An exploratory view into allelic drop-out of sequenced autosomal STRs.

As massively parallel sequencing is implemented in forensic genetics, an understanding of sequence data must accompany these advancements, that is, accurate modeling of data for proper statistical analysis. Allelic drop-out, a common stochastic effect seen in genetic data, is often modeled in statistical analysis of STR results. This proof-of-concept study sequenced several serial dilutions of a standard sample ranging from 4 ng to 7.82 pg to evaluate allelic drop-out trends on a select panel of autosomal STRs using the ForenSeq™ DNA Signature Prep Kit, Primer Set A on the Illumina MiSeq FGx. Parameters assessed included locus, profile, and run specific information. A majority of the allelic drop-out occurred in DNA concentrations less than 31.25 pg. Statistical results indicated a need for locus-specific modeling based on STR descriptors, like simple versus compound repeat patterns. No correlation was seen between average read count of scored alleles and allelic drop-out at a locus. A statistical correlation was observed between the amount of allelic drop-out and the starting amount of DNA in a sample, average read count of a sample, and total read count generated on a flow cell. This study supports using common allelic drop-out factors used in fragment length analysis on sequenced STRs while including additional locus, sample, and run specific information. Results demonstrate multiple factors that can be considered when developing probability of allelic drop-out models for sequenced autosomal STRs including locus-specific analysis, total read count of a profile, and total read count sequenced on a flow cell.

Applied Biosystems™ PrepFiler™ Forensic DNA Extraction Kit (Manual and Semi-automated via AutoMate Express™).

The PrepFiler™ Forensic DNA Extraction Kits allow for optimal genomic DNA isolation and purification from forensic samples through a bind-wash-elute-based technique that can be performed manually or robotically using the Applied Biosystems AutoMate Express™ Forensic DNA Extraction System. The extraction kits come in two formats: the standard kit used for common case type samples, like bodily fluid swabs or stains, and a BTA kit for more challenging evidence sample types that can be submitted for analysis, like bones, teeth, and adhesive-type samples. Both forms of extraction, manual and semi-automated, require an initial manual incubation step using a lysis buffer to release the DNA into solution. If following the semi-automated protocol, the lysate can be purified and eluted on the AutoMate Express™. After lysis, the DNA binds to magnetic beads in the presence of a chaotropic salt and is washed multiple times with an ethanol-based wash buffer to purify the sample and remove potential PCR inhibitors. After removing the wash liquid, elution buffer is added to the tube containing the DNA-bound magnetic beads and heated, which disrupts the bonding between the DNA and beads. The DNA is then concentrated in the final tube and can be moved forward through the DNA analysis workflow. This chapter describes a manual DNA isolation method and the extraction procedures following both manual and robotic methods using the PrepFiler™ chemistries in conjunction with the AutoMate Express™ Forensic DNA Extraction System.

Applied Biosystems' GlobalFiler™ PCR Amplification Kit.

The GlobalFiler™ PCR Amplification Kit is one of the most sensitive kits that exist today that makes the PCR amplification of human DNA possible. PCR amplification using this specific kit makes millions of copies of 24 specific target sequences in the DNA, called markers or loci. This kit is a 6-dye, short tandem repeat (STR) multiplex assay kit that has a synthetic mix of primers and single-stranded oligonucleotides that are combined with DNA samples and then subjected to 29 or 30 cycles of denaturing, annealing, and extension, as per laboratory protocol. Methods for instrument operation will vary depending on the thermal cycler instrument model that is used. Nevertheless, the GlobalFiler™ PCR Amplification Kit has proven to be a very useful tool to DNA analysts, amplifying extremely low quantities of DNA, making it possible to detect partial, if not full, genetic profiles from a wide range of sample types. This chapter discusses the typical preparation and PCR amplification of human forensic DNA samples, using the GlobalFiler™ PCR Amplification Kit.

Likelihood Ratio Calculation Using LRmix Studio.

LRmix Studio performs statistical analyses on forensic casework samples by calculating a likelihood ratio (LR) following a semi-continuous, unrestricted approach. The software utilizes a basic probabilistic model allowing the comparison of two alternative hypotheses regarding the evidence profile to include known and/or unknown contributors, for a maximum of a 4-person mixture. Other statistical factors that are included in this model are the incorporation of multiple probability of drop-out values, probability of drop-in, a correction factor for population substructure, assumed contributor inclusion, and inclusion of an unknown relative in the defense hypothesis. A range of plausible probability of drop-out values can be calculated for various contributors and hypotheses based on a Monte Carlo probability method and included in the likelihood ratio calculation. The software also includes several ways to test the validity and robustness of the probabilistic model. A sensitivity analysis can be performed by calculating likelihood ratios for the given profile against a range of drop-out values. Additionally, a non-contributor test can be performed on the crime scene sample and the chosen LR parameters to test the robustness of the model. This can give a point of comparison of the likelihood ratio generated for the person of interest (POI) compared to "random man" profiles generated from uploaded allelic frequencies. Finally, the analysis can be printed in a well-structured and user-friendly report that includes all analysis parameters. Within this chapter, the reader will learn the steps to calculate a likelihood ratio using the semi-continuous software, LRmix Studio. Additional tools supplied through the software will also be explained and demonstrated.

Rapid DNA Profile Development with Applied Biosystems RapidHIT™ ID System.

The RapidHIT™ ID System by Applied Biosystems allows the generation of a CODIS compatible STR profile in 90 min. The preloaded cartridges, fully automated workflow, and user-friendly computer interface allow for quick and simple single sample processing both in the laboratory and outside by non-laboratory personnel, like law enforcement officers. DNA processing utilizes a direct amplification workflow to generate an STR profile targeting the CODIS or ESS core loci. In conjunction with the RapidLINK™ Software, the system performs an initial analysis, flagging any profiles that do not meet single-source full profile parameters. Additionally, the RapidLINK™ allows for users to manage a multi-instrument/multi-location Rapid DNA system and view results in real-time. This gives users off-site the ability to track and even analyze results. The system allows for rapid reference sample analysis in locations like booking stations and national or border security agencies to obtain quick feedback of database hits for investigative leads while the subject is still in custody. RapidHIT™ ID DNA systems can also be set up at sites to aid in victim identification during mass disasters. The following chapter describes the process of generating a forensic DNA profile using the RapidHIT™ ID instrumentation from start to finish. Additionally, basic use and analysis using the RapidLINK™ and GeneMarker™ HID software is included.

Next-Generation Sequencing: ForenSeq™ DNA Signature Prep Kit with the Illumina MiSeq FGx.

Sequencing forensic DNA samples that are amplified and prepared with the ForenSeq™ DNA Signature Prep Kit allows for the simultaneous targeting of forensically relevant STR and SNP markers. The MiSeq™ FGx system allows massively parallel sequencing of these markers in a single analysis. The library preparation targets autosomal, Y-, and X-STRs, as well as identity SNPs. The kit can also be used to generate investigative information regarding the DNA contributor by analyzing phenotypic SNPs to predict hair color, eye color, and ancestry SNPs.Through two rounds of amplification, all loci are amplified and tagged with individualizing barcodes for sequencing capture and identification. Using bead-based technology, the libraries are purified by the removal of left-over amplification reagents and then normalized to ensure equal representation of all samples during sequencing. The individual libraries are then pooled for insertion into the MiSeq FGx. The pooled libraries are then added to a pre-packaged cartridge that contains all reagents necessary for optimal sequencing. Libraries are captured on a flow cell and undergo bridge amplification for the generation of individual clusters. Sequencing of each cluster is performed using a Sequence-By-Synthesis technology. The following chapter describes the methodology and process of library preparation of samples using the ForenSeq™ DNA Signature Prep Kit Primer Set A and B. Once completed, the chapter then focuses on the setup of a sequencing run on the MiSeq FGx and the sequencing methodology employed by the instrument.

Life history strategies among soil bacteria-dichotomy for few, continuum for many.

Study of life history strategies may help predict the performance of microorganisms in nature by organizing the complexity of microbial communities into groups of organisms with similar strategies. Here, we tested the extent that one common application of life history theory, the copiotroph-oligotroph framework, could predict the relative population growth rate of bacterial taxa in soils from four different ecosystems. We measured the change of in situ relative growth rate to added glucose and ammonium using both 18O-H2O and 13C quantitative stable isotope probing to test whether bacterial taxa sorted into copiotrophic and oligotrophic groups. We saw considerable overlap in nutrient responses across most bacteria regardless of phyla, with many taxa growing slowly and few taxa that grew quickly. To define plausible life history boundaries based on in situ relative growth rates, we applied Gaussian mixture models to organisms' joint 18O-13C signatures and found that across experimental replicates, few taxa could consistently be assigned as copiotrophs, despite their potential for fast growth. When life history classifications were assigned based on average relative growth rate at varying taxonomic levels, finer resolutions (e.g., genus level) were significantly more effective in capturing changes in nutrient response than broad taxonomic resolution (e.g., phylum level). Our results demonstrate the difficulty in generalizing bacterial life history strategies to broad lineages, and even to single organisms across a range of soils and experimental conditions. We conclude that there is a continued need for the direct measurement of microbial communities in soil to advance ecologically realistic frameworks.

A global snapshot of current opinions of next-generation sequencing technologies usage in forensics.

The future of forensic DNA testing is being shaped by the research and usage of next-generation systems, which have increased the multiplexing capabilities of the field and the type and amount of genetic data that can be utilized for investigations. The NGS adoption for casework has been slow, albeit the plethora of data that has been published. This study evaluated the current opinions on sequencing in forensics. A 20-question online-survey focusing on NGS knowledge, training, and usage was distributed to 6001 forensic DNA researchers and practitioners worldwide. A total of 367 responses were obtained from all continents (North/South America (69.8%), Europe (21.2%), Asia (5.5%), Oceania (2.5%), and Africa (1%)). The respondents consisted of 50% practitioners, 31% researchers, and 19% both. Of these, 38% already own a next-gen sequencing instrument, and 13% are planning to purchase one. Overall, there exists an extensive knowledge on next-gen sequencing within the forensic community, including among laboratories that have not yet implemented this high-throughput technology in their workflows. Current usage focuses primarily on SNP analysis for investigative leads and mitochondrial DNA analysis while future applications included both STR and SNP testing applied to general casework. The major overall concerns respondents have for implementing a sequencing instrument include limited funding, staffing, lack of time, and the cost-effectiveness of providing this service. Specific technical concerns that the respondents had are the lack of training, statistical applications, bioinformatics support, and of rigorous guidelines and recommendations. Most of the respondents do believe there will be a technology shift from using CE only to the use of NGS on casework in 5-10 years. In addition, around 66% of respondents believe that it is moderately to very likely that the court will accept sequencing analysis. Sixteen percent fell in the middle, and the remaining 15% believe it is more unlikely, with 3% of respondents believing it is very unlikely. In conclusion, this work outlines current analytical challenges experienced by the global forensic DNA community and addresses different strategies for the implementation of next-gen sequencing technologies in casework.

Quantitative Stable-Isotope Probing (qSIP) with Metagenomics Links Microbial Physiology and Activity to Soil Moisture in Mediterranean-Climate Grassland Ecosystems.

The growth and physiology of soil microorganisms, which play vital roles in biogeochemical cycling, are shaped by both current and historical soil environmental conditions. Here, we developed and applied a genome-resolved metagenomic implementation of quantitative stable isotope probing (qSIP) with an H218O labeling experiment to identify actively growing soil microorganisms and their genomic capacities. qSIP enabled measurement of taxon-specific growth because isotopic incorporation into microbial DNA requires production of new genome copies. We studied three Mediterranean grassland soils across a rainfall gradient to evaluate the hypothesis that historic precipitation levels are an important factor controlling trait selection. We used qSIP-informed genome-resolved metagenomics to resolve the active subset of soil community members and identify their characteristic ecophysiological traits. Higher year-round precipitation levels correlated with higher activity and growth rates of flagellar motile microorganisms. In addition to heavily isotopically labeled bacteria, we identified abundant isotope-labeled phages, suggesting phage-induced cell lysis likely contributed to necromass production at all three sites. Further, there was a positive correlation between phage activity and the activity of putative phage hosts. Contrary to our expectations, the capacity to decompose the diverse complex carbohydrates common in soil organic matter or oxidize methanol and carbon monoxide were broadly distributed across active and inactive bacteria in all three soils, implying that these traits are not highly selected for by historical precipitation. IMPORTANCE Soil moisture is a critical factor that strongly shapes the lifestyle of soil organisms by changing access to nutrients, controlling oxygen diffusion, and regulating the potential for mobility. We identified active microorganisms in three grassland soils with similar mineral contexts, yet different historic rainfall inputs, by adding water labeled with a stable isotope and tracking that isotope in DNA of growing microbes. By examining the genomes of active and inactive microorganisms, we identified functions that are enriched in growing organisms, and showed that different functions were selected for in different soils. Wetter soil had higher activity of motile organisms, but activity of pathways for degradation of soil organic carbon compounds, including simple carbon substrates, were comparable for all three soils. We identified many labeled, and thus active bacteriophages (viruses that infect bacteria), implying that the cells they killed contributed to soil organic matter. The activity of these bacteriophages was significantly correlated with activity of their hosts.

Life and death in the soil microbiome: how ecological processes influence biogeochemistry.

Soil microorganisms shape global element cycles in life and death. Living soil microorganisms are a major engine of terrestrial biogeochemistry, driving the turnover of soil organic matter - Earth's largest terrestrial carbon pool and the primary source of plant nutrients. Their metabolic functions are influenced by ecological interactions with other soil microbial populations, soil fauna and plants, and the surrounding soil environment. Remnants of dead microbial cells serve as fuel for these biogeochemical engines because their chemical constituents persist as soil organic matter. This non-living microbial biomass accretes over time in soil, forming one of the largest pools of organic matter on the planet. In this Review, we discuss how the biogeochemical cycling of organic matter depends on both living and dead soil microorganisms, their functional traits, and their interactions with the soil matrix and other organisms. With recent omics advances, many of the traits that frame microbial population dynamics and their ecophysiological adaptations can be deciphered directly from assembled genomes or patterns of gene or protein expression. Thus, it is now possible to leverage a trait-based understanding of microbial life and death within improved biogeochemical models and to better predict ecosystem functioning under new climate regimes.

Effects of organic acids and common household products on the occurrence of false positive test results using immunochromatographic assays.

Forensic serological analyses often rely on lateral flow immunochromatographic assays to detect proteins that are characteristic of forensically relevant body fluids. In this study, we demonstrate that a positive result, however, is not limited to target protein binding. Citric and lactic acids at various pH levels were tested using 9 different commercial immunochromatographic assays. Varying rates of false positive results were observed with commercial serological assays irrespective of brand or target biological fluid over a wide pH range. The use of kit specific buffers were only partially effective in mitigating the occurrence of organic acid-associated false positive results. Common household products containing organic acids were also tested and found to produce non-specific binding events. This is not to suggest that immunochromatographic assays are not useful as presumptive indicators of bodily fluids. Rather, this study provides a cautionary demonstration of the ease with which organic acids in common household products can generate false positives results. This finding underscores the presumptive nature of these antibody-based lateral flow assay systems.