Development of a novel five dye insertion/deletion (INDEL) panel for ancestry determination.

The use of genetic markers, specifically Short Tandem Repeats (STRs), has been a valuable tool for identifying persons of interest. However, the ability to analyze additional markers including Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletion (INDELs) polymorphisms allows laboratories to explore other investigative leads. INDELs were chosen in this study because large panels can be differentiated by size, allowing them to be genotyped by capillary electrophoresis. Moreover, these markers do not produce stutter and are smaller in size than STRs, facilitating the recovery of genetic information from degraded samples. The INDEL Ancestry Informative Markers (AIMs) in this study were selected from the 1000 Genomes Project based on a fixation index (FST) greater than 0.50, high allele frequency divergence, and genetic distance. A total of 25 INDEL-AIMs were optimized and validated according to SWGDAM guidelines in a five-dye multiplex. To validate the panel, genotyping was performed on 155 unrelated individuals from four ancestral groups (Caucasian, African, Hispanic, and East Asian). Bayesian clustering and principal component analysis (PCA) were performed revealing clear separation among three groups, with some observed overlap within the Hispanic group. Additionally, the PCA results were compared against a training set of 793 samples from the 1000 Genomes Project, demonstrating consistent results. Validation studies showed the assay to be reproducible, tolerant to common inhibitors, robust with challenging casework type samples, and sensitive down to 125 pg. In conclusion, our results demonstrated the robustness and effectiveness of a 25 loci INDEL system for ancestry inference of four ancestries commonly found in the United States.

Development of a novel five-dye panel for human identification insertion/deletion (INDEL) polymorphisms.

DNA analysis of forensic case samples relies on short tandem repeats (STRs), a key component of the combined DNA index system (CODIS) used to identify individuals. However, limitations arise when dealing with challenging samples, prompting the exploration of alternative markers such as single nucleotide polymorphisms (SNPs) and insertion/deletion (INDELs) polymorphisms. Unlike SNPs, INDELs can be differentiated easily by size, making them compatible with electrophoresis methods. It is possible to design small INDEL amplicons (<200 bp) to enhance recovery from degraded samples. To this end, a set of INDEL Human Identification Markers (HID) was curated from the 1000 Genomes Project, employing criteria including a fixation index (FST) ≤ 0.06, minor allele frequency (MAF) >0.2, and high allele frequency divergence. A panel of 33 INDEL-HIDs was optimized and validated following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, utilizing a five-dye multiplex electrophoresis system. A small sample set (n = 79 unrelated individuals) was genotyped to assess the assay's performance. The validation studies exhibited reproducibility, inhibition tolerance, ability to detect a two-person mixture from a 4:1 to 1:6 ratio, robustness with challenging samples, and sensitivity down to 125 pg of DNA. In summary, the 33-loci INDEL-HID panel exhibited robust recovery with low-template and degraded samples and proved effective for individualization within a small sample set.

Developmental validation of the ForenSeq MainstAY kit, MiSeq FGx sequencing system and ForenSeq Universal Analysis Software.

For human identification purposes, forensic genetics has primarily relied upon a core set of autosomal (and to a lesser extent Y chromosome) short tandem repeat (STR) markers that are enriched by amplification using the polymerase chain reaction (PCR) that are subsequently separated and detected using capillary electrophoresis (CE). While STR typing conducted in this manner is well-developed and robust, advances in molecular biology that have occurred over the last 15 years, in particular massively parallel sequencing (MPS) [1-7], offer certain advantages as compared to CE-based typing. First and foremost is the high throughput capacity of MPS. Current bench top high throughput sequencers enable larger batteries of markers to be multiplexed and multiple samples to be sequenced simultaneously (e.g., millions to billions of nucleotides can be sequenced in one run). Second, compared to the length-based CE approach, sequencing STRs increases discrimination power, enhances sensitivity of detection, reduces noise due to instrumentation, and improves mixture interpretation [4,8-23]. Third, since detection of STRs is based on sequence and not fluorescence, amplicons can be designed that are shorter in length and of similar lengths among loci, where possible, which can improve amplification efficiency and analysis of degraded samples. Lastly, MPS offers a single format approach that can be applied to analysis of a wide variety of genetic markers of forensic interest (e.g., STRs, mitochondrial DNA, single nucleotide polymorphisms, insertion/deletions). These features make MPS a desirable technology for casework [14,15,24,25-48]. The developmental validation of the ForenSeq MainstAY library preparation kit with the MiSeq FGx Sequencing System and ForenSeq Universal Software is reported here to assist with validation of this MPS system for casework [49]. The results show that the system is sensitive, accurate and precise, specific, and performs well with mixtures and mock case-type samples.

The effectiveness of various strategies to improve DNA analysis of formaldehyde-damaged tissues from embalmed cadavers for human identification purposes.

Formalin-fixed tissues provide the medical and forensic communities with alternative and often last resort sources of DNA for identification or diagnostic purposes. The DNA in these samples can be highly degraded and chemically damaged, making downstream genotyping using short tandem repeats (STRs) challenging. Therefore, the use of alternative genetic markers, methods that pre-amplify the low amount of good quality DNA present, or methods that repair the damaged DNA template may provide more probative genetic information. This study investigated whether whole genome amplification (WGA) and DNA repair could improve STR typing of formaldehyde-damaged (FD) tissues from embalmed cadavers. Additionally, comparative genotyping success using bi-allelic markers, including INDELs and SNPs, was explored. Calculated random match probabilities (RMPs) using traditional STRs, INDEL markers, and two next generation sequencing (NGS) panels were compared across all samples. Overall, results showed that neither WGA nor DNA repair substantially improved STR success rates from formalin-fixed tissue samples. However, when DNA from FD samples was genotyped using INDEL and SNP-based panels, the RMP of each sample was markedly lower than the RMPs calculated from partial STR profiles. Therefore, the results of this study suggest that rather than attempting to improve the quantity and quality of severely damaged and degraded DNA prior to STR typing, a more productive approach may be to target smaller amplicons to provide more discriminatory DNA identifications. Furthermore, an NGS panel with less loci may yield better results when examining FD samples, due to more optimized chemistries that result in greater allelic balance and amplicon coverage.

Assessment of the ForenSeq mtDNA control region kit and comparison of orthogonal technologies.

The ForenSeq® mtDNA Control Region Kit, MiSeq FGx®, and Universal Analysis Software (UAS) were assessed to better define the performance and limitations of the system with forensically relevant samples to provide data for its transition into practice. A total of six MiSeq FGx sequencing runs of ForenSeq mtDNA Control Region kit, three runs of additional orthogonal sequencing chemistries, and Sanger sequencing results for 14 samples were used to test for concordance. Sensitivity, reproducibility, mixture detection studies, as well as studies to measure the performance of amplification and sequencing controls were performed. The use and reliability of the UAS for data analysis was also examined. With a variety of sample types and controls representing many mitochondrial haplotypes, the recently developed mtDNA Control Region Kit, with the MiSeq FGx and UAS, was found to be fit for purpose as reliable, reproducible, and robust. Sensitivity down to 1 pg of input genomic DNA was demonstrated, which allows the system to offer low limits of detection for better interrogation of potential heteroplasmy in samples. Concerns for implementing next generation sequencing (NGS) for mtDNA in laboratories were addressed in this research, including initial template quantification and confirmation of haplotypes generated by UAS software regarding length-based polymorphisms. To improve performance with forensic samples, laboratories could implement mitochondrial-specific qPCR assays for quantification and perform the optional manual normalization protocol. Additional optimization on sample multiplexing can provide methods that either increase sensitivity or cost efficiency of the assay.

Performance characteristics of chimerism testing by next generation sequencing.

Chimerism testing provides informative clinical data regarding the status of a biological sample mixture. For years, this testing was achieved by measuring the peaks of informative short tandem repeat (STR) loci using capillary electrophoresis (CE). With the advent of next generation sequencing (NGS) technology, the quantification of the percentage of donor/recipient mixtures is more easily done using sequence reads in large batches of samples run on a single flow cell. In this study, we present data on using a FORENSIC NGS chimerism platform to accurately measure the percentage of donor/recipient mixtures. We were able to detect chimerism to a limit threshold of 1% using both STR and single nucleotide polymorphism (SNP) informative loci. Importantly, a significant correlation was observed between NGS and CE chimerism methods when compared at donor detection ranges from 1% to 10%. Furthermore, 100% accuracy was achieved through proficiency testing over six surveys. Its usefulness was expanded beyond this to help identify suitable donors for solid organ transplant patients using ancestry SNP profiles. In summary, the NGS method provides a sensitive and reliable alternative to traditional CE for chimerism testing of clinical samples.

Novel extraction chemistry and alternative amplification strategies for use with rootless hair shafts.

Rootless hair shafts are often considered unsuitable for STR genotyping due to the known high failure rate. The same samples can be reliably processed with mitochondrial sequencing. However, the minimal discriminatory power of widely implemented control region mitochondrial sequencing techniques limits its utility in some forensic casework. In this research, multiple variables were tested to provide information on rootless hair shaft sample genotyping success. Results showed external decontamination procedures decreased drop-in alleles but also greatly reduced profile recovery. The novel InnoXtract™ chemistry was comparable to automated EZ1 DNA Investigator extraction. With thoroughly decontaminated hairs, InnoTyper® 21 amplification generated random match probabilities higher than STR chemistry in 71.875% of samples and 18.75% of samples benefitted from the use of InnoTyper® 21 amplification compared with estimated mtDNA profile rarity. Compared with the capillary electrophoresis-based amplification chemistries tested, the ForenSeq™ DNA Signature Prep chemistry paired with massively parallel sequencing was the most discriminatory amplification strategy tested.

Evaluation of 19 short tandem repeat markers for individualization of Papaver somniferum.

Papaver somniferum, commonly known as opium poppy, is the source of natural opiates, which are used as analgesics or as precursors in the creation of semi-synthetic opioids such as heroin. An increase in opioid addiction in the United States has resulted in high rates of illicit opioid use and overdoses. It has recently been shown that P. somniferum DNA suitable for genetic analysis can be recovered from heroin samples. The development of a comprehensive genetic individualization tool for opium poppy could serve to link cases and strengthen programs such as the Drug Enforcement Administration's (DEA) Heroin Signature Program, which seeks to combat rising opioid use. The purpose of this study was to develop a quantitative real-time PCR (qPCR) method for the quantification of opium poppy DNA, compare three commercial DNA extraction kits for their ability to isolate DNA from poppy seeds, and evaluate nineteen opium poppy short tandem repeat (STR) markers for their use in a forensic identification panel. Such a panel could be used for individualizing samples and determining the geographic origin in heroin or poppy seed tea cases. The qPCR method was proven to be reproducible and reliable, specific for P. somniferum, and sensitive enough for forensic case-type samples. Of the three kits tested, the nexttec™ one-step DNA Isolation Kit for Plants was the optimal method and facilitated rapid extraction of DNA from poppy seeds. The majority of evaluated STR primer sets were unreliable or had low discriminatory power, limiting their use for individualization of poppy samples. A six-locus STR multiplex was developed and evaluated according to Scientific Working Group on DNA Analysis Methods (SWGDAM) and International Society of Forensic Genetics (ISFG) guidelines, including the use of a sequenced allelic ladder. The multiplex was found to have low discriminatory power, with greater than two-thirds of samples analyzed having just two different genotypes. The multiplex was determined to be unsuitable for individualization; however, a genotype map was developed as a proof of concept that these markers may be useful for determining the biogeographical origin of samples. Searching the poppy genome for new STR markers and developing new primer sets may be necessary for the creation of a powerful genetic tool for the individualization of P. somniferum.

Efficacy of "touch" DNA recovery and room-temperature storage from assault rifle magazines.

Crimes committed with assault rifles are becoming increasingly prevalent in the United States. In the absence of other evidence, DNA analysis can often provide informative leads. Unfortunately, any DNA transferred to rifle components left behind at a crime scene is likely to be low in quantity and/or quality. Furthermore, collected evidence is unlikely to be processed immediately and may require storage. Long-term storage can subject DNA to damage and degradation, which ultimately affects DNA profile interpretation and may prevent the identification of potential suspects. This study assessed the ability of a new swab storage device, the SwabSaver®, to preserve "touch" DNA from AR-15 magazine rifles using three different collection devices. Three volunteers loaded bullet cartridges into plastic polymer and aluminum AR-15 magazines. DNA was collected with traditional cotton swabs, layered cotton paper swabs, or nylon-flocked swabs. Collection devices were then stored at room-temperature for up to two months in either the SwabSaver® device or an empty centrifuge tube. The results suggest that substrate and swab type had less of an effect on profile completeness than storage type. Furthermore, SwabSaver® storage yielded DNA quantities comparable to "touch" DNA extracted after 24 h.

Collection and direct amplification methods using the GlobalFiler™ kit for DNA recovered from common pipe bomb substrates.

When analyzing DNA from exploded pipe bombs, quantities are often in trace amounts, making DNA typing extremely difficult. Amplifying minute amounts of DNA can cause stochastic effects resulting in partial or uninterpretable profiles. Therefore, the initial DNA collection from "touch" evidence must be optimized to maximize the amount of DNA available for analysis. This proof-of-concept study evaluated two different swab types with two direct amplification strategies to identify the most effective method for recovering DNA from common pipe bomb substrates. PVC and steel pipes, electrical tape, and copper wire spiked with epithelial cells were swabbed with cotton or microFLOQ® Direct Swabs and amplified directly or via a pre-treatment prior to STR amplification. Not only was the microFLOQ® Direct Swab protocol the quickest method with the least risk of contamination, but in combination with direct amplification, the microFLOQ® Direct Swabs also generated the most complete STR profiles.

The stability and persistence of blood and semen mRNA and miRNA targets for body fluid identification in environmentally challenged and laundered samples.

The identification of body fluids in evidentiary stains may provide investigators with probative information during an investigation. In this study, quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays were performed to detect the presence of mRNA and miRNA in fresh and environmentally challenged samples. Blood, semen, and reference markers were chosen for both mRNA/miRNA testing. Samples of blood and semen were exposed to heat, humidity, and sunlight, and controlled conditions (room temperature, low humidity, and darkness) for 6 months. All mRNA targets were observed through six months under controlled conditions, but were undetected after 30 days in experimental conditions. However, miRNA targets persisted under all test conditions for the duration of the study. Additionally, cotton stained with blood or semen was laundered using a liquid detergent in various washing and drying conditions. An unstained cutting was evaluated for potential transfer. Both miRNA targets were observed in all stained samples regardless of the wash protocol used. Of the mRNA markers, HBB was detected in all bloodstained samples and PRM1 persisted in all but one semen stained sample. The unstained samples showed transfer of at least one body fluid specific miRNA marker in all samples and at least one body fluid specific mRNA in approximately half of the samples. These results support that RNA markers can be used for body fluid identification in challenging samples, and that miRNA markers may be more persistent than mRNA for blood and semen stains. However, some caution is warranted with laundered items due to possible transfer.

Correction to: Nuclear, chloroplast, and mitochondrial data of a US cannabis DNA database.

The original version of this article contained a mistake. In page 10 of the original article, the significant level (p > 0.01) is incorrect. The correct significant level is (p < 0.01). The original article has been corrected.

Nuclear, chloroplast, and mitochondrial data of a US cannabis DNA database.

As Cannabis sativa (marijuana) is a controlled substance in many parts of the world, the ability to track biogeographical origin of cannabis could provide law enforcement with investigative leads regarding its trade and distribution. Population substructure and inbreeding may cause cannabis plants to become more genetically related. This genetic relatedness can be helpful for intelligence purposes. Analysis of autosomal, chloroplast, and mitochondrial DNA allows for not only prediction of biogeographical origin of a plant but also discrimination between individual plants. A previously validated, 13-autosomal STR multiplex was used to genotype 510 samples. Samples were analyzed from four different sites: 21 seizures at the US-Mexico border, Northeastern Brazil, hemp seeds purchased in the US, and the Araucania area of Chile. In addition, a previously reported multi-loci system was modified and optimized to genotype five chloroplast and two mitochondrial markers. For this purpose, two methods were designed: a homopolymeric STR pentaplex and a SNP triplex with one chloroplast (Cscp001) marker shared by both methods for quality control. For successful mitochondrial and chloroplast typing, a novel real-time PCR quantitation method was developed and validated to accurately estimate the quantity of the chloroplast DNA (cpDNA) using a synthetic DNA standard. Moreover, a sequenced allelic ladder was also designed for accurate genotyping of the homopolymeric STR pentaplex. For autosomal typing, 356 unique profiles were generated from the 425 samples that yielded full STR profiles and 25 identical genotypes within seizures were observed. Phylogenetic analysis and case-to-case pairwise comparisons of 21 seizures at the US-Mexico border, using the Fixation Index (F ST ) as genetic distance, revealed the genetic association of nine seizures that formed a reference population. For mitochondrial and chloroplast typing, subsampling was performed, and 134 samples were genotyped. Complete haplotypes (STRs and SNPs) were observed for 127 samples. As expected, extensive haplotype sharing was observed; five distinguishable haplotypes were detected. In the reference population, the same haplotype was observed 39 times and two unique haplotypes were also detected. Haplotype sharing was observed between the US border seizures, Brazil, and Chile, while the hemp samples generated a distinct haplotype. Phylogenetic analysis of the four populations was performed, and results revealed that both autosomal and lineage markers could discern population substructure.

Internal validation of the RapidHIT® ID system.

Traditionally, forensic DNA analysis has required highly skilled forensic geneticists in a dedicated laboratory to generate short tandem repeat (STR) profiles. STR profiles are routinely used either to associate or exclude potential donors of forensic biological evidence. The typing of forensic reference samples has become more demanding, especially with the requirement in some jurisdictions to DNA profile arrestees. The Rapid DNA (RDNA) platform, the RapidHIT® ID (IntegenX®, Pleasanton, CA), is a fully automated system capable of processing reference samples in approximately 90min with minimal human intervention. Thus, the RapidHIT ID instrument can be deployed to non-laboratory environments (e.g., booking stations) and run by trained atypical personnel such as law enforcement. In order to implement the RapidHIT ID platform, validation studies are needed to define the performance and limitations of the system. Internal validation studies were undertaken with four early-production RapidHIT ID units. Reliable and concordant STR profiles were obtained from reference buccal swabs. Throughout the study, no contamination was observed. The overall first-pass success rate with an "expert-like system" was 72%, which is comparable to another current RDNA platform commercially available. The system's second-pass success rate (involving manual interpretation on first-pass inconclusive results) increased to 90%. Inhibitors (i.e., coffee, smoking tobacco, and chewing tobacco) did not appear to affect typing by the instrument system; however, substrate (i.e., swab type) did impact typing success. Additionally, one desirable feature not available with other Rapid systems is that in the event of a system failed run, a swab can be recovered and subsequently re-analyzed in a new sample cartridge. Therefore, rarely should additional sampling or swab consumption be necessary. The RapidHIT ID system is a robust and reliable tool capable of generating complete STR profiles within the forensic DNA typing laboratory or with proper training in decentralized environments by non-laboratory personnel.

Analysis of DNA from post-blast pipe bomb fragments for identification and determination of ancestry.

Improvised explosive devices (IEDs) such as pipe bombs are weapons used to detrimentally affect people and communities. A readily accessible brand of exploding targets called Tannerite® has been identified as a potential material for abuse as an explosive in pipe bombs. The ability to recover and genotype DNA from such weapons may be vital in the effort to identify suspects associated with these devices. While it is possible to recover DNA from post-blast fragments using short tandem repeat markers (STRs), genotyping success can be negatively affected by low quantities of DNA, degradation, and/or PCR inhibitors. Alternative markers such as insertion/null (INNULs) and single nucleotide polymorphisms (SNPs) are bi-allelic genetic markers that are shorter genomic targets than STRs for amplification, which are more likely to resist degradation. In this study, we constructed pipe bombs that were spiked with known amounts of biological material to: 1) recover "touch" DNA from the surface of the device, and 2) recover traces of blood from the ends of wires (simulated finger prick). The bombs were detonated with the binary explosive Tannerite® using double-base smokeless powder to initiate the reaction. DNA extracted from the post-blast fragments was quantified with the Quantifiler® Trio DNA Quantification Kit. STR analysis was conducted using the GlobalFiler® Amplification Kit, INNULs were amplified using an early-access version of the InnoTyper™ 21 Kit, and SNP analysis via massively parallel sequencing (MPS) was performed using the HID-Ion Ampliseq™ Identity and Ancestry panels using the Ion Chef and Ion PGM sequencing system. The results of this study showed that INNUL markers resulted in the most complete genetic profiles when compared to STR and SNP profiles. The random match probabilities calculated for samples using INNULs were lower than with STRs when less than 14 STR alleles were reported. These results suggest that INNUL analysis may be well suited for low-template and/or degraded DNA samples, and may be used to supplement incomplete or failed STR analysis. Human identification using SNP analysis via MPS showed variable success with low-level post-blast samples in this study (<150pg). While neat DNA samples (6µL input as recommended) resulted in <50% of SNP calls, samples that were concentrated from 15µL to 6µL (15µL was added for STR and INNUL typing) resulted in more complete SNP profiles. Five out of six blood samples recovered from the wires attached to the pipe-bombs resulted in the correct ancestry predictions.

Massively parallel sequencing of 68 insertion/deletion markers identifies novel microhaplotypes for utility in human identity testing.

Short tandem repeat (STR) loci are the traditional markers used for kinship, missing persons, and direct comparison human identity testing. These markers hold considerable value due to their highly polymorphic nature, amplicon size, and ability to be multiplexed. However, many STRs are still too large for use in analysis of highly degraded DNA. Small bi-allelic polymorphisms, such as insertions/deletions (INDELs), may be better suited for analyzing compromised samples, and their allele size differences are amenable to analysis by capillary electrophoresis. The INDEL marker allelic states range in size from 2 to 6 base pairs, enabling small amplicon size. In addition, heterozygote balance may be increased by minimizing preferential amplification of the smaller allele, as is more common with STR markers. Multiplexing a large number of INDELs allows for generating panels with high discrimination power. The Nextera™ Rapid Capture Custom Enrichment Kit (Illumina, Inc., San Diego, CA) and massively parallel sequencing (MPS) on the Illumina MiSeq were used to sequence 68 well-characterized INDELs in four major US population groups. In addition, the STR Allele Identification Tool: Razor (STRait Razor) was used in a novel way to analyze INDEL sequences and detect adjacent single nucleotide polymorphisms (SNPs) and other polymorphisms. This application enabled the discovery of unique allelic variants, which increased the discrimination power and decreased the single-locus random match probabilities (RMPs) of 22 of these well-characterized INDELs which can be considered as microhaplotypes. These findings suggest that additional microhaplotypes containing human identification (HID) INDELs may exist elsewhere in the genome.

Selection of highly informative SNP markers for population affiliation of major US populations.

Ancestry informative markers (AIMs) can be used to detect and adjust for population stratification and predict the ancestry of the source of an evidence sample. Autosomal single nucleotide polymorphisms (SNPs) are the best candidates for AIMs. It is essential to identify the most informative AIM SNPs across relevant populations. Several informativeness measures for ancestry estimation have been used for AIMs selection: absolute allele frequency differences (δ), F statistics (F ST), and informativeness for assignment measure (In). However, their efficacy has not been compared objectively, particularly for determining affiliations of major US populations. In this study, these three measures were directly compared for AIMs selection among four major US populations, i.e., African American, Caucasian, East Asian, and Hispanic American. The results showed that the F ST panel performed slightly better for population resolution based on principal component analysis (PCA) clustering than did the δ panel and both performed better than the In panel. Therefore, the 23 AIMs selected by the F ST measure were used to characterize the four major American populations. Genotype data of nine sample populations were used to evaluate the efficiency of the 23-AIMs panel. The results indicated that individuals could be correctly assigned to the major population categories. Our AIMs panel could contribute to the candidate pool of AIMs for potential forensic identification purposes.

Underlying Data for Sequencing the Mitochondrial Genome with the Massively Parallel Sequencing Platform Ion Torrent™ PGM™.

BACKGROUND: Massively parallel sequencing (MPS) technologies have the capacity to sequence targeted regions or whole genomes of multiple nucleic acid samples with high coverage by sequencing millions of DNA fragments simultaneously. Compared with Sanger sequencing, MPS also can reduce labor and cost on a per nucleotide basis and indeed on a per sample basis. In this study, whole genomes of human mitochondria (mtGenome) were sequenced on the Personal Genome Machine (PGMTM) (Life Technologies, San Francisco, CA), the out data were assessed, and the results were compared with data previously generated on the MiSeqTM (Illumina, San Diego, CA). The objectives of this paper were to determine the feasibility, accuracy, and reliability of sequence data obtained from the PGM. RESULTS: 24 samples were multiplexed (in groups of six) and sequenced on the at least 10 megabase throughput 314 chip. The depth of coverage pattern was similar among all 24 samples; however the coverage across the genome varied. For strand bias, the average ratio of coverage between the forward and reverse strands at each nucleotide position indicated that two-thirds of the positions of the genome had ratios that were greater than 0.5. A few sites had more extreme strand bias. Another observation was that 156 positions had a false deletion rate greater than 0.15 in one or more individuals. There were 31-98 (SNP) mtGenome variants observed per sample for the 24 samples analyzed. The total 1237 (SNP) variants were concordant between the results from the PGM and MiSeq. The quality scores for haplogroup assignment for all 24 samples ranged between 88.8%-100%. CONCLUSIONS: In this study, mtDNA sequence data generated from the PGM were analyzed and the output evaluated. Depth of coverage variation and strand bias were identified but generally were infrequent and did not impact reliability of variant calls. Multiplexing of samples was demonstrated which can improve throughput and reduce cost per sample analyzed. Overall, the results of this study, based on orthogonal concordance testing and phylogenetic scrutiny, supported that whole mtGenome sequence data with high accuracy can be obtained using the PGM platform.

High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing.

STR typing in forensic genetics has been performed traditionally using capillary electrophoresis (CE). However, CE-based method has some limitations: a small number of STR loci can be used; stutter products, dye artifacts and low level alleles. Massively parallel sequencing (MPS) has been considered a viable technology in recent years allowing high-throughput coverage at a relatively affordable price. Some of the CE-based limitations may be overcome with the application of MPS. In this study, a prototype multiplex STR System (Promega) was amplified and prepared using the TruSeq DNA LT Sample Preparation Kit (Illumina) in 24 samples. Results showed that the MinElute PCR Purification Kit (Qiagen) was a better size selection method compared with recommended diluted bead mixtures. The library input sensitivity study showed that a wide range of amplicon product (6-200ng) could be used for library preparation without apparent differences in the STR profile. PCR sensitivity study indicated that 62pg may be minimum input amount for generating complete profiles. Reliability study results on 24 different individuals showed that high depth of coverage (DoC) and balanced heterozygote allele coverage ratios (ACRs) could be obtained with 250pg of input DNA, and 62pg could generate complete or nearly complete profiles. These studies indicate that this STR multiplex system and the Illumina MiSeq can generate reliable STR profiles at a sensitivity level that competes with current widely used CE-based method.

An evaluation of the RapidHIT(®) system for reliably genotyping reference samples.

Short tandem repeat (STR) typing is used routinely for associating or excluding individuals with biological evidence left at a crime scene. Improvements have been made to reduce the turnaround time and labor involved with profile generation, but there is still some lag time between sample collection and interpretation of results. The RapidHIT(®) (IntegenX; Pleasanton, CA, USA) system is an automated instrument that is configured to perform DNA extraction, bead-based DNA normalization, amplification, electrophoresis of PCR amplicons, and data analysis of five reference swabs simultaneously. The RapidHIT system provided reliable STR profiles from reference buccal swabs in approximately 90min with nominal "hands-on" sample loading time with no evidence of contamination between samples. The overall success rate of typing buccal swabs was comparable to standard typing systems. In the event of a failed run due to instrument failure, the swab can be removed from the cartridge and reanalyzed in the RapidHIT system or with standard STR genotyping workflows.