126th SEMI-ANNUAL SEMINAR (Fall 2015)
CALIFORNIA ASSOCIATION OF CRIMINALISTS
September 21-25, 2015
Burlingame, California

THIRTY YEARS OF FORENSICS DNA ANALYSIS: A PERSPECTIVE (FOUNDER'S LECTURE)
Henry Erlich, Ph.D., Children's Hospital Oakland Research Institute

In 1985, two papers reported technical developments that revolutionized the field of forensics genetics. The analysis by Southern Blotting of Variable Number Tandem Repeat (VNTR) regions of human genomic DNA, known as "DNA Fingerprinting, was used for individual identification and in vitro method of enzymatic amplification, termed Polymerase Chain Reaction or PCR, was used to amplify and genotype a single gene (beta-globin) from human genomic DNA. PCR amplification of the polymorphic HLA-DQA1 gene with a thermostable DNA polymerase provided evidence in the first U.S. DNA case in 1986 (Pennsylvania vs Pestinikis) as well as the first exoneration (Illinois vs Dotson) in 1987. This assay, based on genotyping with immobilized hybridization probes, became the basis for the first commercial PCR test in 1991 and, along with the SNP-based test, Polymarker, introduced shortly thereafter, was widely used in forensics analysis. The introduction of commercial PCR-based kits to analyze a specific set of short tandem repeats (STRs) using capillary electrophoresis provided greatly enhanced discrimination potential. These STR systems are the basis of current casework and database genotyping and, in a sense, represent the "fusion" of the two technical developments reported 30 years ago. The history of forensic DNA analysis in criminal cases and missing person identification, including a discussion of the so-called "DNA Wars" period during which the admissibility of DNA evidence was hotly debated, will be reviewed. Finally, recent developments in the application of "Next Generation Sequencing" technology using mitochondrial DNA to the analysis of particularly challenging forensic specimens, such as mixed samples, will be discussed.


LOOKING FOR "LOST SHARKS": A JOURNEY OF DISCOVERY
David A. Ebert, Ph.D., Program Director / Pacific Shark Research Center; Victoria Vasquez, Graduate Student / Pacific Shark Research Center

The public's perception of sharks often conjures up images of a large, fearsome, toothy predator, with its large dorsal fin cutting its way through the waters' surface. However, the reality is that sharks come in a variety of sizes and shapes, from the whale shark (Rhincodon typus), the world's largest fish, to the dwarf pygmy sharks (Squaliolus spp.), these enigmatic fishes occupy most marine, and some freshwater, habitats. In addition, the batoids and chimaeras, along with the sharks, form a distinctive group of fishes collectively referred to as the Chondrichthyans. There are more than 500 species of sharks, along with nearly 650 batoid and 50 chimaera species, bringing the overall total to about 1200 species of sharks and shark-like fishes. The diversity of sharks and their relatives has increased exponentially over the past decade with more than 230 new species having been described over the past decade. This represents nearly 20% of all shark species that have been described. Most of these new discoveries have come from the Indo-Australian region, followed by the Western Indian Ocean and western North Pacific regions. However, despite such a rich and diverse fauna, the majority of sharks and their relatives have largely been "lost" in a hyper-driven media age whereby a few large charismatic shark mega-stars overshadow the majority of shark species. While these mega-star's, such the Great White Shark (Carcharodon carcharias), receive much media adulation and are the focus of numerous conservation and "scientific" efforts the "Lost Sharks" remains largely unknown not only to the public, but also to the scientific and conservation communities. A review of the Red List status of Chondrichthyans indicates that 17.4% are threatened and nearly half (46.8%) are Data Deficient or have not been assessed. It is these "Lost Sharks" sharks that will be the focus of my presentation.


FAMILIAL DNA LINKS SUSPECT IN THE NORTH COUNTY CREEPER CASE
Jennifer Mertz, Bureau of Forensic Services Richmond DNA Laboratory; Jason Rouse, Escondido Police Department; Jeffrey Udvarhelyi, Escondido Police Department; Shelly Webster, San Diego County Sheriff's Department

In the summer of 2013, San Diego County was having a heat wave in the North County. Many homes had their windows open at night for relief; thus making it easy for hot prowls to occur. One individual (North County Creeper) was climbing in open windows. He was molesting innocent children, while they were sleeping. This happened in multiple jurisdiction with numerous houses. A team approach was taken to catch this molester. After a year and half with no suspect, a familial hit occurred. This lead to an arrest of the North County Creeper in Feb.2015.

This presentation will take you back to summer of 2013. We will follow the cases from the perspectives of the lead detectives, the casework DNA analyst, and the familial DNA analyst. It will show the importance of teamwork among investigators and forensic scientists.


CRIME SCENE BRAVOS AND BLUNDERS
Kirsten Fraser, Los Angeles County Sheriff's Department

Crime scene investigation is a delicate process and one that is just as reliant on human diligence as it is susceptible to human error. This presentation will discuss some important successes over the years as well as some funny, albeit embarrassing, blunders that have occurred at crime scenes.


COMMENTS ON THE RESPONSES TO THE PROPOSED NATIONAL CODE OF ETHICS AND PROFESSIONAL RESPONSIBILITY FOR THE FORENSIC SCIENCES
Donald Jones, San Bernadino Sheriff's Department (Retired)

Under the auspices of the National Institute of Standards and Technology the Interim Solutions Subcommittee of the National Commission of Forensic Science drafted a proposed National Code of Ethics and Professional Responsibility for the Forensic Sciences (Code). In April 2015 this draft was made available for one month for the solicitation of comments and feedback. In all, twenty seven responses were posted. They contained specific concerns relating to protection of defense analysts, lab managers, and whistle-blowers to whether certification should be required to properly defining terms to grammatical corrections. In general, it appears the majority of input was from attorneys, professors, and lab management personnel. It seemed there were very few comments from actual forensic laboratory practitioners. While this was only a starting point for a Code, many commented on the need for an enforcement process, a fact already recognized by the subcommittee up front. A close look at some of the Code requirements for forensic service providers (both analysts and managers) should cause some concern regarding the scope of intentions for a National Code of Ethics.


HILLSDALE HIGH SCHOOL ATTACK
Jeff Gilbert, Lead Principal - Hillsdale High School; Kevin Raeffaelli, Inspector - County of San Mateo District Attorney's Office; Christina Richardson, Criminalist - County of San Mateo Sheriff's Office; Brian Hester, Special Agent - Bureau of Alcohol, Tobacco, Firearms and Explosives; Karen Guidotti, Chief Deputy - County of San Mateo District Attorney's Office

On August 24 of 2009, a 17 year old former student of Hillsdale High School in San Mateo, CA walked onto the campus with the intent to kill his teachers. He had a well designed and practiced plan of attack and was heavily and uniquely armed for the assault. Alert and courageous school staff and first responders, coupled with the assailant's poor plan execution, thwarted the attack and averted potential tragedy.

A panel, consisting of the school principal, law enforcement incident commander, explosives expert, forensic laboratory / CSI team member, and prosecuting attorney will discuss the unusual event circumstances and lessons learned. This is a unique opportunity to hear from those directly involved in the detection and intervention of the unsuccessful attack and its subsequent investigation and prosecution.


ASIANA AIRLINES FLIGHT 214 PLANE CRASH
Robert Foucrault, Coroner - County of San Mateo Coroner's Office

On July 6, 2013 at 11:30 a.m. the San Mateo County Coroner's Office was notified of a plane crash at San Francisco International Airport. The airport is located in the north end of the county and is one of the busiest airports in the United States.

This presentation will take you through the process and procedures followed in responding to this disaster. The aircraft was a Boeing 777 and was reported to have 307 passengers on board. As the investigation progressed we were able to confirm 2 deaths at the scene. One additional victim was seriously injured and subsequently died at San Francisco General Hospital 6 days later.

During our investigation, we were able to confirm one of the deaths occurred from fatal injuries that were unfortunately caused by a responding San Francisco fire truck.

I will discuss how our investigation began and some of the details that led us to our conclusions regarding the decedents as well as the complications associated with a multi-agency involvement and parallel investigations.


CONFIRMATION BIAS: A LARGE SCALE EVALUATION OF PAST FIREARMS COMPARISON CASEWORK
Todd Weller, Oakland Police Department

The concept of confirmation bias and its influence on forensic science is not new. Past studies indicate that confirmation bias is real has the potential to influence forensic science outcomes. In a nutshell, the fear is that close proximity to police personnel and access to case information will result in a higher portion of identifications (or misidentifications). However, it has been our experience at the Oakland Police Department Criminalistics Firearms and Toolmark Unit that a significant portion of our casework results in eliminations. We examined several years of past firearms casework and tabulated the numbers of identifications and eliminations being performed at the Oakland Police Criminalistics Laboratory. We purposely focused on casework where confirmation bias would be influential: requests for direct comparisons of firearms to recovered evidence. We found that we are reporting more eliminations than identifications. Furthermore we realized it was important to present this data since the justice system may not be aware of the frequency of these eliminations.


IAI / NIST STIPEND FOR ABSTRACT SUBMISSION TO MAKE ORAL PRESENTATION AT 2016 IAI CONFERENCE
Gregory Laskowski, Criminalistics Services International, LLC

Interested parties in the fields of criminalistics (crime scenes, bloodstain pattern analysis, and footwear/tire track analysis being excluded) are invited to submit abstracts to the International Association for Identification for oral presentation at next years annual educational conference in Las Vegas, NV. Two presenters will be selected to have their registration, travel, and lodging paid by NIST. The deadline to submit abstracts is December 31. Information will be given on how to submit abstracts for oral presentation.


DRIVING UNDER THE INFLUENCE OF CANNABIS VERSUS DRIVING AND DYING UNDER THE INFLUENCE OF CANNABIS
Nikolas P. Lemos, San Francisco Office of Chief Medical Examiner

Cannabis intoxication in living and deceased drivers is an important medicolegal topic, but a limited number of studies review cannabinoid concentrations in both living and deceased humans.

California is currently one of 25 U.S. states and territories to have existing or pending legislation permitting the use of cannabis for medicinal purposes, the State having enacted this legislation since 1996.

The present study examines and compares cannabinoid concentrations measured in two groups of vehicle operators in San Francisco: (1) arrested operators of vehicles who allegedly operated their vehicles in San Francisco while impaired by cannabis, and (2) deceased operators of vehicles involved in fatal traffic accidents whose postmortem bloods were found to contain cannabinoids.

The goals of the study were to determine blood cannabinoid concentrations and to better characterize any differences between these two groups of drivers, who theoretically have access and use similar cannabis preparations available in the City and County of San Francisco, thus removing any bias based on geo-location and cannabis product availability, and whose bloods were analyzed by the same ABFT-accredited laboratory, thus removing any bias based on analytical capability differences.

In addition, the Huestis Predictive Models I II were applied and evaluated in a subgroup of living drivers DUID suspects for whom we were able to identify the time interval between the driving incident (and therefore the last time they had the opportunity to be exposed to cannabis) and the time of blood draw. This portion of the study was designed to determine the consistency between the predicted times of cannabis exposure provided by the Huestis' models and the true (time of alleged driving incident to time of blood draw) obtained from police reports and chain of custody records, in order to assess the usefulness of these research-derived, plasma-based predictive models in a forensic toxicology, whole blood setting after converting whole blood concentrations to plasma equivalent concentrations using various plasma to whole blood ratios and including the 95% Confidence Intervals (CIs) for each of these cases.

From 2010-2013, there were 318 cannabis-positive DUID cases (mean, median THC: 4.9, 3); 88 had cannabis-only in their bloods (mean, median THC: 5.8, 4). In 23 DUID cases, Huestis' Predictive Models with 95% confidence intervals were applied and evaluated, demonstrating that the actual case time points in all 23 cases fell within the predicted time ranges. Among deceased drivers, 19 had cannabis-positive toxicology (mean, median THC: 11.7, 4.5) and 8 had cannabis-only (mean, median THC: 20.3, 19.5). Motorcyclists and bicyclists comprised the majority of deceased vehicle operators, with bicyclists averaging the highest mean and median THC concentrations overall. The ANOVA, between living and deceased drivers' cannabinoid concentrations demonstrated that THC-OH and THC-COOH concentrations are not statistically different between the two groups, but that THC concentrations are, making it difficult to correlate or compare postmortem to antemortem THC concentrations in living and deceased drivers.


CURRENT TRENDS IN MEDICAL CANNABIS FROM THEVIEWPOINT OF A CANNABIS TESTING LAB
Joshua Wurzer, SC Laboratories Inc.

Medical Cannabis is quickly gaining legitimacy in the medical and scientific community. With many states also legalizing recreational Cannabis, and California poised to do the same, many issues are raised. How do Medical Cannabis and Recreational Cannabis coexist in California? This presentation would discuss the current products in the Medical Cannabis market, their uses, and recent medical research regarding Cannabis and cannabinoids. It will also touch on numerous issues surrounding the current Medical Cannabis system and those that will arise in the near future from the vantage point of a testing laboratory.

We will also discuss what our laboratory does and the history of Cannabis testing worldwide which has its roots in forensic testing for Law Enforcement but has taken on a new meaning since independent labs began opening in the San Francisco Bay Area. Now, quality and safety testing have become mandatory in most states which have legalized medical or recreational Cannabis.

Lastly, we will cover the most common tests offered by Cannabis labs, as well as, methodology and techniques and discuss the challenges the industry is facing in adopting contaminant tolerances in the absence of federal guidance.


A COMPARISON OF COLLECTION METHODS FOR TOUCH DNA SAMPLES ON STEERING WHEELS OF VEHICLES
Irina Kirgiz, UC Davis Forensic Science Graduate Program

The purpose of this research was to directly compare alternative methods for collecting touch DNA from steering wheels. Tape lifting and FTA paper scraping methods were compared to a traditional double swabbing DNA collection method. 35 cars were used in the double swabbing vs. tape lifting study. One half of a steering wheel of each car was sampled using two sterile cotton swabs, while the other side of a steering wheel was sampled using 3M™ Water-Soluble Wave Solder Tape 5414. Another 35 cars were used in the double swabbing vs. FTA paper scraping study. Cotton swabs were used to sample one side of a steering wheel, and the Whatman WB120205 FTA Classic Cards were used to sample the other side. The sides were frequently alternated in both studies.

QIAGEN® QIAamp DNA Kit was used to extract DNA from all samples. Promega® PlexorHY Human Quantitation kit was used to quantitate each sample. Statistical analysis of data showed no significant difference in yields between double swabbing and tape lifting techniques. It has also shown that the difference in yield between FTA scraping and double swabbing is statistically significant, with FTA paper collecting more touch DNA. AmpFLSTR Identifiler™ kit was used to generate a driver's profile from 20 randomly selected samples. STR analysis has shown that most of the samples contained two persons' mixtures, where the owner (the most recent driver) is the major donor. The results of this research indicated that FTA paper scraping method has high potential and should be further studied.


A BRIEF HISTORY OF FORENSIC SCIENCES FROM GENESIS TO TODAY
Gregory Laskowski, Criminalistics Services International, LLC

The history of forensic science and its sub discipline criminalistics will be explored from the time of genesis as described in the Old Testament of the Bible to today. The audience will be introduced to early pioneers in the field, including Locard, Bertillon, Gross, and Orphilia as well as new pioneers, Goddard, Landsteiner, McDonnell, Wraxell, Culliford, Jeffreys, and Mullis in the form of biographical sketches. This presentation will also examine the many specialty areas in forensic science such as crime scene analysis toxicology, firearms, fire debris analysis, trace evidence, impression evidence, serology, and DNA highlighting the milestones and the technology employed in the course of history. In addition, the effects of television in the genres of crime dramas, docudramas and documentaries will be explored.


POSTERS

CHARACTERIZING THE FREQUENCY OF HETEROPLASMY IN MTDNA OF TISSUES USING NEXT-GENERATION SEQUENCING
Janice Lin, Erin Laurie, George Sensabaugh, Cassandra Calloway; UC Davis Forensic Science Graduate Program

Mitochondrial DNA (mtDNA) is often used for analysis in forensic and mass disaster cases involving small and degraded tissue samples. Tissues can degrade from exposure to various weather and environmental conditions. High temperatures and level of destruction can also cause tissues to fuse together or commingle with other remains, resulting in mixtures that can make analysis and interpretation difficult. While the quality and level of nuclear DNA is low in these cases, mtDNA has a high copy number, increasing the possibility of obtaining enough DNA from compromised samples for fragment-size analysis of short tandem repeats. In addition, the control region of mtDNA contains highly polymorphic hypervariable regions I/II (HVI/HVII) where heteroplasmic mutations have been observed. It is important to understand the nature of heteroplasmy across various tissue types because the presence of heteroplasmy in mtDNA can affect interpretation and analysis.

While Sanger sequencing is a standard approach for mtDNA analysis, it has a number of limitations that prevent a comprehensive analysis of mtDNA in forensic samples and mixtures. Sanger sequencing electropherograms display peak heights that do not always reflect the ratio of mixture components, making it difficult to determine individual mtDNA haplotypes in a mixed sample. Moreover, minor components that make up less than 10% of a DNA mixture are not detected with Sanger sequencing. This research project aims to address these common issues in forensic mtDNA analysis by using a sensitive Next Generation Sequencing (NGS) method to characterize low levels of heteroplasmy in brain, heart, muscle, and blood tissues.

The Roche 454 NGS technology overcomes limitations of Sanger sequencing in forensic mtDNA analysis with its high and massively-parallel throughput, clonal amplification, and pyrosequencing chemistry. Through these features, the 454 GS Junior can separate individual components of a mixture, provide a quantifiable estimate of the ratio of mixture components, and analyze low frequency of heteroplasmy in mtDNA. In comparison to Sanger sequencing data on the same tissue samples, data from this research project has detected heteroplasmy occurring at frequencies as low as 1%. These results support the high sensitivity of 454 GS Junior to not only detect low levels of heteroplasmy but also reveal additional heteroplasmic sites in the HVI/HVII regions. In addition to confirming heteroplasmy previously detected by Sanger sequencing, the more sensitive NGS has detected additional heteroplasmy that were not previously observed in Sanger sequencing. For example, preliminary NGS data shows that heteroplasmy was observed at a low level in the muscle tissue at hot spot HVII position 189 in at least one individual. Furthermore, NGS provides a quantitative assessment of heteroplasmy by establishing frequencies of the two bases that occur at one site. This research will help establish NGS sensitivity thresholds for varying levels of heteroplasmy in different sample types. Moreover, the results demonstrate the potential of NGS to improve interpretation guidelines and increase the efficiency of forensic mtDNA analysis, especially with limited and degraded DNA samples.


WHOLE MITOCHONDRIAL GENOME SEQUENCING METHOD FOR LIMITED AND HIGHLY DEGRADED BONE SAMPLES FOR USE IN FORENSIC CASEWORK
Rachel Gordon, Sarah Copeland, George Sensabaugh, D. Crim, Henry Erlich and Cassandra D. Calloway, PhD1 Children's Hospital Oakland Research Center

DNA samples of limited quantity and quality are often encountered in forensic casework and are at times difficult to analyze with standard STR markers. Mitochondrial DNA analysis is most useful in these cases because of its high copy number per cell (200 copies/cell). Currently, the standard approach is to analyze the hypervariable regions (HVI/HVII) of the mitochondrial genome using Sanger sequencing. However, Sanger sequencing has limited sensitivity and often fails when the DNA is highly degraded (<250bp). Given the limitations associated with the current standard method, there is a need for the development of an improved system for limited and degraded DNA analysis in the forensic field.

We have developed an optimized whole mitochondrial genome probe capture enrichment method coupled with Next Generation Sequencing (NGS) on the Illumina platform to overcome these limitations. Mitochondrial DNA whole genome analysis is an alternative approach and can be used for increased genotyping success particularly with forensic samples where nuclear DNA is limited or degraded. We present here results demonstrating the potential application of this optimized NGS whole mitochondrial genome enrichment method for sequence analysis of difficult forensic samples. This robust method was successfully used for sequencing the entire mitochondrial genome of highly degraded bone samples, limited DNA samples, as well as samples from different population groups.

We tested the sensitivity of the assay by reducing the starting amount of a control DNA from 1 ng to 10 pg. All limiting dilution samples led to full (100%) coverage of the whole mitochondrial genome with a specificity of 96% average on target rate using the optimized protocols. We also tested DNA samples (n=20) from 4 different population groups (Caucasian, African-American, Hispanic, and Japanese), which led to 100% coverage of the whole Mitochondrial Genome with a specificity of 95% average on target rate. DNA from <50 year old femur was also processed and sequenced successfully with 100% coverage. A mock degradation experiment was also performed where DNA was mechanically sheared to 150 bp on average and then processed using the 250 bp protocol and compared to a DNA control processed only at 250 bp shearing protocol. The samples were successfully captured and sequenced showing the method is independent of quality. We have also applied this method to DNA from >1000 year old bones. Preliminary results show ~99% mitochondrial genome coverage demonstrating the application of this method to highly degraded DNA samples.

Our results show our optimized method for whole mitochondrial genome analysis to be robust with increased specificity and sensitivity. Standard NGS methods require starting DNA amounts higher than a typical forensic sample, however, we have successfully captured and sequenced with significantly less starting material (10pg). Our optimized whole mitochondrial genome NGS method has the potential to greatly improve the discrimination power compared to current mitochondrial HVI/HVII sequence analysis as well as to increase the number of limited and degraded samples successfully genotyped.


INCREASING THE DETECTION OF A MINOR CONTRIBUTOR IN MIXTURES USING QIAGENS INVESTIGATOR 24PLEX QS STR MULTIPLEX PCR ASSAY
Dukes, M.J.; D. Muller; A. Prochnow; M. Scherer; R. Perist; John Haley, Pamela Jarman; Qiagen

In response to the FBI expanded CODIS core loci, QIAGEN developed and validated two Multiplex PCR kits for reliable genotyping of human DNA. The two kits, Investigator® 24plex QS Kit and Investigator® GO! Kit, were developed for forensic DNA casewrk and database type samples, respectively. Both kits contain a patent pending and novel Quality Sensor.

The Quality Sensor is unique, and patent pending, in that the Quality Sensor resultant alleles, "Q" and "S", allows differentiation between:

  1. Robust amplification
  2. Degradation
  3. Inhibition
  4. Lack of amplification of DNA
  5. Failed PCR amplification

The QIAGEN Investigator® 24plex kit was evaluated at the U.S. Army Criminal Investigation Laboratory (USACIL) Defense Forensic Science Center (DFSC) and ultimately requested for NDIS approval. The 24-plex QS kit was approved as an accepted kit by National DNA Index System (NDIS) on June 29, 2015. Analysis of DNA samples from forensic casework must contain the ability to differentiate mixtures of DNA from more than one contributor. It has often been the task to increase the total amount of DNA input in the PCR to boost the detection of the minor contributor. This study addresses same ratios of DNA, but variable total templates (0.5ng vs. 1.0ng) in the PCR. This presentation will provide a glimpse into the mixture studies designed and performed, and the resultant data obtained and submitted for NDIS approval.