Search

Next-generation sequencing (NGS) technologies have revolutionized phylogenomics by decreasing the cost and time required to generate sequence data from multiple markers or whole genomes. Further, the fragmented DNA of biological specimens collected decades ago can be sequenced with NGS, reducing the need for collecting fresh specimens. Sequence capture, also known as anchored hybrid enrichment, is a method to produce reduced representation libraries for NGS sequencing. The technique uses single-stranded oligonucleotide probes that hybridize with pre-selected regions of the genome that are sequenced via NGS, culminating in a dataset of numerous orthologous loci from multiple taxa. Phylogenetic analyses using these sequences have the potential to resolve deep and shallow phylogenetic relationships. Identifying the factors that affect sequence capture success could save time, money, and valuable specimens that might be destructively sampled despite low likelihood of sequencing success. We investigated the impacts of specimen age, preservation method, and DNA concentration on sequence capture (number of captured sequences and sequence quality) while accounting for taxonomy and extracted tissue type in a large-scale butterfly phylogenomics project. This project used two probe sets to extract 391 loci or a subset of 13 loci from over 6,000 butterfly specimens. We found that sequence capture is a resilient method capable of amplifying loci in samples of varying age (0–111 years), preservation method (alcohol, papered, pinned), and DNA concentration (0.020 ng/μl - 316 ng/ul). Regression analyses demonstrate that sequence capture is positively correlated with DNA concentration. However, sequence capture and DNA concentration are negatively correlated with sample age and preservation method. Our findings suggest that sequence capture projects should prioritize the use of alcohol-preserved samples younger than 20 years old when available. In the absence of such specimens, dried samples of any age can yield sequence data, albeit with returns that diminish with increasing age.

Advances in the field of museomics have promoted a high sampling demand for natural history collections (NHCs), eventually resulting in damage to invaluable resources to understand historical biodiversity. It is thus essential to achieve a consensus about which historical tissues present the best sources of DNA. In this study, we evaluated the performance of different historical tissues from Iberian wolf NHCs in genome-wide assessments. We targeted three tissues—bone (jaw and femur), maxilloturbinal bone, and skin—that have been favored by traditional taxidermy practices for mammalian carnivores. Specifically, we performed shotgun sequencing and target capture enrichment for 100,000 single nucleotide polymorphisms (SNPs) selected from the commercial Canine HD BeadChip across 103 specimens from 1912 to 2005. The performance of the different tissues was assessed using metrics based on endogenous DNA content, uniquely high-quality mapped reads after capture, and enrichment proportions. All samples succeeded as DNA sources, regardless of their collection year or sample type. Skin samples yielded significantly higher amounts of endogenous DNA compared to both bone types, which yielded equivalent amounts. There was no evidence for a direct effect of tissue type on capture efficiency; however, the number of genotyped SNPs was strictly associated with the starting amount of endogenous DNA. Evaluation of genotyping accuracy for distinct minimum read depths across tissue types showed a consistent overall low genotyping error rate (

The extraction of nucleic acids is one of the most routine procedures used in molecular biology laboratories, yet kit performance may influence the downstream processing of samples, particularly for samples which are degraded, and in low concentrations. Here we tested several commercial kits for specific use on commonly sampled mammalian museum specimens to evaluate the yield, size distribution, and endogenous content. Samples were weighed and had approximately equal input material for each extraction. These sample types are typical of natural history repositories ranged from 53 to 130 years old. The tested protocols spanned spin-column based extractions, magnetic bead purification, phenol/chloroform isolation, and specific modifications for ancient DNA. Diverse types of mammalian specimens were tested including adherent osteological material, bone and teeth, skin, and baleen. The concentration of DNA was quantified via fluorometry, and the size distributions of extracts visualized on an Agilent TapeStation. Overall, when DNA isolation was successful, all methods had quantifiable concentrations, albeit with variation across extracts. The length distributions varied based on the extraction protocol used. Shotgun sequencing was performed to evaluate if the extraction methods influenced the amount of endogenous versus exogenous content. The DNA content was similar across extraction methods indicating no obvious biases for DNA derived from different sources. Qiagen kits and phenol/chloroform isolation outperformed the Zymo magnetic bead isolations in these types of samples. Statistical analyses revealed that extraction method only explained 5% of the observed variation, and that specimen age explained variation (29%) more effectively.

Museum material is an important source of metadata for past and recent biological events. With current sequencing technologies, it is possible to obtain historical DNA (hDNA) from older material and/or endangered species to answer taxonomic, systematic, and biogeographical questions. However, hDNA from museum collections is often highly degraded, making it difficult to assess relationships at or above the species level. We therefore studied two probably extinct gastropod species of the genus Laevicaspia, which were collected ∼140 years ago in the Caspian Sea, to map “standard” mitochondrial and nuclear markers and assess both the sequencing depth and the proportion of ambiguous sites as an indicator for the phylogenetic quality of the NGS data. Our study resulted in the first phylogenetically informative mitochondrial and nuclear markers for L. caspia. Assessment of both sequencing depth (mean coverage) and proportion of ambiguous sites suggests that our assembled consensus sequences are reliable for this species. In contrast, no informative gastropod-specific DNA was obtained for L. conus, likely due to a high degree of tissue digestion and contamination with non-gastropod DNA. Nevertheless, our results show that hDNA may in principle yield high-quality sequences for species-level phylogenetic analyses, which underlines the importance of museum collections as valuable archives of the biological past.

Biological specimens in natural history collections worldwide are increasingly being used in biogeographical, environmental, and taxonomic studies. For their meaningful use, correct species identification is crucial. For example, clarifying if a species is new to science requires an overview of what has already been described. This includes comparisons with existing authoritative specimens (types). Most type specimens are rather old and their DNA expected to be degraded to various extents. Comparative DNA sequence analysis is in regular use in taxonomic research of today and is essential for identifying and delimiting species. In this study, we focus on lichenized fungi (lichens), in which many species groups are highly inconspicuous and impossible to identify to species based on morphology alone. Our aim was to test the non-mutually exclusive hypotheses that DNA quality of lichens depends on (1) time since collection, (2) taxonomic affinity, and/or (3) habitat/ecology. We included two species from each of four different lichen genera (i.e., Cladonia, Nephroma, Peltigera, and Ramalina), each species pair with a different autecology. For each species, we included samples from approximately every 25 years from present to about 150 years back in time. We used a two-step PCR-based approach followed by sequencing on an Ion Torrent PGM to produce target sequences (mtSSU) of degraded DNA. We received satisfactory DNA sequence information for 54 of 56 specimens. We recovered full-length sequences for several more than 100-years-old specimens, including a 127-years-old specimen, and retrieved enough sequence information for species identification of a 150-years-old specimen. As expected, sequencing success was negatively correlated with age of the specimens. It also varied with taxonomic affinity. We found no significant correlation between sequencing success and habitat ecology of the investigated specimens. The herein tested Ion Torrent sequencing approach outperformed Sanger sequencing with regard to sequencing success and efficiency. We find the protocol used herein highly suitable for obtaining sequences from both young and old lichen specimens and discuss potential improvements to it.