Fur and faeces: an experimental assessment of non-invasive DNA sampling for the European pine marten

Non-invasive genetic sampling using materials such as faeces or hair can be used to monitor wildlife populations, although DNA quality is often poor. Improving sampling efficiency and minimising factors that reduce DNA quality are therefore critical. After a severe decline, the European pine marten, Martes martes, has reclaimed much of its former range in Scotland, UK. Recording this rapid range expansion requires developing techniques for accurate monitoring, but this is hampered by the species' elusive behaviour. We tested two sampling methods, hair collected from hair tubes and faeces (scat) collected along tracks, to assess the effects of key environmental and sampling variables on DNA quality and sampling efficiency. For hair, we tested the influence of hair tube location (distance from forest tracks) on collection rate and sex ratio of animals successfully sampled. For scats, we assessed the effect of time since defecation (1 to 16days) on genotyping error rates and success under two contrasting environmental conditions (exposed to rainfall or sheltered). We found no bias in the collection rate or sex ratio of animals detected by hair samples with differing proximity to forest tracks. DNA amplification failure for scats exposed to rainfall increased from 28 to 65% over the 16-day experimental period. During periods of low rainfall, the length of collection sessions could therefore be extended to increase sample number without risk of DNA degradation. Lack of bias in hair collection rates with proximity to forest tracks provides justification for tube placement close to tracks, as this reduces survey effort. These findings provide guidance for the development of efficient and cost-effective non-invasive sampling of Scottish pine martens

Non-invasive genotyping and spatial mark-recapture methods to estimate European pine marten density in forested landscapes

Accurate population density estimates are important for conservation but can be difficult to obtain where species are elusive or rare. Non-invasive genotyping from hair or faeces has provided a promising solution and allowed individual identification from genotypes to inform population assessment models. We use individual genotypes derived from hair samples and Spatially Explicit Capture Recapture (SECR) models to estimate the population density of European pine marten (Martes martes) in three Scottish forests, then examine the effects of forest fragmentation on population size. Relative trends in pine marten abundance can be observed via changes in the number of scats in an area through time, but the link between this measure and population density remains unclear. We provide the first calibration of scat counts to enable population density estimation without the need for genetic analysis. Population density estimates ranged from 0.07 km-2 (95% CI 0.03 – 0.16) to 0.38 km-2 (95% CI 0.11 – 1.07), which were mid to low compared to other estimates from the Scottish population. An unequal sex ratio was found in one of the three forests. We found support for the previous finding that pine marten density in Scotland increases with forest fragmentation up to a threshold level (20 – 35% forest cover), beyond which it decreases. Our calibration suggests a non-linear relationship between scat counts and population density, although relatively small changes in population density result in marked changes in scat number. Following the recent re-introduction of pine martens to Wales, non-invasive genetic sampling for population estimation may provide an effective way of monitoring their progress

Energy spread of ultracold electron bunches extracted from a laser cooled gas

Ultrashort and ultracold electron bunches created by near-threshold femtosecond photoionization of a laser-cooled gas hold great promise for single-shot ultrafast diffraction experiments. In previous publications the transverse beam quality and the bunch length have been determined. Here the longitudinal energy spread of the generated bunches is measured for the first time, using a specially developed Wien filter. The Wien filter has been calibrated by determining the average deflection of the electron bunch as a function of magnetic field. The measured relative energy spread $\frac{\sigma_{U}}{U} = 0.64 \pm 0.09\%$ agrees well with the theoretical model which states that it is governed by the width of the ionization laser and the acceleration length

Increasing quality, throughput and speed of sample preparation for strand-specific messenger RNA sequencing

Background RNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming. Methods Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. Results This work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments. Conclusions These developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25 ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types.Medicine, Faculty ofScience, Faculty ofNon UBCMedical Genetics, Department ofMicrobiology and Immunology, Department ofReviewedFacult

Automated high throughput nucleic acid purification from formalin-fixed paraffin-embedded tissue samples for next generation sequence analysis

<div><p>Curation and storage of formalin-fixed, paraffin-embedded (FFPE) samples are standard procedures in hospital pathology laboratories around the world. Many thousands of such samples exist and could be used for next generation sequencing analysis. Retrospective analyses of such samples are important for identifying molecular correlates of carcinogenesis, treatment history and disease outcomes. Two major hurdles in using FFPE material for sequencing are the damaged nature of the nucleic acids and the labor-intensive nature of nucleic acid purification. These limitations and a number of other issues that span multiple steps from nucleic acid purification to library construction are addressed here. We optimized and automated a 96-well magnetic bead-based extraction protocol that can be scaled to large cohorts and is compatible with automation. Using sets of 32 and 91 individual FFPE samples respectively, we generated libraries from 100 ng of total RNA and DNA starting amounts with 95–100% success rate. The use of the resulting RNA in micro-RNA sequencing was also demonstrated. In addition to offering the potential of scalability and rapid throughput, the yield obtained with lower input requirements makes these methods applicable to clinical samples where tissue abundance is limiting.</p></div

Automated high throughput FormaPure-based extraction protocol.

<p>(<b>A</b>) Work flow illustration of sample acquisition, upstream sample processing and extraction. Note that a separate high temperature incubation step is added to facilitate the reversal of remaining crosslinks. The upstream processes are manual in the original protocol whereas those steps are modified to be suitable for automation in the modified protocol. The in-house on-deck heating blocks were instrumental in rendering the lysis/deparaffinization steps automatable. Acquisition of samples in SBS format matrix tubes with their automated capping and decapping were also further measures that allowed the entire process to be amenable for automated liquid handling. (<b>B</b>) gDNA yield. Historical gDNA yield data from the Qiagen/High Pure protocol (Q; n = 142) using equivalent sizes of numerous FFPE samples of lymphoma origin was compared with that of the FormaPure protocol (F; n-91). (C) RNA yield. Comparison of the Qiagen-High Pure (Q-H), and FormaPure (F) protocols are shown. N = 142 for Q-H and N = 44 for F.</p

Suitability of the FormaPure extracted RNA for FFPE strand-specific RNA-seq.

<p>(<b>A)</b> Strand-specific libraries were generated from four different FormaPure extracted human FFPE samples (FFPE A-D) and UHR fresh RNA. Two different total (DNase-treated) RNA input amounts were used (100 and 200 ng, respectively). Final library yield (nM) (left panel) and % duplicates (middle panel) as well as the distribution of aligned reads to various regions of the transcriptome (right panel) are shown graphically. These libraries were sequenced as a pool at PE75 bp. (<b>B</b>) Comparison of Qiagen and FormaPure extraction protocols using mouse FFPE scrolls. Final library yield (nM) (Left panel) and % duplicates, % aligned, and the distribution of aligned reads to various regions of the transcriptome (middle panel) as well as number of genes with 1x coverage (right panel) are shown graphically.</p