James M. Robertson

An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil

Aims:  To evaluate six commercial DNA extraction kits for their ability to isolate PCR‐quality DNA from Bacillus spores in various soil samples.

Use of Fatty Acid Methyl Ester Profiles for Discrimination of Bacillus cereus T-Strain Spores Grown on Different Media

ABSTRACT The goal of this study was to determine if cellular fatty acid methyl ester (FAME) profiling could be used to distinguish among spore samples from a single species (Bacillus cereus T strain) that were prepared on 10 different medium formulations. To analyze profile differences and identify FAME biomarkers diagnostic for the chemical constituents in each sporulation medium, a variety of statistical techniques were used, including nonmetric multidimensional scaling (nMDS), analysis of similarities (ANOSIM), and discriminant function analysis (DFA). The results showed that one FAME biomarker, oleic acid (18:1 ω9c), was exclusively associated with spores grown on Columbia agar supplemented with sheep blood and was indicative of blood supplements that were present in the sporulation medium. For spores grown in other formulations, multivariate comparisons across several FAME biomarkers were required to discern profile differences. Clustering patterns in nMDS plots and R values from ANOSIM revealed that dissimilarities among FAME profiles were most pronounced when spores grown with disparate sources of complex additives or protein supplements were compared (R > 0.8), although other factors also contributed to FAME differences. DFA indicated that differentiation could be maximized with a targeted subset of FAME variables, and the relative contributions of branched FAME biomarkers to group dissimilarities changed when different media were compared. When taken together, these analyses indicate that B. cereus spore samples grown in different media can be resolved with FAME profiling and that this may be a useful technique for providing intelligence about the production methods of Bacillus organisms in a forensic investigation.

Comparison and evaluation of RNA quantification methods using viral, prokaryotic, and eukaryotic RNA over a 104 concentration range

Quantification of RNA is essential for various molecular biology studies. In this work, three quantification methods were evaluated: ultraviolet (UV) absorbance, microcapillary electrophoresis (MCE), and fluorescence-based quantification. Viral, bacterial, and eukaryotic RNA were measured in the 500 to 0.05-ng microl(-1) range via an ND-1000 spectrophotometer (UV), Agilent RNA 6000 kits (MCE), and Quant-iT RiboGreen assay (fluorescence). The precision and accuracy of each method were assessed and compared with a concentration derived independently using inductively coupled plasma-optical emission spectroscopy (ICP-OES). Cost, operator time and skill, and required sample volumes were also considered in the evaluation. Results indicate an ideal concentration range for each quantification technique to optimize accuracy and precision. The ND-1000 spectrophotometer exhibits high precision and accurately quantifies a 1-microl sample in the 500 to 5-ng microl(-1) range. The Quant-iT RiboGreen assay demonstrates high precision in the 1 to 0.05-ng microl(-1) range but is limited to lower RNA concentrations and is more costly than the ND-1000 spectrophotometer. The Agilent kits exhibit less precision than the ND-1000 spectrophotometer and Quant-iT RiboGreen assays in the 500 to 0.05-ng microl(-1) range. However, the Agilent kits require 1 microl of sample and can determine the integrity of the RNA, a useful feature for verifying whether the isolation process was successful.

Evaluation of circular DNA substrates for whole genome amplification prior to forensic analysis

Forensic biological evidence often contains low quantities of DNA or substantially degraded DNA which makes samples refractory to genotype analysis. One approach that shows promise to overcome the limited quantity of DNA is whole genome amplification (WGA). One WGA technique, termed rolling circle amplification (RCA), involves the amplification of circular DNA fragments and this study evaluates a single-stranded (ss) DNA ligase enzyme for generating circular DNA templates for RCA WGA. Fast, efficient ligation of several sizes of ssDNA templates was achieved. The enzyme also ligated double-stranded (ds) DNA templates, a novel activity not previously reported. Adapter sequences containing optimal terminal nucleotide ends for increased ligation efficiency were designed and ligation of adapters to template DNA was optimized. Increased amplification of DNA templates was observed following WGA; however, no amplification advantage for ssDNA ligase treatment of templates was evident compared to linear templates. A multi-step process to utilize ssDNA ligase prior to WGA was developed and short tandem repeat (STR) analysis of simulated low template (LT) and fragmented DNA was evaluated. The process resulted in the loss of template DNA and failed STR analysis whereas input of linear genomic DNA template directly into WGA prior to STR analysis improved STR genotyping results compared to non-WGA treated samples. Inclusion of an extreme thermostable single-stranded DNA binding protein (SSB) during WGA also increased DNA yields. While STR artifacts such as peak imbalance, drop-in, and dropout persisted, WGA shows potential for successful genetic profiling of LT and fragmented DNA samples. Further research and development is warranted prior to use of WGA in forensic casework.

52 additional reference population samples for the 55 AISNP panel

Ancestry inference for a person using a panel of SNPs depends on the variation of frequencies of those SNPs around the world and the amount of reference data available for calculation/comparison. The Kidd Lab panel of 55 AISNPs has been incorporated in commercial kits by both Life Technologies and Illumina for massively parallel sequencing. Therefore, a larger set of reference populations will be useful for researchers using those kits. We have added reference population allele frequencies for 52 population samples to the 73 previously entered so that there are now allele frequencies publicly available in ALFRED and FROG-kb for a total of 125 population samples.

Assessing PreCR™ repair enzymes for restoration of STR profiles from artificially degraded DNA for human identification ☆

Forensic scientists have used several approaches to obtain short tandem repeat (STR) profiles from compromised DNA samples, including supplementing the polymerase chain reaction (PCR) with enhancers and using procedures yielding reduced-length amplicons. For degraded DNA, the peak intensities of the alleles separated by electrophoresis generally decrease as the length of the allele increases. When the intensities of the alleles decrease below an established threshold, they are described as drop-outs, thus contributing to a partial STR profile. This work assesses the use of repair enzymes to improve the STR profiles from artificially degraded DNA. The commercial PreCR™ repair kit of DNA repair enzymes was tested on both purified DNA and native DNA in body fluids exposed to oxidizing agents, hydrolytic conditions, ultraviolet (UV) and ionizing radiation, and desiccation. The strategy was to restrict the level of DNA damage to that which yields partial STR profiles in order to test for allele restoration as opposed to simple allele enhancement. Two protocols were investigated for allele restoration: a sequential protocol using the manufacturers repair procedure and a modified protocol reportedly designed for optimal STR analysis of forensic samples. Allele restoration was obtained with both protocols, but the peak height appeared to be higher for the modified protocol (determined by Mann-Kendall Trend Test). The success of the approach using the PreCR™ repair enzymes was sporadic; it led to allele restoration as well as allele drop-out. Additionally, allele restoration with the PreCR™ enzymes was compared with restoration by alternative, but commonly implemented approaches using Restorase™, PCRBoost™, bovine serum albumin (BSA) and the Minifiler™ STR system. The alternative methods were also successful in improving the STR profile, but their success also depended on the quality of the template encountered. Our results indicate the PreCR™ repair kit may be useful for restoring STR profiles from damaged DNA, but further work is required to develop a generalized approach.

The Microbial Rosetta Stone: a database system for tracking infectious microorganisms

The Microbial Rosetta Stone (MRS) database system was developed to support the law enforcement community by providing a comprehensive and connected microbial pathogen data–information repository. To handle the myriad types of pathogen information required to support law enforcement and intelligence community investigations, a data model previously developed for medical and epidemiological information was enhanced. The data contained in MRS are a broad collection of expert-curated microbial pathogen information, but given the multitude of potential microbes and toxins that may be used in a biocrime or bioterrorism act continual information collection and updating are required. The MRS currently relates governmental community-specific pathogen priority lists, sequence metadata, taxonomic classifications, and diseases to strain collections, specific detection and treatment protocols, and experimental results for biothreat agents. The system contains software tools that help to load, curate, and connect the data. A shared MRS database can be populated in real time by multiple users in multiple locations. Querying tools also provide simple and powerful means to access the data in any part of the database.

The Microbial Rosetta Stone Central Agricultural Database: An Information Resource on High-Consequence Plant Pathogens

Microbial pathogens of humans, animals, and plants can serve as potential agents of biowarfare, bioterrorism, and biocrime. Previously, the Microbial Rosetta Stone (MRS) Central database, an easily accessible informational resource tool, was developed to assist law enforcement personnel in the event of a disease investigation by providing key information on pathogens of concern. Although the database already contained information on a few high-profile plant pathogens, the coverage was insufficient considering the large number of plant pathogens that pose a threat, not only to agricultural production but also to natural plant resources such as forests and rangelands. In this project, 100 plant pathogens of high consequence were selected for study, existing literature on these agents was reviewed, and both the sources and key pathogen information provided therein were curated in the new Agricultural Database (AgDB), an accessory to the existing MRS Central Database. Chosen for inclusion in the MRS Central AgDB were plant pathogens having significant potential for damage to U.S. agricultural and natural ecosystems. The selection process included review of several previously developed plant-pathogen threat lists and recommendations from experts within the U.S. plant biosecurity community. Pathogen information was collected by searching a number of relevant literature databases, sites on the World Wide Web, and other resources. For inclusion in the MRS, the information was curated into categories: pathogen taxonomy, nomenclature synonyms, disease symptoms and geographic distribution, plant hosts, insect vectors, detection and diagnostic methods, laboratory and field protocols, sample collection, and epidemiology. The resulting AgDB enhances the MRS Central Database by summarizing and linking key information on high-threat plant diseases and their causal agents to relevant scientific literature and internet resources. The AgDB contains critical, key information on high-consequence plant pathogens, curated in a format that is readily accessible and easily searched. The resource enhances the existing MRS Central Database and provides law enforcement, forensic, and investigative personnel with an additional tool with which to respond to microbial emergencies, particularly those affecting the agricultural and environmental sectors.

Evaluation of the Precision ID Identity Panel for the Ion Torrent™ PGM™ sequencer

In cases where only a partial or incomplete STR profile is obtained from a sample, information contained in single nucleotide polymorphisms (SNPs) can prove informative for human identification. Thermo Fisher Scientific, which developed the high throughput Ion Torrent™ PGM™ sequencer, released the Precision ID Identity Panel, a multiplex SNP panel for human identity. We evaluated the reproducibility and sensitivity of this multiplex, which contains primers for the amplification of 90 autosomal SNPs and 34 Y-clade SNPs. The manufacturers protocol was tested using five commercially available pure native DNAs and six forensic type samples at a range of DNA input amounts (0.2-1.0ng; n, 90). In addition to analyzing the data using the manufacturers software, HID SNP Genotyper (v4.3.1), we also used CLC Genomics Workbench (Qiagen). Although library yields and templating of ion sphere particles (ISPs) were low, downstream sequencing was still successful. Across all samples, only 1.5% of all possible quality control (QC) flags were raised by both the plugin QC filter and CLC; 85% of those flags were raised as the SNP had a major allele frequency outside the thresholds specified by the manufacturer. For the remaining SNPs, coverage of >1500 X and >780 X was obtained for autosomal and Y-clade SNPs respectively, and 100% congruence among genotype calls from both analysis programs was observed. Our results demonstrate that it is possible to obtain reliable and reproducible genotypes using the Precision ID Identity Panel, when using low quantities (≥0.2ng) of either pure native DNA or forensic type DNA samples.

Isolation of Mitochondrial DNA from Single, Short Hairs without Roots Using Pressure Cycling Technology

Hairs are commonly submitted as evidence to forensic laboratories, but standard nuclear DNA analysis is not always possible. Mitochondria (mt) provide another source of genetic material; however, manual isolation is laborious. In a proof-of-concept study, we assessed pressure cycling technology (PCT; an automated approach that subjects samples to varying cycles of high and low pressure) for extracting mtDNA from single, short hairs without roots. Using three microscopically similar donors, we determined the ideal PCT conditions and compared those yields to those obtained using the traditional manual micro-tissue grinder method. Higher yields were recovered from grinder extracts, but yields from PCT extracts exceeded the requirements for forensic analysis, with the DNA quality confirmed through sequencing. Automated extraction of mtDNA from hairs without roots using PCT could be useful for forensic laboratories processing numerous samples.