broaden the scope of population genetic research by enhancing our understanding of evolutionary processes and anthropogenic influences on natural populations. Genotyping historical and low-quality samples has been plagued by challenges associated with low amounts of template DNA and the potential for pre-existing DNA contamination among samples. We describe a two-step process designed to (i) accurately genotype large numbers of historical low-quality scale samples in a high-throughput format and (ii) screen samples for pre-existing DNA contamination. First, we describe how an efficient multiplex preamplification PCR of 45 single nucleotide polymorphisms (SNPs) can generate highly accurate genotypes with low failure and error rates in subsequent SNP genotyping reactions of individual historical scales from sockeye salmon (Oncorhynchus nerka). Second, we demonstrate how the method can be modified for the amplification of microsatellite loci to detect pre-existing DNA contamination. A total of 760 individual historical scale and 182 contemporary fin clip samples were genotyped and screened for contamination. Genotyping failure and error rates were exceedingly low and similar for both historical and contemporary samples. Pre-existing contamination in 21% of the historical samples was successfully identified by screening the amplified microsatellite loci. The advantages of automation, low failure and error rates, and ability to multiplex both the preamplification and subsequent genotyping reactions combine to make the protocol ideally suited for efficiently genotyping large numbers of potentially contaminated low-quality sources of DNA.     

Detecting the immune system response of a 500 year-old inca mummy

Disease detection in historical samples currently relies on DNA extraction and amplification, or immunoassays. These techniques only establish pathogen presence rather than active disease. We report the first use of shotgun proteomics to detect the protein expression profile of buccal swabs and cloth samples from two 500-year-old Andean mummies. The profile of one of the mummies is consistent with immune system response to severe pulmonary bacterial infection at the time of death. Presence of a probably pathogenic Mycobacterium sp. in one buccal swab was confirmed by DNA amplification, sequencing, and phylogenetic analyses. Our study provides positive evidence of active pathogenic infection in an ancient sample for the first time. The protocol introduced here is less susceptible to contamination than DNA-based or immunoassay-based studies. In scarce forensic samples, shotgun proteomics narrows the range of pathogens to detect using DNA assays, reducing cost. This analytical technique can be broadly applied for detecting infection in ancient samples to answer questions on the historical ecology of specific pathogens, as well as in medico-legal cases when active pathogenic infection is suspected.     

PCR-DGGE Comparison of Bacterial Community Structure in Fresh and Archived Soils Sampled along a Chihuahuan Desert Elevational Gradient

The polymerase chain reaction coupled with denaturing gradient gel electrophoresis (PCR-DGGE) has been used widely to determine species richness and structure of microbial communities in a variety of environments. Researchers commonly archive soil samples after routine chemical or microbial analyses, and applying PCR-DGGE technology to these historical samples offers evaluation of long-term patterns in bacterial species richness and community structure that was not available with previous technology. However, use of PCR-DGGE to analyze microbial communities of archived soils has been largely unexplored. To evaluate the stability of DGGE patterns in archived soils in comparison with fresh soils, fresh and archived soils from five sites along an elevational gradient in the Chihuahuan Desert were compared using PCR-DGGE of 16S rDNA. DNA from all archived samples was extracted reliably, but DNA in archived soils collected from a closed-canopy oak forest site could not be amplified. DNA extraction yields were lower for most archived soils, but minimal changes in bacterial species richness and structure due to archiving were noted in bacterial community profiles from four sites. Use of archived soils to determine long-term changes in bacterial community structure via PCR-DGGE appears to be a viable option for addressing microbial community dynamics for particular ecosystems or landscapes.     

Successful extraction of DNA from archived alcohol-fixed white-eye fish specimens using an ancient DNA protocol

A protocol used routinely for rapid ancient DNA extraction was applied to fish tissue archived over 80 years ago. The method proved successful, whereas other extraction protocols failed. Researchers working on DNA from older archived fish samples are encouraged to continue to concentrate their efforts on 'white-eye' specimens, which indicate an alcohol-based fixative and are thus likely to yield viable DNA.     

Simultaneous assessment of average fragment size and amount in minute samples of degraded DNA

There is currently no method allowing routine characterization of minute amounts of degraded DNA samples such as those encountered in forensic science, archived tissues, ancient DNA, extracellular or stool DNA, and processed food. Here we describe and directly validate such a method based, on the one hand, on a generalized DNA random fragmentation model and, on the other, on two quantitative polymerase chain reaction (PCR) experiments using two different target sizes. The model also makes it possible to determine the minimum sample amount, the minimum mass average fragment size, and the maximum degradation time necessary to obtain a positive PCR.     

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.     

Source-Sink Estimates of Genetic Introgression Show Influence of Hatchery Strays on Wild Chum Salmon Populations in Prince William Sound,Alaska

The extent to which stray, hatchery-reared salmon affect wild populations is much debated. Although experiments show that artificial breeding and culture influence the genetics of hatchery salmon, little is known about the interaction between hatchery and wild salmon in a natural setting. Here, we estimated historical and contemporary genetic population structures of chum salmon (Oncorhynchus keta) in Prince William Sound (PWS), Alaska, with 135 single nucleotide polymorphism (SNP) markers. Historical population structure was inferred from the analysis of DNA from fish scales, which had been archived since the late 1960’s for several populations in PWS. Parallel analyses with microsatellites and a test based on Hardy-Weinberg proportions showed that about 50% of the fish-scale DNA was cross-contaminated with DNA from other fish. These samples were removed from the analysis. We used a novel application of the classical source-sink model to compare SNP allele frequencies in these archived fish-scales (1964–1982) with frequencies in contemporary samples (2008–2010) and found a temporal shift toward hatchery allele frequencies in some wild populations. Other populations showed markedly less introgression, despite moderate amounts of hatchery straying. The extent of introgression may reflect similarities in spawning time and life-history traits between hatchery and wild fish, or the degree that hybrids return to a natal spawning area. The source-sink model is a powerful means of detecting low levels of introgression over several generations.     

Partial uracil–DNA–glycosylase treatment for screening of ancient DNA

The challenge of sequencing ancient DNA has led to the development of specialized laboratory protocols that have focused on reducing contamination and maximizing the number of molecules that are extracted from ancient remains. Despite the fact that success in ancient DNA studies is typically obtained by screening many samples to identify a promising subset, ancient DNA protocols have not, in general, focused on reducing the time required to screen samples. We present an adaptation of a popular ancient library preparation method that makes screening more efficient. First, the DNA extract is treated using a protocol that causes characteristic ancient DNA damage to be restricted to the terminal nucleotides, while nearly eliminating it in the interior of the DNA molecules, allowing a single library to be used both to test for ancient DNA authenticity and to carry out population genetic analysis. Second, the DNA molecules are ligated to a unique pair of barcodes, which eliminates undetected cross-contamination from this step onwards. Third, the barcoded library molecules include incomplete adapters of short length that can increase the specificity of hybridization-based genomic target enrichment. The adapters are completed just before sequencing, so the same DNA library can be used in multiple experiments, and the sequences distinguished. We demonstrate this protocol on 60 ancient human samples.     

Extensive human DNA contamination in extracts from ancient dog bones and teeth

Ancient DNA (aDNA) sequences, especially those of human origin, are notoriously difficult to analyze due to molecular damage and exogenous DNA contamination. Relatively few systematic studies have focused on this problem. Here we investigate the extent and origin of human DNA contamination in the most frequently used sources for aDNA studies, that is, bones and teeth from museum collections. To distinguish contaminant DNA from authentic DNA we extracted DNA from dog (Canis familiaris) specimens. We monitored the presence of a 148-bp human-specific and a 152-bp dog-specific mitochondrial DNA (mtDNA) fragment in DNA extracts as well as in negative controls. The total number of human and dog template molecules were quantified using real-time polymerase chain reaction (PCR), and the sequences were characterized by amplicon cloning and sequencing. Although standard precautions to avoid contamination were taken, we found that all samples from the 29 dog specimens contained human DNA, often at levels exceeding the amount of authentic ancient dog DNA. The level of contaminating human DNA was also significantly higher in the dog extracts than in the negative controls, and an experimental setup indicated that this was not caused by the carrier effect. This suggests that the contaminating human DNA mainly originated from the dog bones rather than from laboratory procedures. When cloned, fragments within a contaminated PCR product generally displayed several different sequences, although one haplotype was often found in majority. This leads us to believe that recognized criteria for authenticating aDNA cannot separate contamination from ancient human DNA the way they are presently used.     

Long-term effective population sizes, temporal stability of genetic composition and potential for local adaptation in anadromous brown trout (Salmo trutta) populations

We examined the long-term temporal (1910s to 1990s) genetic variation at eight microsatellite DNA loci in brown trout (Salmo trutta L) collected from five anadromous populations in Denmark to assess the long-term stability of genetic composition and to estimate effective population sizes (Ne). Contemporary and historical samples consisted of tissue and archived scales, respectively. Pairwise thetaST estimates, a hierarchical analysis of molecular variance (amova) and multidimensional scaling analysis of pairwise genetic distances between samples revealed much closer genetic relationships among temporal samples from the same populations than among samples from different populations. Estimates of Ne, using a likelihood-based implementation of the temporal method, revealed Ne >or= 500 in two of three populations for which we have historical data. A third population in a small (3 km) river showed Ne >or= 300. Assuming a stepping-stone model of gene flow we considered the relative roles of gene flow, random genetic drift and selection to assess the possibilities for local adaptation. The requirements for local adaptation were fulfilled, but only adaptations resulting from strong selection were expected to occur at the level of individual populations. Adaptations resulting from weak selection were more likely to occur on a regional basis, i.e. encompassing several populations. Ne appears to have declined recently in at least one of the studied populations, and the documented recent declines of many other anadromous brown trout populations may affect the persistence of local adaptation.     

Screening of DNA polymorphisms in samples of archived scales from New Zealand snapper

Primers and protocols were developed to screen effciently DNA sequence polymorphism of nuclear and mitochondrial loci in samples of dried archived scales collected over the last 50 years from the New Zealand snapper Pagrus auratus (Sparidae).     

Identification of high-confidence somatic mutations in whole genome sequence of formalin-fixed breast cancer specimens

The utilization of archived, formalin-fixed paraffin-embedded (FFPE) tumor samples for massive parallel sequencing has been challenging due to DNA damage and contamination with normal stroma. Here, we perform whole genome sequencing of DNA isolated from two triple-negative breast cancer tumors archived for >11 years as 5 μm FFPE sections and matched germline DNA. The tumor samples show differing amounts of FFPE damaged DNA sequencing reads revealed as relatively high alignment mismatch rates enriched for C · G > T · A substitutions compared to germline samples. This increase in mismatch rate is observable with as few as one million reads, allowing for an upfront evaluation of the sample integrity before whole genome sequencing. By applying innovative quality filters incorporating global nucleotide mismatch rates and local mismatch rates, we present a method to identify high-confidence somatic mutations even in the presence of FFPE induced DNA damage. This results in a breast cancer mutational profile consistent with previous studies and revealing potentially important functional mutations. Our study demonstrates the feasibility of performing genome-wide deep sequencing analysis of FFPE archived tumors of limited sample size such as residual cancer after treatment or metastatic biopsies.     

Ancient DNA analysis of human populations

The use of ancient DNA (aDNA) in the reconstruction of population origins and evolution is becoming increasingly common. The resultant increase in number of samples and polymorphic sites assayed and the number of studies published may give the impression that all technological hurdles associated with aDNA technology have been overcome. However, analysis of aDNA is still plagued by two issues that emerged at the advent of aDNA technology, namely the inability to amplify a significant number of samples and the contamination of samples with modern DNA. Herein, we analyze five well-preserved skeletal specimens from the western United States dating from 800-1600 A.D. These specimens yielded DNA samples with levels of contamination ranging from 0-100%, as determined by the presence or absence of New World-specific mitochondrial markers. All samples were analyzed by a variety of protocols intended to assay genetic variability and detect contamination, including amplification of variously sized DNA targets, direct DNA sequence analysis of amplification products and sequence analysis of cloned amplification products, analysis of restriction fragment length polymorphisms, quantitation of target DNA, amino acid racemization, and amino acid quantitation. Only the determination of DNA sequence from a cloned amplification product clearly revealed the presence of both ancient DNA and contaminating DNA in the same extract. Our results demonstrate that the analysis of aDNA is still an excruciatingly slow and meticulous process. All experiments, including stringent quality and contamination controls, must be performed in an environment as free as possible of potential sources of contaminating DNA, including modern DNA extracts. Careful selection of polymorphic markers capable of discriminating between ancient DNA and probable DNA contaminants is critical. Research strategies must be designed with a goal of identifying all DNA contaminants in order to differentiate convincingly between contamination and endogenous DNA.     

Tracking down human contamination in ancient human teeth

DNA contamination arising from the manipulation of ancient calcified tissue samples is a poorly understood, yet fundamental, problem that affects the reliability of ancient DNA (aDNA) studies. We have typed the mitochondrial DNA hypervariable region I of the only 6 people involved in the excavation, washing, and subsequent anthropological and genetic study of 23 Neolithic remains excavated from Granollers (Barcelona, Spain) and searched for their presence among the 572 clones generated during the aDNA analyses of teeth from these samples. Of the cloned sequences, 17.13% could be unambiguously identified as contaminants, with those derived from the people involved in the retrieval and washing of the remains present in higher frequencies than those of the anthropologist and genetic researchers. This finding confirms, for the first time, previous hypotheses that teeth samples are most susceptible to contamination at their initial excavation. More worrying, the cloned contaminant sequences exhibit substitutions that can be attributed to DNA damage after the contamination event, and we demonstrate that the level of such damage increases with time: contaminants that are >10 years old have approximately 5 times more damage than those that are recent. Furthermore, we demonstrate that in this data set, the damage rate of the old contaminant sequences is indistinguishable from that of the endogenous DNA sequences. As such, the commonly used argument that miscoding lesions observed among cloned aDNA sequences can be used to support data authenticity is misleading in scenarios where the presence of old contaminant sequences is possible. We argue therefore that the typing of those involved in the manipulation of the ancient human specimens is critical in order to ensure that generated results are accurate.     

Technical note: PCR analysis of minimum target amount of ancient DNA

The study of ancient DNA plays an important role in archaeological and palaeontological research as well as in pathology and forensics. Here, we present a new tool for ancient DNA analysis, which overcomes contamination problems, DNA degradation, and the negative effects of PCR inhibitors while reducing the amount of starting target material in the picogram range. Ancient bone samples from four Egyptian mummies were examined by combining laser microdissection, conventional DNA extraction, and low‐volume PCR. Initially, several bone particles (osteons) in the micrometer range were extracted by laser microdissection. Subsequently, ancient DNA amplification was performed to verify our extraction method. Amelogenin and β‐actin gene specific fragments were amplified via low‐volume PCR in a total reaction volume of 1 μl. Results of microdissected mummy DNA samples were compared to mummy DNA, which was extracted using a standard DNA extraction method based on pulverization of bone material. Our results highlight the combination of laser microdissection and low‐volume PCR as a promising new technique in ancient DNA analysis. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

Cryptic contamination and phylogenetic nonsense

Ancient human DNA has been treated cautiously ever since the problems related to this type of material were exposed in the early 1990s, but as sequential genetic data from ancient specimens have been key components in several evolutionary and ecological studies, interest in ancient human DNA is on the increase again. It is especially tempting to approach archaeological and anthropological questions through this type of material, but DNA from ancient human tissue is notoriously complicated to work with due to the risk of contamination with modern human DNA. Various ways of authenticating results based on ancient human DNA have been developed to circumvent the problems. One commonly used method is to predict what the contamination is expected to look like and then test whether the ancient human DNA fulfils this prediction. If it does, the results are rejected as contamination, while if it does not, they are often considered authentic. We show here that human contamination in ancient material may well deviate from local allele frequencies or the distributions to be found among the laboratory workers and archaeologists. We conclude that it is not reliable to authenticate ancient human DNA solely by showing that it is different from what would be expected from people who have handled the material.     

Using in silico predicted ancestral genomes to improve the efficiency of paleogenome reconstruction

Paleogenomics is the nascent discipline concerned with sequencing and analysis of genome‐scale information from historic, ancient, and even extinct samples. While once inconceivable due to the challenges of DNA damage, contamination, and the technical limitations of PCR‐based Sanger sequencing, following the dawn of the second‐generation sequencing revolution, it has rapidly become a reality. However, a significant challenge facing ancient DNA studies on extinct species is the lack of closely related reference genomes against which to map the sequencing reads from ancient samples. Although bioinformatic efforts to improve the assemblies have focused mainly in mapping algorithms, in this article we explore the potential of an alternative approach, namely using reconstructed ancestral genome as reference for mapping DNA sequences of ancient samples. Specifically, we present a preliminary proof of concept for a general framework and demonstrate how under certain evolutionary divergence thresholds, considerable mapping improvements can be easily obtained.     

Hypersensitive PCR, ancient human mtDNA, and contamination

When highly efficient polymerase was used with high cycle numbers (50-60), strong amplifications were observed, but negative controls were also unexpectedly amplified in a study of ancient human mtDNA from 2000-year-old skeletons. The results of a series of tests revealed that the hypersensitive polymerase chain reaction (PCR) generated by higher cycles and the presence of contaminant DNA (though at extremely low levels) should be responsible for the amplification of negative controls. We suggest that PCR sensitivity be optimized to take advantage of highly efficient polymerase and at the same time prevent "background DNA" from becoming "contaminant DNA" and obscuring the analysis of authentic ancient DNA. We propose the use of multiple positive controls when amplifying ancient human mtDNA samples to indicate the sensitivity of individual PCR amplifications and to monitor the contamination levels of modern human DNA. This study provides some suggestions as to how to amplify and analyze ancient human mtDNA when unavoidable and extremely tiny amounts of modern human DNA exist.     

Museum genomics: low-cost and high-accuracy genetic data from historical specimens

Natural history collections are unparalleled repositories of geographical and temporal variation in faunal conditions. Molecular studies offer an opportunity to uncover much of this variation; however, genetic studies of historical museum specimens typically rely on extracting highly degraded and chemically modified DNA samples from skins, skulls or other dried samples. Despite this limitation, obtaining short fragments of DNA sequences using traditional PCR amplification of DNA has been the primary method for genetic study of historical specimens. Few laboratories have succeeded in obtaining genome-scale sequences from historical specimens and then only with considerable effort and cost. Here, we describe a low-cost approach using high-throughput next-generation sequencing to obtain reliable genome-scale sequence data from a traditionally preserved mammal skin and skull using a simple extraction protocol. We show that single-nucleotide polymorphisms (SNPs) from the genome sequences obtained independently from the skin and from the skull are highly repeatable compared to a reference genome.