DNA Barcoding and Metabarcoding Protocols for Species Identification.

The article presents the several steps to be performed on a plant, fungal, insect, or soil sample to obtain DNA sequences for DNA barcode analysis. The chapter begins with a description of sample preparation including procedures for cleaning and proceeds to DNA extraction with methods adapted for the specific type of sample. Next, DNA quantification is described so the proper amount is used for the amplification of the selected barcode regions. Information is provided for reaction mixes and amplification conditions for several referenced barcode primer pairs tuned for the individual sample of interest. This is followed by a description of procedures to access the success of amplification, cleanup, and quantification of the product ready for either Sanger sequencing or library preparation for massive parallel sequencing (MPS). Finally, procedures are provided for Sanger sequencing, library preparation, and MPS sequencing. The chapter provides several references of barcode regions for different sample types.

Guidelines for the Analysis of DNA Barcoding/Metabarcoding Sequencing Data and Interpretation of Publicly Available Databases.

This chapter describes procedures for the use of DNA sequence data to obtain and compare taxonomic identification using the public databases GenBank and Barcode of Life Data System (BOLD). The chapter begins by describing procedures used to prepare quality sequences for uploading into GenBank and BOLD. Next, steps used to query the DNA sequences against the public databases are described using GenBank BLAST and BOLD identification engines. Interpretation guidelines for the taxonomic identification assignments are presented. Finally, a procedure for evaluating the accuracy and reliability of sequences from GenBank and BOLD is provided.

Evaluation of Library Preparation Workflows and Applications to Different Sample Types Using the PowerSeq® 46GY System with Massively Parallel Sequencing.

This project evaluated the prototype PowerSeq® 46GY System using donor DNA and casework-type samples. The goal of this study was to determine whether modifications to the manufacturer's protocol could increase read coverage and improve sample results. Buccal and casework-type libraries were prepared using the TruSeq® DNA PCR-Free HT kit or the KAPA HyperPrep kit. Both kits were evaluated unmodified, and by substituting AMPure® XP beads for the beads of the most optimal kit. Two qPCR kits, the PowerSeq® Quant MS System and KAPA Library Quantification Kit, were also evaluated along with a KAPA size-adjustment workbook, which was compared as a third quantification method. Libraries were sequenced using the MiSeq® FGx and data were analyzed with STRait Razor. Results suggested that all three quantification methods overestimated library concentration, but the PowerSeq kit was most accurate. Samples prepared with the TruSeq library kit provided the highest coverage and the fewest instances of dropout and below-threshold alleles compared with the KAPA kit. Additionally, all bone and hair samples demonstrated full profile completeness, with bone samples yielding a higher average coverage than hair samples. Overall, our study demonstrated that the 46GY manufacturer's protocol produced the best quality results compared to alternative library preparation options.

Assessment of detection limits for dyed and mounted textile fibers using Raman spectroscopy.

Trace evidence in the form of textile fibers can be used to link objects and places during an investigation. Raman spectroscopy is a well-established technique that has been used for the examination of various pigments, paints, inks, and dyes. The objective of this study was to determine the capability of Raman spectroscopy to detect several different dye classes and colors on a variety of textile fibers. To test this, four categories of dyes, reactive, disperse, acid, and direct were examined with Raman microscopy while applied to one of five fiber types (cotton, polyester, nylon, wool, and rayon). Each dye category was tested using four colors, black, blue, red, and yellow, while at four concentrations of dye (w/w), 4% (black only), 1%, 0.5%, and 0.05% (blue, red, and yellow). Finally, each dye, fiber, color, and dye concentration combination were examined with Raman using one of two laser excitation sources (532 nm and 780 nm) while mounted in one of two mounting media, Permount™ and Entellan® new, as well as unmounted. Raman spectroscopy could detect some dyes at low concentrations (0.5% and 0.05%) even when mounted in mounting media and covered with a glass coverslip. Excitation source, dye category, dye concentration, fiber type, and mounting method all influence the ability to detect any given dye. These results support the continued study of Raman as a tool for the examination of fiber dyes as it has shown the potential to be effective even under constraints experienced by forensic examiners.

Recovery of DNA from fired and unfired cartridge casings: comparison of two DNA collection methods.

For over 10 years, various studies have attempted to increase the recovery of DNA from ammunition by modifying the DNA collection, extraction, purification, and amplification procedures, with varying levels of success. This study focused on the "soaking" method of Montpetit & O'Donnell [1] and the "rinse-and-swab" method of Bille et al. [2]. First, testing for the presence of exogenous DNA, 210 boxed cartridges (brass, steel, and nickel-plated) from nine manufacturers were swabbed and DNA was extracted, concentrated, and quantified. Extracts that quantified > 0 ng/µL (44 of 210) were amplified and genotyped with GlobalFiler™. Of those, only one extract yielded two alleles indicating that the manufacturing and packaging of ammunition was virtually DNA free. Next, to obtain a baseline comparison of two DNA collection methods on a non-metallic substrate and identify a suitable number of cells to spot on cartridges, different DNA input amounts of primary human adult epidermal keratinocytes (HEKa) were tested. Thereafter, 300 brass and 300 nickel-plated, cartridges were spotted with HEKa cells containing ~5 ng of DNA, fired or unfired, and processed with either method. Finally, five methods representing hybrids of the soaking and rinse-and-swab methods were tested to determine if variations of those methods could be used to increase DNA yield and recovery. The results show that the soaking method consistently yielded more DNA than the rinse-and-swab method from a non-metallic substrate. However, the comparison study demonstrated that both methods performed comparably for cartridges. On average, the soaking method recovered 0.25 ng of DNA (5.1% recovery) and the rinse-and-swab method recovered 0.28 ng (5.8% recovery). However, average recoveries were significantly different among three analysts and considerable variation in yields were observed, possibly due to storage time. Furthermore, consistent with prior reports, the DNA recovered from brass casings was only 16% of that recovered from nickel-plated casings and the average yield of DNA from fired casings was reduced to 67% of unfired casings. Moreover, DNA extracts from brass or nickel-plated casings did not appear to contain amplification inhibitors and only 30/596 appeared severely degraded. Finally, both the published rinse-and-swab and soaking methods yielded more DNA than all modifications of the two methods. Overall, both methods yielded equivalent DNA quantities. Additionally, recovery of DNA from any given cartridge casing may be dependent on storage time as well as the skill, proficiency, and experience of the analyst and may reflect stochastic effects, particularly for casings containing low copy and/or degraded DNA.

Mitochondrial DNA analysis of the putative skeletal remains of Sieur de Marle: Genetic support for anthropological assessment of biogeographic ancestry.

In 1932, seven burials were discovered on a Texas plantation that was originally the site of a 17th-century Caddo Indian village. Of the seven excavated graves, one set of remains (an adult male) was notably buried in a manner inconsistent with traditional Caddoan burial practices and has long been purported to be the remains of Sieur de Marle (a member of the French explorer La Salle's last expedition). Diary accounts of La Salle's expedition scribe report that Sieur de Marle died along a river near an Indian village during a trek to Canada to find help for colonists left behind at the ill-fated Fort St. Louis. Additionally, two lead projectiles recovered from the grave were ballistically analyzed and determined to be consistent with ammunition used in 17th-century weaponry. In the 1980s, anthropologists requested access to the remains for study, but the skull was missing. Cranial measurements recorded in 1940 and 1962 (by two independent anthropologists) were used to investigate the ancestry of this individual; and the Giles-Elliot (G-E) discriminant function was calculated to be 18.1, within the Anglo-European range. Dietary isotope testing on non-cranial skeletal elements determined that this unknown male's diet was rich in animal/marine protein sources, which differs appreciably from Caddo Indian populations of that time period. In order to genetically assess this individual's biogeographic ancestry and to provide further support that this individual is of European descent, mitochondrial DNA (mtDNA) sequencing was performed using the Applied Biosystems™ Precision ID mtDNA Whole Genome Panel. mtDNA sequencing of multiple sections from two different long bones yielded compiled results consistent with either Haplogroup H or R, both predominantly European mtDNA haplogroups. Further anthropological calculations were conducted using cranial measurements, FORDISC™ software, and discriminant function analysis. Two-way, four-way, and multigroup discriminant function analyses further classify this set of unidentified remains as being White (European) in origin, with posterior probabilities of 0.999, 0.881 and 0.986, respectively. Combined with historical records of Sieur de Marle's death, as well as overlays of historical and contemporary maps which demonstrate that the plantation site aligns with Joutel's diary accounts of de Marle's burial, these collective results support that these remains are of a European male and may possibly belong to this prominent member of La Salle's expedition team.

Utility of the Ion S5™ and MiSeq FGx™ sequencing platforms to characterize challenging human remains.

Often in missing persons' and mass disaster cases, the samples remaining for analysis are hard tissues such as bones, teeth, nails, and hair. These remains may have been exposed to harsh environmental conditions, which pose challenges for downstream genotyping. Short tandem repeat analysis (STR) via capillary electrophoresis (CE) is still the gold standard for DNA typing; however, a newer technology known as massively parallel sequencing (MPS) could improve upon our current techniques by typing different and more markers in a single analysis, and consequently improving the power of discrimination. In this study, bone and tooth samples exposed to a variety of DNA insults (cremation, embalming, decomposition, thermal degradation, and fire) were assessed and sequenced using the Precision ID chemistry and a custom AmpliSeq™ STR and iiSNP panel on the Ion S5™ System, and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™ system, as well as the GlobalFiler™ PCR Amplification Kit on the 3500™ Genetic Analyzer. The results demonstrated that using traditional CE-based genotyping performed as expected, producing a partial or full DNA profile for all samples, and that both sequencing chemistries and platforms were able to recover sufficient STR and SNP information from a majority of the same challenging samples. Run metrics including profile completeness and mean read depth produced good results with each system, considering the degree of damage of some samples. Most sample insults (except decomposed) produced similar numbers of alleles for both MPS systems. Comparable markers produced full concordance between the two platforms.

Assessment of impact of DNA extraction methods on analysis of human remain samples on massively parallel sequencing success.

Skeletal remains recovered from missing persons' cases are often exposed to harsh environmental conditions resulting in the DNA being damaged, degraded, and/or the samples containing PCR inhibitors. In this study, the efficacy of common extraction methods was evaluated to remove high levels of PCR inhibitors commonly encountered with human remains, and their downstream compatibility with the two leading sequencing chemistries and platforms for human identification purposes. Blood, hair, and bone samples were spiked with high levels of inhibitors commonly identified in each particular substrate in order to test the efficiency of various DNA extraction methods prior to sequencing. Samples were extracted using three commercial extraction kits (DNA IQ™, DNA Investigator, and PrepFiler® BTA), organic (blood and hair only), and two total demineralization protocols (bone only)). Massively parallel sequencing (MPS) was performed using two different systems: Precision ID chemistry and a custom AmpliSeq™ STR and iiSNP panel on the Ion S5™ System and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™. The overall results showed that all DNA extraction methods were efficient and are fully compatible with both MPS systems. Key performance indicators such as STR and SNP reportable alleles, read depth, and heterozygote balance were comparable for each extraction method. In samples where CE-based STRs yielded partial profiles (bone), MPS-based STRs generated more complete or full profiles. Moreover, MPS panels contain more STR loci than current CE-based STR kits and also include SNPs, which can further increase the power of discrimination obtained from these samples, making MPS a desirable choice for the forensic analysis of such challenging samples.

Comparative sensitivity and inhibitor tolerance of GlobalFiler® PCR Amplification and Investigator® 24plex QS kits for challenging samples.

In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, been buried, decomposed, and/or contain inhibitory substances. This study compares the performance of a relatively new STR kit in the US market (Investigator® 24plex QS kit; Qiagen) with the GlobalFiler® PCR Amplification kit (Thermo Fisher Scientific) when genotyping highly inhibited and low level DNA samples. In this study, DNA samples ranging from 1 ng to 7.8 pg were amplified to define the sensitivity of two systems. In addition, DNA (1 ng and 0.1 ng input amounts) was spiked with various concentrations of five inhibitors common to human remains (humic acid, melanin, hematin, collagen, calcium). Furthermore, bone (N = 5) and tissue samples from decomposed human remains (N = 6) were used as mock casework samples for comparative analysis with both STR kits. The data suggest that the GlobalFiler® kit may be slightly more sensitive than the Investigator® kit. On average STR profiles appeared to be more balanced and average peak heights were higher when using the GlobalFiler® kit. However, the data also show that the Investigator® kit may be more tolerant to common PCR inhibitors. While both STR kits showed a decrease in alleles as the inhibitor concentration increased, more complete profiles were obtained when the Investigator® kit was used. Of the 11 bone and decomposed tissue samples tested, 8 resulted in more complete and balanced STR profiles when amplified with the GlobalFiler® kit.

Comparative tolerance of two massively parallel sequencing systems to common PCR inhibitors.

Human remains can be severely affected by the environment, and the DNA may be damaged, degraded, and/or inhibited. In this study, a DNA sample (at 1 ng DNA target input in triplicate) was spiked with five concentrations of five inhibitors (humic acid, melanin, hematin, collagen, and calcium) and sequenced with both the HID-Ion AmpliSeq™ Library Kit and ID panel on the Ion PGM™ System and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™. The objective of this study was to compare the baseline tolerance of the two sequencing chemistries and platforms to common inhibitors encountered in human remains recovered from missing person cases. The two chemistries generally were comparable but not always susceptible to the same inhibitors or at the same capacity. The HID-Ion AmpliSeq™ Library Kit and ID panel and the ForenSeq DNA Signature Prep Kit both were susceptible to humic acid, melanin, and collagen; however, the ForenSeq kit showed greater inhibition to melanin and collagen than the AmpliSeq™ kit. In contrast, the ForenSeq kit was resistant to the effects of hematin and calcium, whereas the AmpliSeq™ kit was highly inhibited by hematin. Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) showed the same trend among inhibitors when using the ForenSeq kit. Generally, locus read depth, heterozygote allele balance, and the numbers of alleles typed were inversely correlated with increasing inhibitor concentration. The larger STR loci were affected more so by the presence of inhibitors compared to smaller STR amplicons and SNP loci. Additionally, it does not appear that sequence noise is affected by the inhibitors. The noise percentage, however, does increase as the inhibitor concentration increases, due to the decrease in locus read depth and not likely because of chemistry effects.

Evaluation of four commercial quantitative real-time PCR kits with inhibited and degraded samples.

DNA quantification is a vital step in forensic DNA analysis to determine the optimal input amount for DNA typing. A quantitative real-time polymerase chain reaction (qPCR) assay that can predict DNA degradation or inhibitors present in the sample prior to DNA amplification could aid forensic laboratories in creating a more streamlined and efficient workflow. This study compares the results from four commercial qPCR kits: (1) Investigator® Quantiplex® Pro Kit, (2) Quantifiler® Trio DNA Quantification Kit, (3) PowerQuant® System, and (4) InnoQuant® HY with high molecular weight DNA, low template samples, degraded samples, and DNA spiked with various inhibitors.The results of this study indicate that all kits were comparable in accurately predicting quantities of high quality DNA down to the sub-picogram level. However, the InnoQuant(R) HY kit showed the highest precision across the DNA concentration range tested in this study. In addition, all kits performed similarly with low concentrations of forensically relevant PCR inhibitors. However, in general, the Investigator® Quantiplex® Pro Kit was the most tolerant kit to inhibitors and provided the most accurate quantification results with higher concentrations of inhibitors (except with salt). PowerQuant® and InnoQuant® HY were the most sensitive to inhibitors, but they did indicate significant levels of PCR inhibition. When quantifying degraded samples, each kit provided different degradation indices (DI), with Investigator® Quantiplex® Pro indicating the largest DI and Quantifiler® Trio indicating the smallest DI. When the qPCR kits were paired with their respective STR kit to genotype highly degraded samples, the Investigator® 24plex QS and GlobalFiler® kits generated more complete profiles when the small target concentrations were used for calculating input amount.