Can models for forest attributes based on airborne laser scanning be generalized for different silvicultural management systems?

In Finland, interest in continuous cover forestry (CCF) has increased rapidly in recent years. During those years CCF has been examined from various viewpoints but not from the perspective of forest inventories. This holds especially true for applications based on remote sensing. Conversely, airborne laser scanning (ALS) data have been widely used to predict forest characteristics such as size distribution and vertical forest structure, which are closely related to the forest information needs of CCF. In this study we used the area-based approach to predict a set of stand attributes from ALS data (5 pulses per m2) in a CCF forest management experiment in Katajama & BULL;ki, eastern Finland. In addition to the CCF stands, the experiment included shelterwood stands and untreated stands. The predicted attributes included volume, biomass, basal area, number of stems, mean diameter, Lorey's height, dominant height, standing dead wood volume, parameters of the theoretical stem diameter distribution model, understory height and number of understory stems. Our main aim was to test whether the same model could be used across different management systems. The accuracy of the attributes predicted for the CCF stands was compared with the predictions for the other management systems in the same experiment. We also compared and discussed our results in relation to the even-aged stand attribute predictions that were conducted by using separate operational forest data collected from sites surrounding Katajama & BULL;ki. The results showed that forest data from the different management systems could be combined into a single model of a stand attribute, i.e., ALS metrics were found to be suitable for comparing different management systems in regard to differences in forest structure. The accuracy of the predicted attributes in the CCF plots was comparable to that of the other management alternatives in the experiment. The accuracy was also comparable to that of even-aged forests. The results of this study were promising; the stand attributes of CCF-managed forests could be predicted analogously to those of other management systems. This indicates that for the purposes of forest inventories there may not be a need to stratify forest lands by management system. It should be noted, however, that the study area was relatively small, that the forest stands were harvested in the 1980 s, and that the attributes may not have been completely exhaustive for CCF

A Rapid, Cost-Effective Method of Assembly and Purification of Synthetic DNA Probes >100 bp

Here we introduce a rapid, cost-effective method of generating molecular DNA probes in just under 15 minutes without the need for expensive, time-consuming gel-extraction steps. As an example, we enzymatically concatenated six variable strands (50 bp) with a common strand sequence (51 bp) in a single pool using Fast-Link DNA ligase to produce 101 bp targets (10 min). Unincorporated species were then filtered out by passing the crude reaction through a size-exclusion column (<5 min). We then compared full-length product yield of crude and purified samples using HPLC analysis; the results of which clearly show our method yields three-quarters that of the crude sample (50% higher than by gel-extraction). And while we substantially reduced the amount of unligated product with our filtration process, higher purity and yield, with an increase in number of stands per reaction (>12) could be achieved with further optimization. Moreover, for large-scale assays, we envision this method to be fully automated with the use of robotics such as the Biomek FX; here, potentially thousands of samples could be pooled, ligated and purified in either a 96, 384 or 1536-well platform in just minutes

PathogenMip Assay: A Multiplex Pathogen Detection Assay

The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe

Comparing mutation calls in fixed tumour samples between the Affymetrix OncoScan® Array and PCR based next-generation sequencing

Background: The importance of accurate and affordable mutation calling in fixed pathology samples is becoming increasingly important as we move into the era of personalised medicine. The Affymetrix OncoScan® Array platform is designed to produce actionable mutation calls in archival material. Methods: We compared calls made using the OncoScan platform with calls made using a custom designed PCR panel followed by next-generation sequencing (NGS), in order to benchmark the sensitivity and specificity of the OncoScan calls in a large cohort of fixed tumour samples. 392 fixed, clinical samples were sequenced, encompassing 641 PCR regions, 403 putative positive calls and 1528 putative negative calls. Results: A small number of mutations could not be validated, either due to large indels or pseudogenes impairing parts of the NGS pipeline. For the remainder, if calls were filtered according to simple quality metrics, both sensitivity and specificity for the OncoScan platform were over 98%. This applied even to samples with poorer sample quality and lower variant allele frequency (5–10%) than product claims indicated. Conclusions: This benchmarking study will be useful to users and potential users of this platform, who wish to compare technologies or interpret their own results

High Throughput Automated Allele Frequency Estimation by Pyrosequencing

Pyrosequencing is a DNA sequencing method based on the principle of sequencing-by-synthesis and pyrophosphate detection through a series of enzymatic reactions. This bioluminometric, real-time DNA sequencing technique offers unique applications that are cost-effective and user-friendly. In this study, we have combined a number of methods to develop an accurate, robust and cost efficient method to determine allele frequencies in large populations for association studies. The assay offers the advantage of minimal systemic sampling errors, uses a general biotin amplification approach, and replaces dTTP for dATP-apha-thio to avoid non-uniform higher peaks in order to increase accuracy. We demonstrate that this newly developed assay is a robust, cost-effective, accurate and reproducible approach for large-scale genotyping of DNA pools. We also discuss potential improvements of the software for more accurate allele frequency analysis

High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays

BACKGROUND:A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small (~40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue.RESULTS:Using an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%), with only a modest loss in performance in FFPE.CONCLUSION:MIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at [email protected]

Connector Inversion Probe Technology: A Powerful One-Primer Multiplex DNA Amplification System for Numerous Scientific Applications

We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods. Furthermore, we introduce the concept of two-dimension informational barcodes, i.e. “multiplex multiplexing padlocks” (MMPs). For the readers' convenience, we also provide an on-line tutorial with user-interface software application CIP creator 1.0.1, for custom probe generation from virtually any new or established primer-pairs

Abrupt global events in the Earth's history: a physics perspective

The timeline of the Earth's history reveals quasi-periodicity of the geological record over the last 542 Myr, on timescales close, in the order of magnitude, to 1 Myr. What is the origin of this quasi-periodicity? What is the nature of the global events that define the boundaries of the geological time scale? I propose that a single mechanism is responsible for all three types of such events: mass extinctions, geomagnetic polarity reversals, and sea-level fluctuations. The mechanism is fast, and involves a significant energy release. The mechanism is unlikely to have astronomical causes, both because of the energies involved, and because it acts quasi-periodically. It must then be sought within the Earth itself. And it must be capable of reversing the Earth's magnetic field. The last requirement makes it incompatible with the consensus model of the origin of the geomagnetic field - the hydromagnetic dynamo operating in the Earth's fluid core. In the second part of the paper, I show that a vast amount of seemingly unconnected geophysical and geological data can be understood in a unified way if the source of the Earth's main magnetic field is a ~200-km-thick lithosphere, repeatedly magnetized as a result of methane-driven oceanic eruptions, which produce ocean flow capable of dynamo action. The eruptions are driven by the interplay of buoyancy forces and exsolution of dissolved gas, which accumulates in the oceanic water masses prone to stagnation and anoxia. Polarity reversals, mass extinctions, and sequence boundaries are consequences of these eruptions. Unlike the consensus model of geomagnetism, this scenario is consistent with the paleomagnetic data showing that "directional changes during a [geomagnetic polarity] reversal can be astonishingly fast, possibly occurring as a nearly instantaneous jump from one inclined dipolar state to another in the opposite hemisphere".Comment: Final journal version. New title, significant changes. Supersedes v.

Single nucleotide polymorphism genotyping in polyploid wheat with the Illumina GoldenGate assay

Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapping and construction of high-density genetic maps. These applications usually require genotyping of thousands of SNPs in a large number of individuals. Although a number of SNP genotyping assays are available, most of them are designed for SNP genotyping in diploid individuals. Here, we demonstrate that the Illumina GoldenGate assay could be used for SNP genotyping of homozygous tetraploid and hexaploid wheat lines. Genotyping reactions could be carried out directly on genomic DNA without the necessity of preliminary PCR amplification. A total of 53 tetraploid and 38 hexaploid homozygous wheat lines were genotyped at 96 SNP loci. The genotyping error rate estimated after removal of low-quality data was 0 and 1% for tetraploid and hexaploid wheat, respectively. Developed SNP genotyping assays were shown to be useful for genotyping wheat cultivars. This study demonstrated that the GoldenGate assay is a very efficient tool for high-throughput genotyping of polyploid wheat, opening new possibilities for the analysis of genetic variation in wheat and dissection of genetic basis of complex traits using association mapping approach