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Concept: Combined DNA Index System

194

BACKGROUND: DNA analysis of ancient skeletal remains is invaluable in evolutionary biology for exploring the history of species, including humans. Contemporary human bones and teeth, however, are relevant in forensic DNA analyses that deal with the identification of perpetrators, missing persons, disaster victims or family relationships. They may also provide useful information towards unravelling controversies that surround famous historical individuals. Retrieving information about a deceased person’s externally visible characteristics can be informative in both types of DNA analyses. Recently, we demonstrated that human eye and hair colour can be reliably predicted from DNA using the HIrisPlex system. Here we test the feasibility of the novel HIrisPlex system at establishing eye and hair colour of deceased individuals from skeletal remains of various post-mortem time ranges and storage conditions. METHODS: Twenty-one teeth between 1 and approximately 800 years of age and 5 contemporary bones were subjected to DNA extraction using standard organic protocol followed by analysis using the HIrisPlex system. RESULTS: Twenty-three out of 26 bone DNA extracts yielded the full 24 SNP HIrisPlex profile, therefore successfully allowing model-based eye and hair colour prediction. HIrisPlex analysis of a tooth from the Polish general W[latin small letter l with stroke]adys[latin small letter l with stroke]aw Sikorski (1881 to 1943) revealed blue eye colour and blond hair colour, which was positively verified from reliable documentation. The partial profiles collected in the remaining three cases (two contemporary samples and a 14th century sample) were sufficient for eye colour prediction. CONCLUSIONS: Overall, we demonstrate that the HIrisPlex system is suitable, sufficiently sensitive and robust to successfully predict eye and hair colour from ancient and contemporary skeletal remains. Our findings, therefore, highlight the HIrisPlex system as a promising tool in future routine forensic casework involving skeletal remains, including ancient DNA studies, for the prediction of eye and hair colour of deceased individuals.

Concepts: DNA, Molecular biology, Full genome sequencing, Identification, Eye color, DNA profiling, National DNA database, Combined DNA Index System

150

DNA testing is an established part of the investigation and prosecution of sexual assault. The primary purpose of DNA evidence is to identify a suspect and/or to demonstrate sexual contact. However, due to highly uneven proportions of female and male DNA in typical stains, routine autosomal analysis often fails to detect the DNA of the assailant. To evaluate the forensic efficiency of the combined application of autosomal and Y-chromosomal short tandem repeat (STR) markers, we present a large retrospective casework study of probative evidence collected in sexual-assault cases. We investigated up to 39 STR markers by testing combinations of the 16-locus NGMSElect kit with both the 23-locus PowerPlex Y23 and the 17-locus Yfiler kit. Using this dual approach we analyzed DNA extracts from 2077 biological stains collected in 287 cases over 30 months. To assess the outcome of the combined approach in comparison to stand-alone autosomal analysis we evaluated informative DNA profiles. Our investigation revealed that Y-STR analysis added up to 21% additional, highly informative (complete, single-source) profiles to the set of reportable autosomal STR profiles for typical stains collected in sexual-assault cases. Detection of multiple male contributors was approximately three times more likely with Y-chromosomal profiling than with autosomal STR profiling. In summary, 1/10 cases would have remained inconclusive (and could have been dismissed) if Y-STR analysis had been omitted from DNA profiling in sexual-assault cases.

Concepts: DNA, Molecular biology, Identification, Short tandem repeat, Genealogical DNA test, DNA profiling, National DNA database, Combined DNA Index System

31

Dipterous fly larvae (maggots) are frequently collected from a corpse during a criminal investigation. Previous studies showed that DNA analysis of the gastrointestinal contents of maggots might be used to reveal the identity of a victim. However, this approach has not been used to date in legal investigations, and thus its practical usefulness is unknown. A badly burned body was discovered with its face and neck colonized by fly larvae. Given the condition of the body, identification was not possible. Short tandem repeat (STR) typing was performed using the gastrointestinal contents of maggots collected from the victim and was compared to STR profiles obtained from the alleged father. The probability of paternity was 99.685%. Thus, this comparative DNA test enabled the conclusive identification of the remains. This is the first reported case of analysis of human DNA isolated from the gastrointestinal tract of maggots used to identify a victim in a criminal case.

Concepts: DNA, Full genome sequencing, Identification, Short tandem repeat, Fly, DNA profiling, National DNA database, Combined DNA Index System

30

Comparison of DNA profiles is often used in verifying the identification of deceased human beings when other easier, quicker, and less expensive means to identification are not possible. Fifty-five adult subjects divided into 3 groups provided a used toothbrush along with a small bloodstain control for DNA analysis and comparison. Results indicate that there is no significant difference in the quantity and quality of DNA recovered from a toothbrush that has been used for 1 month versus 3 months versus random periods. The results of this study confirm earlier conclusions that a used toothbrush is a reliable source of antemortem DNA from a putative decedent. The use of aviation snips to remove a small portion of the toothbrush head provides an easy, inexpensive method of obtaining a sample for DNA extraction. The authors recommend this method as a standardized technique for use in forensic DNA laboratories.

Concepts: DNA, Molecular biology, Full genome sequencing, Identification, DNA profiling, National DNA database, Combined DNA Index System, Thomas Jefferson

28

Traditional forensic DNA interpretation methods are restricted as they are unable to deal completely with complex low level or mixed DNA profiles. This type of data has become more prevalent as DNA typing technologies become more sensitive. In addition they do not make full use of the information available in peak heights. Existing methods of interpretation are often described as binary which describes the fact that the probability of the evidence is assigned as 0 or 1 (hence binary) (see for example [1] at 7.3.3). These methods are being replaced by more advanced interpretation methods such as continuous models. In this paper we describe a series of models that can be used to calculate expected values for allele and stutter peak heights, and their ratio SR. This model could inform methods which implement a continuous method for the interpretation of DNA profiling data.

Concepts: DNA, Full genome sequencing, DNA profiling, National DNA database, Combined DNA Index System, Alec Jeffreys

28

While the analysis of human DNA has been the focus of large-scale collaborative endeavors, non-human forensic DNA analysis has not benefited from the same funding streams and coordination of effort. Consequently, the development of standard marker panels, allelic ladders and allele-specific sequence data comparable to those established for human forensic genetics has lagged. To meet that need for domestic dogs, we investigated sequence data provided by the published 7.6X dog genome for novel short tandem repeat markers that met our criteria for sensitivity, stability, robustness, polymorphic information content, and ease of scoring. Fifteen unlinked tetranucleotide repeat markers were selected from a pool of 3113 candidate markers and assembled with a sex-linked marker into a multiplex capable of generating a full profile with as little as 60pg of nuclear DNA. An accompanying allelic ladder was assembled and sequenced to obtain detailed repeat motif data. Validation was carried out according to SWGDAM guidelines, and the DogFiler panel has been integrated into forensic casework and accepted in courts across the U.S. Applying various formulae for calculating random match probabilities for inbred populations, estimates for this panel of markers have proven to be comparable to those obtained in human forensic genetics. The DogFiler panel and the associated allelic ladder represent the first published non-human profiling system to fully address all SWGDAM recommendations.

Concepts: DNA, Genetics, Molecular biology, Full genome sequencing, DNA profiling, National DNA database, Combined DNA Index System, Thomas Jefferson

18

DNA fingerprinting, one of the great discoveries of the late 20th century, has revolutionized forensic investigations. This review briefly recapitulates 30 years of progress in forensic DNA analysis which helps to convict criminals, exonerate the wrongly accused, and identify victims of crime, disasters, and war. Current standard methods based on short tandem repeats (STRs) as well as lineage markers (Y chromosome, mitochondrial DNA) are covered and applications are illustrated by casework examples. Benefits and risks of expanding forensic DNA databases are discussed and we ask what the future holds for forensic DNA fingerprinting.

Concepts: DNA, Full genome sequencing, Identification, DNA profiling, National DNA database, Combined DNA Index System, Alec Jeffreys

14

In this interview we talk with Professor Sir Alec Jeffreys about DNA fingerprinting, his wider scientific career, and the past, present and future of forensic DNA applications.The podcast with excerpts from this interview is available at: http://www.biomedcentral.com/biome/alec-jeffreys.

Concepts: DNA, Full genome sequencing, DNA profiling, National DNA database, Combined DNA Index System, Alec Jeffreys, Colin Pitchfork

10

Identification of foreign biological material by genetic profiling is widely used in forensic DNA testing in different cases of sexual violence, sexual abuse or sexual harassment. In all these kinds of sexual assaults, the perpetrator could constrain the victim to kissing. The value of the victim’s saliva taken after such an assault has not been investigated in the past with currently widely used molecular methods of extremely high sensitivity (e.g. qPCR) and specificity (e.g. multiplex Y-STR PCR). In our study, 12 voluntary pairs were tested at various intervals after intense kissing and saliva samples were taken from the women to assess the presence of male DNA. Sensitivity-focused assays based on the SRY (single-copy gene) and DYS (multi-copy gene) sequence motifs confirmed the presence of male DNA in female saliva after 10 and even 60min after kissing, respectively. For specificity, standard multiplex Y-STR PCR profiling was performed and male DNA was found in female saliva samples, as the entire Y-STR profile, even after 30min in one sample. Our study confirms that foreign DNA tends to persist for a restricted period of time in the victim’s mouth, can be isolated from saliva after prompt collection and can be used as a valuable source of evidence.

Concepts: DNA, Molecular biology, Full genome sequencing, Identification, Rape, DNA profiling, National DNA database, Combined DNA Index System

3

The turn-around time of urgent crime scene DNA samples is often far longer than desired by law enforcement. Crime scene DNA sample processing involves both complex and routine processing steps. Simplification and integration of the routine steps would dramatically improve turn-around time and reduce the risk of operator contamination. Routine DNA extraction and quantitation is readily available. However, PCR amplification and electrophoretic analysis remain largely manual. Rapid DNA Analysis is a hands-free “swab in - profile out” process which consists of automated DNA extraction, amplification, separation, detection, and allele calling without human intervention. RapidHIT®200 and RapidHIT ID are rapid DNA systems developed by IntegenX (Pleasanton, CA) and validated for the use of buccal swabs. A new generation of the RapidHIT sample cartridge for RapidHIT ID has been designed and tested which allows the loading of extracted and quantified DNA. RapidHIT EXT sample cartridge allows a user to generate a forensic DNA profile from less than 250 pg of extracted and quantified DNA in less than 90 min with less than one-minute hands-on time. Once the sample is loaded in the RapidHIT EXT sample cartridge, a DNA profile is produced after amplification, detection and automated data analysis. We report on sensitivity, reproducibility, concordance, DNA mixtures and carryover for EXT sample cartridges pre-loaded with GlobalFiler®Express and AmpFLSTR®NGM SElect™ Express, (Thermo Fisher Scientific, Waltham, MA) STR chemistries. Purified and quantified DNA from mock crime scene samples were used to demonstrate the utility of these cartridges in an established forensic laboratory.

Concepts: DNA, Polymerase chain reaction, Molecular biology, Full genome sequencing, DNA profiling, National DNA database, Combined DNA Index System, Thomas Jefferson