Sens vitro Polymerase Interrogation and Inflammation of ancient Salmon

Transcription

1 Forensic Science International 228 (2013) Contents lists available at SciVerse ScienceDirect Forensic Science International jou r nal h o mep age: w ww.els evier.co m/lo c ate/fo r sc iin t Evaluating the efficacy of various thermo-stable polymerases against co-extracted PCR inhibitors in ancient DNA samples Cara Monroe a,b,c, Colin Grier b, Brian M. Kemp a,b, * a Department of Anthropology, Washington State University, Pullman, WA , United States b School of Biological Sciences, Washington State University, Pullman, WA , United States c Department of Anthropology, University of California-Santa Barbara, Santa Barbara, CA , United States A R T I C L E I N F O Article history: Received 6 January 2013 Accepted 18 February 2013 Available online Keywords: DNA extraction Ancient DNA Degraded DNA Polymerase chain reaction inhibitors Polymerases Species identification A B S T R A C T DNA from ancient and forensic specimens is often co-extracted with unknown amounts of unknown PCR inhibitors, which can lead to underestimated DNA concentrations, allelic drop-out, and/or false-negative results. It is not surprising, in this case, that numerous methods have been developed to remove PCR inhibitors or subdue their effects. One simple and cost effective approach could be the adoption of a polymerase that overcomes or is less affected by PCR inhibitors. In this study, nine different polymerases were evaluated for their efficacy against PCR inhibitors co-extracted with DNA from 63 ancient salmon vertebrae. These samples were excavated from two archeological sites located at the Dionisio Point locality on the northern end of Galiano Island in coastal southwestern British Columbia, Canada and date to and years before present. Previously, DNA extracts from samples studied from this locality were determined to be largely inhibited to PCR amplification. In the present study, Omni Klentaq LA (DNA Polymerase Technology, Inc.) outperformed the other 8 polymerases in two measures: (1) its success in genetic species identification of these vertebrae, and (2) its ability to amplify an ancient DNA positive control when spiked with a volume of potentially inhibited extract from the vertebrae. ß 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Numerous substances can potentially inhibit PCR and are routinely encountered in both the study of ancient DNA (adna) (see review by [28]) and forensic DNA (see review by [4]). In these types of investigations, if contaminating DNA is sufficiently minimized or fully removed (e.g., [29]), and because the degree of post-mortem damage [37,41] witnessed by a sample cannot be controlled, co-purified PCR inhibitors remain as the greatest threat to the successful study of ancient, degraded, and/or low copy number (LCN) DNA samples. The presence of PCR inhibitors can normally be confirmed visually as DNA extractions ranging from tinged yellow to dark brown in color (Fig. 1). However, lack of coloration does not guarantee that a DNA extract is free of inhibitors [4,28]. The presence and concentration of PCR inhibitors have major consequences for downstream applications and can lead to * Corresponding author at: Department of Anthropology, Washington State University, Pullman, WA , United States. Tel.: ; fax: address: bmkemp@wsu.edu (B.M. Kemp). underestimated DNA concentrations, allelic drop-out, and/or false-negative results [17,34,35,43,44]. Compromised DNA material is particularly prone to inhibition, which can be introduced naturally through soils, plants (e.g. polysaccharides, humic, tannic, and fulvic acids), clothing (e.g. dyes, detergents) or food as well as during laboratory processing (phenol salts, ethanol, isopropanol, EDTA, and chaotrophic salts). Other and more insidious PCR inhibitors are endogenous to the biological samples themselves and include calcium ions and collagen from bone, blood components (billirubin, heme, immunoglobin, lactoferrin), salvia, semen, cervical fluid, bile, feces (polysaccharides, and humic acid), melanin, and myoglobin from muscle tissue among many others [1,7,12,13,33,36,42,48,50,55,56]. Given the importance of removing PCR inhibitors from DNA extracts, a number of techniques have been developed to eliminate this problem. These methods can be generally divided into two groups: (1) those that remove inhibitors during the DNA extraction and purification, and (2) those that diminish the effects of inhibitors by later manipulation of template DNA, PCR reagents, or incorporating PCR additives (reviewed by [4,28]). While most of these techniques have been relatively effective with specific classes of inhibitors, it is difficult to predict which types of inhibitors or groups of inhibitors are present in any given /$ see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved.

2 C. Monroe et al. / Forensic Science International 228 (2013) Fig. 1. Photograph depicting, in the left seven tubes in the top row, DNA extractions from salmonid vertebrae (samples 1, 21, 49, 75, 94, 160, and 167) recovered from the Late period plankhouse at the DgRv-006 site. These samples date to approximately YBP. Dilutions of these samples are found in the left seven tubes of the middle row (1:10) and the bottom row (1:50). The eighth tube in each row represents the extraction negative control and dilutions of that control (1:10 and 1:50). This batch of samples illustrates the range of inhibitors visually observed in this study (and the range we have typically observed in other studies). In the top row, from the left to right the samples were described as follows: (1) light brown, (2) brown, (3) light brown, (4) dark, dark brown, (5) brown, (6) light brown, (7) light brown, and (8) clear. In the middle row, from left to right the 1:10 dilutions were described as follows: (1) clear, (2) light tinge, (3) clear, (4) brown, (5) light tinge, (6) clear, (7) clear, and (8) clear. In the bottom row, from the left to right the 1:50 dilutions were described as follows: (1) clear, (2) clear, (3) clear, (4) tinged, (5) clear, (6) clear, (7) clear, and (8) clear. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) sample [11]. Additionally, methods that remove inhibitors during extraction are associated with DNA loss, especially fragments <200 base pairs in length, which poses a significant problem when working with degraded samples [25]. Moreover, some purification methods may achieve the desired end of removing environmental or endogenous PCR inhibitors while unintentionally incorporating new ones as a result of reagent carry-over [8,10]. Of the second category of techniques, diluting template DNA and/or adding bovine serum albumin (BSA) to the PCR reaction are common remedies [1,22,39,50,54]. In the first instance PCR inhibitors are sufficiently diluted with water to no longer affect the PCR reaction, while at the same time the template DNA is not diluted sufficiently to preclude its amplification. As dilutions maintain a constant ratio of inhibitors to DNA, the effectiveness of this approach must be contingent on either: (1) the existence of an inhibitor threshold level, or (2) creating sufficient distance in the solution between inhibitory molecules (in this case referring to inhibitors not bound directly to the DNA) and template DNA, thus subduing their effects. Overcoming inhibitors through dilution may not be the best approach as it increases the need for experimental PCR reactions in order to determine appropriate dilutions. Moreover, it may be counterproductive to further dilute template DNA already present in low copy number, as is typically recovered from degraded samples [61]. Similarly, BSA blocks PCR inhibitors and, thus indirectly promotes polymerase activity. However, the use of BSA suffers from complications comparable to serial dilutions. Determining the appropriate concentrations is problematic, as various types of inhibitors require differing amounts of BSA in order to overcome PCR inhibition and in some cases BSA is ineffective or can even be detrimental to the success of an amplification [2,3,15,53]. As a result it is preferable to have a generalized technique that decreases time, cost, and labor, and one that eliminates, circumvents, and/or inactivates as many inhibitors as possible while maintaining DNA yield. Adopting such a strategy will ultimately be a cost effective means for working with DNA in the face of PCR inhibitors. A simple solution might be the adoption of the polymerase that performs well in the face of inhibitory molecules. A number of studies have shown that polymerase derived from Thermus aquaticus (Taq) is highly susceptible to inhibitory substances whereas polymerases from Pyrococcus furiosus (Pfu), Pyrococcus woesei (Pwu), Thermus flavus (Tfl), and Thermus thermophilus (Tth and rtth) are less so [2,3,21,24,40,57]. For example, Tth polymerase was used to successfully amplify DNA in the presence of immunoglobin, myoglobin, bile, ocular fluid and phenol [2,3,7,24] and Pwo polymerase was able to overcome inhibition from blood [3]. Eilert and Foran [15] found two polymerases, a variant of Thermus aquaticus (Taq) and Thermus thermophiles (Tth), which had an increased resistance to inhibition from bone, while polymerases from Thermus flavus (Tfl) and Thermus filiformis (Tfi) were prone to inhibition. Additionally, novel mutant Taq and Klentaq had increased amplification in crude blood and soil samples and folvic and humic acid [31,32,40] while 2D9, a chimeric polymerase with components derived from Thermus aquaticus, Thermus oshimai (Tos), Thermus thermophiles (Tth) and Thermus brockianus (Tbr), had a resistance to a variety of inhibitors such as: humic acid, bone dust, coprolite material, peat extract, clay-rich soil, cave sediment, and tar [6]. However, while some polymerases have higher fidelity (e.g., Pfu over Taq polymerase), as well as higher tolerances for certain inhibitors, they can produce lower yields as a consequence of their exonuclease (proofreading) activity [46]. Researchers often overcome this proclivity of Taq polymerase by adding increasing amounts to PCR reactions [14,16,45,52]. While, increasing the amount of Taq polymerase may be an effective means to amplify DNA in the face of inhibition, this practice simultaneously makes a system that is already hypersensitive to the detection of contamination even

3 144 C. Monroe et al. / Forensic Science International 228 (2013) more so [28,60] and, as a result, can become cost and time ineffective. The extent to which alternative forms of polymerase can tolerate inhibitory substances as well as amplify degraded samples is only beginning to be understood in adna and forensic studies. It is therefore compelling to determine which polymerase or manufactured blends of polymerases give the most optimal yield, fidelity, and resistance to a wide-range of inhibitors. In this project we evaluated the overall effectiveness of nine different thermostable polymerases and polymerase blends in their ability to amplify mitochondrial DNA () present in DNA extractions from salmon vertebrae from two archaeological sites (DgRv-003 and DgRv-006). These samples were chosen for two reasons. First, in a previous study of salmon vertebrae from the DgRv-003 site, DNA extracted from the samples was found to be particularly challenging to purify [19], requiring on average 4.62 (SD = 2.31) repeat silica extractions to sufficiently remove the inhibitory effects (following [28]). In our experience, these samples are some of the most inhibited samples with which we have ever worked, rivaling even year old human fecal samples (or coprolites ) from archeological sites located in Southeastern Utah [27]. Secondly, working with non-human, non-domestic animal samples minimizes the influence that contamination might otherwise have had. In the end, we posed a simple question that is, which of a number of commercially available polymerases or blends of polymerases performs best in the study of these highly inhibited samples. Our approach was not exhaustive to the number of polymerases available on the market; those tested represent only a portion of them. Performances were measured very simply as percent return of salmonid sequences and percent of samples that amplified when spiked with an adna positive control. 2. Materials and methods 2.1. Archeological materials Salmon vertebrae were recovered from excavations at two archeological sites located within the Dionisio Point locality on the northern end of Galiano Island in coastal southwestern British Columbia, Canada [19]. Twenty-two of the vertebral elements analyzed in this study derive from excavations within an ancient plankhouse (House 2) at the DgRv-003 (or Dionisio Point ) archeological site, dated to approximately years ago. The other forty-one samples analyzed in this study were recovered from the remains of a second plankhouse (House 1) at the DgRv-006 site, which dates to between 700 and 1000 years ago. The two sites are in adjacent bays and separated by roughly 150 m. The salmon elements analyzed were sampled from much larger zooarchaeological assemblages. On the whole, bone was well preserved at both sites, at least visually. Despite the difference in age, the state of preservation appears comparable between sites. Because of their proximity, soil, moisture conditions and geology are similar, and bone elements were likely subject to similar anthropogenic and taphonomic processes at both sites DNA methods All preparation methods (i.e., extraction and PCR set-up) were conducted in the adna laboratory at Washington State University, one dedicated to the analysis of degraded and low copy number (LCN) DNA. Appropriate measures to minimize contamination and, importantly, to detect it if present, were employed [30] DNA extraction Except in one case, portions of each vertebra, weighing between 12 and 195 mg (Table 1), were carefully removed from the whole using a fresh razor blade on each sample. In the case of sample #410 from DgRv-003, which weighed 10 mg, DNA was extracted from the whole sample. Samples were extracted in batches of seven with an accompanying extraction negative control to which no sample was added. Samples were submerged in 6% (w/v) sodium hypochlorite (Clorox bleach) for four minutes to remove contaminating DNA from the surfaces of the samples [29]. The samples were then rinsed twice with DNA-free ddh 2 O and DNA was extracted according to a modified protocol of Kemp et al. [26] changed specifically in the silica extraction portion of the method as follows: (1) following the isopropanol precipitation, 750 ml of 2% celite in 6 M guanidine HCl 1 and 250 ml of 6 M guanidine HCl were added to samples and vortexed numerous times over a 2 min period, (2) 3 ml of DNA-free ddh 2 O was pulled across the syringe and Promega Wizard 1 Minicolumns to wash them before pulling the samples across the columns, (3) the DNA bound silica was rinsed with 3 ml of 80% isopropanol (versus 2 ml recommended by the manufacturer), (4) 50 ml of 65 8C DNA-free ddh 2 O was added to the column and left for 3 min prior to centrifugation, and this step was repeated again resulting in 100 ml of extracted DNA Polymerases and salmonid PCRs Nine different polymerases were tested in this study: (1) Amplitaq Gold 1 DNA Polymerase (Invitrogen), (2) Herculase II Fusion DNA Polymerase (Agilent), (3) Omni Klentaq LA (DNA Polymerase Technology, Inc.), (4) Phusion 1 High Fidelity DNA Polymerase (Finnzymes), (5) Platinum 1 Taq DNA Polymerase (Invitrogen), (6) Pwo DNA Polymerase (Roche), (7) rtth (Applied Biosystems) (8) Tfl DNA Polymerase (Promega), and (9) Vent R 1 DNA Polymerase (New England Biolabs). From this point onward, these polymerases will simply be referred to as: (1) Amplitaq Gold, (2) Herc II, (3) Klentaq, (4) Phusion, (5) Platinum Taq, (6) Pwo, (7) rtth, (8) Tfl, and (9) Vent. All PCR amplifications were conducted in 15 ml reaction volumes that included 1.5 ml of template DNA. Details of the PCR reactions conducted in this study are summarized in Table 2. Attempts were made to standardize PCR components across kits and make the reactions match as closely to those described by Kemp et al. [26] which used Platinum Taq and has been met with success across a number of additional studies of ancient bones, teeth, and feces (e.g. [9,38,51,58,59]). Complete standardization was not always possible in the cases that cofactors were already premixed in the PCR buffers at various concentrations (i.e., for Herc II and Klentaq, Table 2). Moreover, we did not control for differences between the components found in the PCR buffers supplied with the polymerases, which are highly variable (see respective manufacturer s manuals). A 189 base pair (bp) portion of the 12S mitochondrial gene, used for species identification of salmonids, was PCR amplified in 15 ml reactions according to Table 2, using the primers (called OST12S-F and OST12S-R ) described by Jordan et al. [23]. PCR denaturing and extension conditions varied according the manufacturer s recommendations for the respective polymerases. A rainbow trout (Oncorhynchus mykiss) positive control, added in the post-pcr lab just prior to running the PCRs, accompanied each batch of reactions to preclude PCR failure. The extraction negative controls and PCR negatives were tested in parallel with all rounds of amplification. Four microliters of PCR products were separated on 6% polyacrylamide gels. Gels were stained with ethidium bromide and viewed under UV light to confirm successful amplification. Amplicons were prepared for sequencing by addition of 10 U of ExoI and 2 U of SAP. Reactions were incubated at 378 C for 20 min, followed by 80 8C for 20 min. Sequences were generated in both directions at Elim Biopharm (Hayward, CA). Aligned against a rainbow trout sequence (NCBI Accession DQ288271) in Sequencher (version 4.8), sequences generated in this study were used for species determination according to Jordan et al. [23] Evaluating inhibitory effects Extractions and modified extractions (as described below) were tested for inhibitory effects against the polymerases listed above, following the rationale provided by Kemp et al. [28] and as put into practice by Grier et al. [19] using DNA extracted from 170 to 415 year old goose remains [58] as the positive adna controls. However, in contrast to those previous studies, here batches of seven to thirteen goose bones were extracted, as described above, and pooled into what we call Goose Collective ancient positive controls. The choice to pool these individual extractions was intentionally done to even out variance across samples in both endogenous goose copy number and possible inhibitors co-extracted with the goose DNA. A different Goose Collective was used in each of the three rounds of experiments described below, so that the inhibitory effect of the salmonid extractions is directly comparable within each round. Fifteen microliter PCRs, which included 1.5 ml of Goose Collective DNA, were set up according to Table 2 to amplify a 159 bp portion of goose mitochondrial cytochrome B gene using the primers BSP-I and GooseR described by Wilson et al. [58]. To these reactions, 1.5 ml of the ancient salmon template DNA was added (totaling 16.5 ml reactions). The extraction negative controls were also tested for inhibitors. PCRs were run according to Table 2 in parallel with a goose PCR that contained no salmon DNA extract (this reaction was used as a positive control, which allowed us to preclude PCR failure from contributing to our results [30]). PCR negatives accompanied each round of amplification. If the goose DNA failed to amplify when spiked with ancient salmon DNA extract, we considered the 1 This solution is intended to mimic the Wizard 1 PCR Preps DNA Purification Resin, as best could be ascertained from the material safety data sheet (MSDS). To make this solution, add DNA-free ddh 2 O to 1.25 mg of Celite 1 Analytical Filter Aid II (CAFA II, Sigma) up to 25 ml, vortex and let incubate at room temperature overnight. Pour off the water carefully as to not pour off the celite and add DNA-free ddh 2 O to 5 ml and 6 M guanidine HCl (Teknova) to 50 ml. Vortex thoroughly before each use.

4 C. Monroe et al. / Forensic Science International 228 (2013) Table 1 Samples studied and results. Round 1 Site mg extracted Full concentration extractions Extract color Omni Klen Taq Platinum Taq Pwo Dn/a Polymerase Sample ID 310 DgRv Light tinge O Yes O Yes O Yes 312 DgRv Brown O Yes O Yes O Yes 315 DgRv Brown O Yes O Yes O Yes 317 DgRv Light tinge O Yes O Yes O Yes 375 DgRv Clear O Yes O Yes O Yes 376 DgRv Light brown O Yes O Yes O Yes 377 DgRv Clear O Yes O Yes O Yes EC 1/26/12 n/a n/a Clear O No O No O No 163 DgRv Light brown O Yes O Yes O Yes 330 DgRv Light, light brown O Yes O Yes O Yes 385 DgRv Clear O Yes O Yes O Yes 386 DgRv Brown O Yes O Yes O Yes 388 DgRv Clear O Yes O Yes O Yes 401 DgRv Clear O Yes O Yes O Yes 420 DgRv Light, light brown O Yes O Yes O Yes EC 1/31/12-A n/a n/a Clear O Yes O Yes O Yes 336 DgRv Clear O Yes O Yes O No 337 DgRv Clear O Yes O Yes O No 390 DgRv Brown O Yes O Yes O Yes 391 DgRv Brown O Yes O Yes O Yes 396 DgRv Clear O Yes O Yes O No 405 DgRv Light tinge O Yes O Yes O Yes 416 DgRv Light tinge O Yes O Yes O Yes EC 1/31/12-B n/a n/a Clear O No O No O No Positive Control n/a n/a Clear X X X X X X PCR NEG 2 n/a n/a n/a O n/a O n/a O n/a PCR NEG 3 n/a n/a n/a O n/a O n/a O n/a Round 1 Site mg extracted 1:10 Diluted extractions Extract Color Omni Klen Taq Platinum Taq Pwo Dn/a Polymerase Sample ID 310 DgRv Clear O No O Yes O No 312 DgRv Light tinge O Yes O Yes O Yes 315 DgRv Light tinge O Yes O Yes O Yes 317 DgRv Clear O No O Yes O No 375 DgRv Clear O No O Yes O No 376 DgRv Light, light tinge O Yes O Yes O Yes 377 DgRv Clear O No O Yes O No EC 1/26/12 n/a n/a Clear O No O No O No 163 DgRv Light, light brown O Yes O Yes O Yes 330 DgRv Light, light tinge O Yes O Yes O Yes 385 DgRv Clear O No O Yes O No 386 DgRv Light brown O Yes O Yes O Yes 388 DgRv Clear O No O Yes O No 401 DgRv Clear O No Pink (with damage) Yes O No 420 DgRv Clear Chum Yes Chum Yes O No EC 1/31/12-A n/a n/a Clear O Slightly O No O No 336 DgRv Clear Human No O No O No 337 DgRv Clear O No O No O No 390 DgRv Tinged O Yes O Yes O Yes 391 DgRv Tinged O Yes O Yes O Yes 396 DgRv Clear O No O No O No 405 DgRv Clear Sockeye No O Yes O No 416 DgRv Clear O No O Yes O No EC 1/31/12-B n/a n/a Clear O No O No O No Positive Control n/a n/a Clear X X X X X X PCR NEG 2 n/a n/a n/a O O O n/a O n/a PCR NEG 3 n/a n/a n/a O O O n/a O n/a Round 1 Site mg extracted 1:50 Diluted extractions Extract Color Omni Klen Taq Platinum Taq Pwo DNA Polymerase Sample ID 310 DgRv Clear O No O No O No 312 DgRv Light, light tinge O No O Yes O Slightly

5 146 C. Monroe et al. / Forensic Science International 228 (2013) Table 1 (Continued ) Round 1 Site mg extracted 1:50 Diluted extractions Extract Color Omni Klen Taq Platinum Taq Pwo DNA Polymerase Sample ID 315 DgRv Light, light tinge O No O Yes O No 317 DgRv Clear O No O No O No 375 DgRv Clear O No O No O No 376 DgRv Clear O No O Yes O No 377 DgRv Clear O No O No O No EC 1/26/12 n/a n/a Clear O No O No O No 163 DgRv Clear O No O Yes O Yes 330 DgRv Clear O No O Yes O No 385 DgRv Clear O No O Slightly O No 386 DgRv Light, light tinge O Yes O Yes O Yes 388 DgRv Clear O No O No O No 401 DgRv Clear O No O Yes O No 420 DgRv Clear Chum No Chum Slightly O No EC 1/31/12-A n/a n/a Clear O No O No O No 336 DgRv Clear O No O No O No 337 DgRv Clear O No O No O No 390 DgRv Light tinge O No O Yes O No 391 DgRv Light tinge O No O Yes O Yes 396 DgRv Clear O No O No O No 405 DgRv Clear Sockeye No O No O No 416 DgRv Clear O No O No O No EC 1/31/12-B n/a n/a Clear O No O No O No Positive Control n/a n/a Clear X X X X X X PCR NEG 2 n/a n/a n/a O O Human O O n/a PCR NEG 3 n/a n/a n/a O O O O O n/a Round 2 Site mg extracted Full concentration extractions Extract Color Herculase II Omni Klen Taq Vent Sample ID 6 DgRv Light brown O Yes O Yes O Yes 17 DgRv Light brown O Yes O Yes O Yes 22 DgRv Light brown O Yes O Yes O Yes 34 DgRv Brown O Yes O Yes O Yes 43 DgRv Dark brown O Yes O Yes O Yes 56 DgRv Light brown O Yes O Yes O Yes 63 DgRv Light brown O Yes O Yes O Yes EC 2/10/12-C n/a n/a Clear O No O No O No 47 DgRv Brown O Yes O Yes O Yes 110 DgRv Dark, dark brown O Yes O Yes O Yes 124 DgRv Brown O Yes O Yes O Yes 129 DgRv Dark brown O Yes O Yes O Yes 155 DgRv Light, light tinge O Yes O Yes O Yes 156 DgRv Light tinge O Yes O Yes O Yes 162 DgRv Light brown O Yes O Yes O Yes EC 2/10/12-D n/a n/a Clear O No O No O No 37 DgRv Brown O Yes O Yes O Yes 41 DgRv Brown O Yes O Yes O Yes 65 DgRv Brown O Yes O Yes O Yes 72 DgRv Brown O Yes O Yes O Yes 118 DgRv Brown O Yes O Yes O Yes 137 DgRv Light tinge O Yes O Yes O Yes 142 DgRv Brown O Yes O Yes O Yes EC 2/10/12-E n/a n/a Clear O No O No O No Positive Control n/a n/a n/a X X X X X X Round 2 Site mg extracted 1:10 Diluted extractions Extract Color Herculase II Omni Klen Taq Vent Sample ID 6 DgRv Clear O Yes O Yes O Yes 17 DgRv Clear O Yes Chum Yes O Yes 22 DgRv Clear O Yes O Yes O Yes 34 DgRv Light brown O Yes O Yes O Yes 43 DgRv Light brown O Yes O Yes O Yes 56 DgRv Clear O Yes O Yes O Yes 63 DgRv Clear O Yes O Yes O Yes

6 C. Monroe et al. / Forensic Science International 228 (2013) Table 1 (Continued ) Round 2 Site mg extracted 1:10 Diluted extractions Extract Color Herculase II Omni Klen Taq Vent Sample ID EC 2/10/12-C n/a n/a Clear O No O No O No 47 DgRv Light tinge O Yes O Yes O Yes 110 DgRv Light brown O Yes O Yes O Yes 124 DgRv Light tinge O Yes O Yes O Yes 129 DgRv Light brown O Yes O Yes O Yes 155 DgRv Clear Pink Yes Pink No O Yes 156 DgRv Clear O Yes O Yes O Yes 162 DgRv Clear O Yes O Yes O Yes EC 2/10/12-D n/a n/a Clear O No O No O No 37 DgRv Light Tinge O Slightly O Yes O Yes 41 DgRv Light Tinge O Yes O Yes O Yes 65 DgRv Light tinge O Yes O Yes O Yes 72 DgRv Light tinge O Yes O Yes O Yes 118 DgRv Light Tinge O Yes O Yes O Yes 137 DgRv Clear O Yes O Yes O Yes 142 DgRv Light tinge O Yes O Yes O Yes EC 2/10/12-E n/a n/a Clear O No O No O No Positive Control n/a n/a n/a X X X X X X Round 2 Site mg extracted 1:50 Diluted extractions Extract Color Herculase II Omni Klen Taq Vent Sample ID 6 DgRv Clear O Yes Pink No O Slightly 17 DgRv Clear Chum Yes Chum No O No 22 DgRv Clear O Yes Sockeye No O Yes 34 DgRv Light, light tinge O Yes O Yes O Yes 43 DgRv Light, light tinge O Yes O Yes O Yes 56 DgRv Clear Sockeye Yes Sockeye (with damage) No O No 63 DgRv Clear Chum Yes Chum No O Slightly EC 2/10/12-C n/a n/a Clear O No O No O No 47 DgRv Clear O Yes O Yes O Yes 110 DgRv Light tinge O Yes O Yes O Yes 124 DgRv Clear O Yes O Yes O Yes 129 DgRv Light tinge O Yes O Yes O Yes 155 DgRv Clear Pink No Pink No O No 156 DgRv Clear Chum Yes Chum No O Yes 162 DgRv Clear O Yes Chum No O Yes EC 2/10/12-D n/a n/a Clear O No O No O No 37 DgRv Clear O Yes Chum Slightly O Yes 41 DgRv Clear O Yes O No O Yes 65 DgRv Clear O Yes Chum No O Yes 72 DgRv Clear O Yes double band No O Yes 118 DgRv Clear O Yes O Slightly O Yes 137 DgRv Clear Chum Yes Chum No O No 142 DgRv Clear O Yes Chum No O Yes EC 2/10/12-E n/a n/a Clear O No O No O No Positive Control n/a n/a Clear X X X X X X Round 3 Site mg extracted Full concentration extractions Extract Color Omni Klen Taq Phusion rtth Polymerase Sample ID 10 DgRv Light tinge O Yes O Yes O Yes 38 DgRv Brown O Yes O Yes O Yes 67 DgRv Light brown O Yes O Yes O Yes 139 DgRv Brown O Yes O Yes O Yes 164 DgRv Light brown O Yes O Yes O Yes 166 DgRv Brown O Yes O Yes O Yes 410 DgRv Light tinge O Yes O Yes O Yes EC 2/10/12-F n/a n/a Clear O No O No O Yes 12 DgRv Light, light tinge O Yes O Yes O Yes 29 DgRv Light, light tinge O Yes O Yes O Yes 57 DgRv Light, light brown O Yes O Yes O Yes 100 DgRv Light, light brown O Yes O Yes O Yes 128 DgRv Brown O Yes O Yes O Yes 130 DgRv Dark brown O Yes O Yes O Yes

7 148 C. Monroe et al. / Forensic Science International 228 (2013) Table 1 (Continued ) Round 3 Site mg extracted Full concentration extractions Extract Color Omni Klen Taq Phusion rtth Polymerase Sample ID 152 DgRv Brown O Yes O Yes O Yes EC 4/11/12-G n/a n/a Clear O No O No O No 1 DgRv Light brown O Yes O Yes O Yes 21 DgRv Brown O Yes O Yes O Yes 49 DgRv Light brown O Yes O Yes O Yes 75 DgRv Dark, dark brown O Yes O Yes O Yes 94 DgRv Brown O Yes O Yes O Yes 160 DgRv Light brown O Yes O Yes O Yes 167 DgRv Light brown O Yes O Yes O Yes EC 4/11/12-H n/a n/a Clear O No O No O No Positive Control n/a n/a n/a X X X X X X Round 3 Site mg extracted 1:10 Diluted extractions Extract Color Omni Klen Taq Phusion rtth Polymerase Sample ID 10 DgRv Clear Pink No Pink Yes O Yes 38 DgRv Light tinge O Yes O Yes O Yes 67 DgRv Clear Chum No O Yes O Yes 139 DgRv Light tinge O Yes O Yes O Yes 164 DgRv Clear Pink No O Yes O Yes 166 DgRv Light tinge O Yes O Yes O Yes 410 DgRv Clear O No O Yes O Yes EC 2/10/12-F n/a n/a Clear O No O No O No 12 DgRv Clear Sockeye No Sockeye Yes O Yes 29 DgRv Clear Pink No Pink Yes O Yes 57 DgRv Clear Greenling No O Yes O Yes 100 DgRv Clear Chum No O Yes O Yes 128 DgRv Light tinge O Yes O Yes O Yes 130 DgRv Light tinge O Yes O Yes O Yes 152 DgRv Light tinge O Yes O Yes O Yes EC 4/11/12-G n/a n/a Clear O No O No O No 1 DgRv Clear Coho No O Yes O Yes 21 DgRv Light tinge Chum No O Yes O Yes 49 DgRv Clear Sockeye No O Yes O Yes 75 DgRv Brown O Yes O Yes O Yes 94 DgRv Light tinge O Yes O Yes O Yes 160 DgRv Clear Sockeye No O Yes O Yes 167 DgRv Clear Pink No O Yes O Yes EC 4/11/12-H n/a n/a Clear O No O No O No Positive Control n/a n/a X X X X X X PCR NEG 1 n/a n/a O O O O O O Round 3 Site mg extracted 1:50 Diluted extractions Extract Color Omni Klen Taq Phusion rtth Polymerase Sample ID 10 DgRv Clear Pink No Pink No Pink Slightly 38 DgRv Clear Pink No O Yes O Yes 67 DgRv Clear Chum No Chum Yes O Yes 139 DgRv Light, light tinge Chum No O Yes O Yes 164 DgRv Clear Pink No Pink Yes O Yes 166 DgRv Light, light tinge Chum No O Yes O Yes 410 DgRv Clear O No O No O Yes EC 2/10/12-F n/a n/a Clear O No O No O No 12 DgRv Clear Sockeye No Sockeye No Sockeye Slightly 29 DgRv Clear Pink No Pink No Pink Yes 57 DgRv Clear Greenling No Greenling Yes O Yes 100 DgRv Clear Chum No Chum Yes O Yes 128 DgRv Clear Sockeye No O Yes O Yes 130 DgRv Clear Chum No O Yes O Yes 152 DgRv Clear Chum No O Yes O Yes EC 4/11/12-G n/a n/a Clear O No O Yes O No 1 DgRv Clear Coho No 359 bp amplicon Yes O Yes 21 DgRv Clear Chum No Chum Yes O Yes 49 DgRv Clear Sockeye (with damage) No O Yes O Yes 75 DgRv Tinged O Yes O Yes O Yes

8 C. Monroe et al. / Forensic Science International 228 (2013) Table 1 (Continued ) Round 3 Site mg extracted 1:50 Diluted extractions Extract Color Omni Klen Taq Phusion rtth Polymerase Sample ID 94 DgRv Clear Chum No O Yes O Yes 160 DgRv Clear Sockeye No Sockeye Yes O Yes 167 DgRv Clear Pink No Pink No O Yes EC 4/11/12-H n/a n/a Clear O No O No O No Positive Control n/a n/a Clear X X X X X O Note: X indicates positive amplification, but these amplicons were not sequenced, and O reflects no amplification. polymerase to be inhibited, regardless of whether the ancient salmon amplified as described above. If the goose DNA amplified when spiked with ancient salmon DNA extract, but exhibited a noticeably dimmer band, we considered this to be slightly inhibited, and recorded it as such Experiments During the course of the experiments described below, we recognized that it is crucial that the polymerases tested here are able to amplify the ancient Goose Collective positive control alone. Otherwise, they could not be tested for inhibitors as designed in this study (described above under Section 2.5), regardless of their ability to amplify ancient salmonid. In this case, both Amplitaq Gold and Tfl failed to be able to amplify the Goose Collective positive control and were, therefore, removed from the study. In the first round of experiments, we tested Klentaq, Platinum Taq, and Pwo against one another in their ability amplify ancient salmonid from 21 extracts, and 1:10 and 1:50 dilutions of those extractions. In the second round of experiments, we tested Klentaq (the best performing polymerase in the first round of experiments), Herc II, and Vent against one another in their ability amplify ancient salmonid from an additional 21 extracts, and 1:10 and 1:50 dilutions of those extractions. In the last round of experiments, we tested Klentaq (the best performing polymerase in the first two rounds of experiments), Phusion and rtth against one another in their ability amplify ancient salmonid from a third set of 21 extracts, and 1:10 and 1:50 dilutions of those extractions. In each set of experiments the color of the full concentration and diluted DNA extractions were visually inspected against a plain white paper background and recorded (Fig. 1 and Table 1) Data analysis The success of the various polymerases in all of the experiments was based on two measures: (1) the number of positive returns of salmon species identifications, and (2) the number of extracts that were not completely affected by inhibition (in this case, slightly inhibited results were treated as successes). The number of successes was totaled in each experimental round, across the full concentration extractions and dilutions of those extracts. Two-tailed Fisher s exact tests were used to statistically evaluate differences in performance between the polymerases at the 0.05 level of probability. 3. Results and discussion Because both Amplitaq Gold and Tfl failed in their ability to amplify the Goose Collective positive control they were removed from further consideration in this study. We were particularly surprised that Amplitaq Gold failed in this experiments, as it is a widely used polymerase in adna studies (e.g. [20,47,49,60]) and commercial forensic typing kits such as Promega s Powerplex systems and Applied Biosystem s (Life Technologies) AmpFlSTR and Identifiler products. However, this is not the first reported case of Amplitaq Gold s inability to overcome PCR inhibitors. Al-Soud and Radstrom [2,3] first noted Amplitaq Gold s inefficiency against inhibitors, such as hemoglobin and humic acid, which was only improved with the addition of BSA. Eilert and Foran [15] found similar results with Amplitaq Gold being more susceptible to Ca + ions, humic acid and collagen, with no improvement against collagen in the presence of BSA. In addition Tfl was also particularly prone to PCR inhibition from skeletal remains [15]. Recent research of forensic saliva samples likewise observed a reduced efficacy of Amplitaq Gold [21]. However, this does not preclude the robustness of these polymerases in other applications. Rather, these polymerases were not effective in either fidelity or their ability to resist specific inhibitors commonly found among forensic or degraded samples especially without the presence of BSA Ability to amplify salmonid The results of positive salmon species identifications across all of the experiments are found in Table 1, summarized in Table 3, and discussed in the following section Round 1: Klentaq, Platinum Taq, Pwo In the first round of experiments, of the 21 full concentration extracts, neither Klentaq, Platinum Taq, nor Pwo were capable of amplifying salmonid from the full concentration extractions. In the case of the 1:10 dilutions of the extractions, Klentaq yielded two positive results, one chum salmon (O. keta) and one sockeye salmon (O. nerka). From the extract of sample #336, it also amplified an 183 bp (including primers) section of human DNA corresponding to chromosome 2 [NCBI Blast search [5], Accession number AC ]. This is not surprising, as the primers OST12S-F and OST12S-R ) designed by Jordan et al. [23] have been shown to be capable of amplifying human and mouse (Mus musculus), but not brown bear (Ursus arctos) or dog (Canis lupus familiaris) DNA [19]. Platinum Taq also yielded two positive results, first confirming the observation that sample #420 from DgRv-003 is a chum salmon and secondly identifying sample #401 from DgRv-003 as likely pink salmon (O. gorbuscha). The sequence exhibited a thymine (T) at nucleotide position (np) 660 and a T at np 670, relative to the rainbow trout sequence. Since the 670T has not yet been observed in contemporary [23] or ancient salmonids [19], it likely represents post-mortem genetic damage (the most common form of miscoding lesions leads to artificial C>T transitions [18]). Nevertheless, the sequence exhibits no other known shared-derived mutations that would cause the samples to be identified as any Pacific salmonid species besides pink. Pwo was incapable of amplifying any salmonid DNA in the 1:10 dilutions. In the case of the 1:50 dilutions of the extractions, Klentaq yielded the same two positive results as the 1:10 dilutions, but did not amplify any human contamination from sample #336, whereas Platinum Taq yielded only one positive result and amplified human DNA in one PCR negative control (matching a human BAC clone, according to an NCBI Blast search [5], Accession number AC ). Pwo was again incapable of amplifying any salmonid DNA from these dilutions. Across these extracts and their dilutions, there was no difference in positive returns between Klentaq (4/63 successes) and Platinum Taq (3/63 successes) (p = 1.00). Moreover, both polymerases were used to successfully identify the species of a sample that the other did not produce: (1) sample #405 was identified as sockeye by Klentaq, but not Platinum Taq, and (2) sample #401 as pink by Platinum Taq, but not by Klentaq. Pwo

9 150 C. Monroe et al. / Forensic Science International 228 (2013) Table 2 Specification of PCR components and conditions of the salmonid reactions conducted in this study. All reactions were 15 ml in volume, employed primers OST12S-F and OST12S-R [23], and were run for 60 cycles. The PCR reactions used to test for inhibition were set-up as described for salmonid, employed primers BSP1 and GooseR [58], but were spiked with 1.5 ml of Goose Collective ancient positive control (resulting in total volume of 16.5 ml). Sixty cycles of touch-down PCR, during which the initial annealing temperature of 60 8C was reduced by 0.1 8C each round, were conducted according to Wilson et al. [58]. Amplitaq Gold Herculase II Omni Klentaq Phusion Platinum Taq Pwo rtth Tfl Vent PCR components dntps 0.32 mm 0.32 mm 0.32 mm 0.32 mm 0.32 mm 0.32 mm 0.32 mm 0.32 mm 0.32 mm 1X Phusion 1 HF Buffer 1X PCR Buffer 1X PCR Buffer 1X XL Buffer II 1X Reaction Buffer 1X ThermoPol Reaction Buffer 1X Omni Klentaq Reaction Buffer (including final concentration of 3.5 mm MgCl2) Buffer 1X PCR Gold Buffer 1X Herculase II Reaction Buffer (including final concentration of 2 mm MgCl2) Cofactor 1.5 mm MgCl2 In buffer In buffer 1.5 mm MgCl2 1.5 mm MgCl2 1.5 mm MgSO4 1.5 mm Mg(OAc)2 1.5 mm MgSO4 1.5 mm MgSO4 Primers 0.24 mm each 0.24 mm each 0.24 mm each 0.24 mm each 0.24 mm each 0.24 mm each 0.24 mm each 0.24 mm each 0.24 mm each Polymerase 0.3 U 0.3 U 0.3 U 0.3 U 0.3 U 0.3 U 0.3 U 0.3 U 0.3 U Template DNA 1.5 ml 1.5 ml 1.5 ml 1.5 ml 1.5 ml 1.5 ml 1.5 ml 1.5 ml 1.5 ml PCR reactions Initial Denaturing 95 8C/5 min 95 8C/2 min 94 8C/3 min 98 8C/30 s 94 8C/3 min 94 8C/3 min 94 8C/1 min 94 8C/30 s 958C/2 min Denaturing 95 8C/15 s 95 8C/15 s 95 8C/15 s 98 8C/15 s 94 8C/15 s 94 8C/15 s 94 8C/15s 95 8C/15s 958C/15 s Annealing 55 8C/15 s 55 8C/15 s 55 8C/15 s 55 8C/15 s 55 8C/15 s 55 8C/15 s 55 8C/15s 55 8C/15s 558C/15 s Extension 72 8C/15 s 72 8C/15 s 68 8C/15 s 72 8C/15 s 72 8C/15 s 72 8C/15 s 72 8C/15s 74 8C/15s 728C/15 s Final Extension 72 8C/3 min 72 8C/3 min 68 8C/3 min 72 8C/3 min 72 8C/3 min 72 8C/3 min 72 8C/3 min 74 8C/3 min 72 8C/3 min yielded zero successes in amplifying salmonid in this set of experiments Round 2: Klentaq, Herc II, Vent In the second round of experiments neither Klentaq, Herc II, nor Vent were capable of amplifying salmonid from the full concentration extracts. In the case of the 1:10 dilution of these extracts: (1) Klentaq returned positive results from 2/21 extracts (9.5%), (2) Herc II returned positive results from 1/21 samples (4.8%), and (3) Vent was not capable of amplifying salmonid. In the case of the 1:50 dilution of these extracts Klentaq returned positive results from 12/21 extracts (57.1%). One of the positive results, sample #56 from site DgRv-006 was identified as sockeye salmon, but exhibited a mix of cytosine (C) and T at np 650 relative to the rainbow trout reference sequence. This is likely the result of sequencing a mix of damaged and undamaged template molecules. Klentaq also produced a double-banded amplicon from sample #72. The lower band appeared to be approximately the correct size, with the upper band being 20 bps larger. The sequence returned from these amplicons was unreadable and, thus, not counted toward success for Klentaq. Herc II returned positive results from 6/21 extracts (28.6%). Vent was again not capable of amplifying salmonid from any of the 1:50 dilutions. Across these extracts and their dilutions, Klentaq yielded the highest return on salmon species identifications (14/63 successes), however it was not statically greater than the return from Herc II (7/63) (p = ). Yet, in no case was Herc II used to make a species identification that was not made by Klentaq. Conversely, the species of six salmon vertebrae were uniquely determined with Klentaq in this round of experiments Round 3: Klentaq, Phusion, rtth In the third round of experiments, of the 21 full concentration extracts, neither Klentaq, Phusion, nor rtth were capable of amplifying salmonid. In the case of the 1:10 dilution of these extracts, Klentaq was used to identify 11/21 samples (52.4%) as salmonids. In addition, sample #57 produced a sequence that is seven mutational steps away from a number of different fishes according to an NCBI Blast search [5]. However, this sample is tentatively typed as Pacific greenling (Hexagrammos sp.) and this observation is, thus, treated as a positive result (totaling 12/21, or 57.1%). Phusion returned positive results from 3/21 samples (14.3%) and rtth was not capable of amplifying salmonid from any of the 1:10 dilutions. In the case of the 1:50 dilution of these extracts, Klentaq was used to identify 18/21 samples (85.7%) as being salmonids. Sample #49 was identified as sockeye salmon, but exhibits additional mutations at nps 690A and 703A relative to the rainbow trout sequence. As Klentaq was used to make this same species identification from the 1:10 dilution of this extract and that sequence did not exhibit the 690A and 703A mutations, it is concluded that these mutations are due to damaged template molecules detected in the 1:50 dilution. Since these artifactual mutations appear without competing secondary peaks, it suggests that that PCR was initiated from very few molecules. Given that this extract is five times more dilute than the 1:10 diluted extract, this observation is not unreasonable to expect. Sample #57 produced again the Pacific greenling-like sequence, identical to that observed in the 1:10 diluted extract, bringing the total success for Klentaq to 19/21 (90.5%). Phusion returned positive results from 10/21 samples (47.6%, including samples #57 with an identical Pacific greenling-like sequence). In addition it produced a 359 bp amplicon (including primers) from sample #1, which was unidentifiable according to a NCBI Blast search [5] and, thus, not treated as a success. rtth returned positive results from 3/21 of the 1:50 dilute extracts (14.3%).

10 C. Monroe et al. / Forensic Science International 228 (2013) Table 3 Summary of result presented in Table 1. Polymerase Full concentration 1:10 Dilutions 1:50 Dilutions Total Count (of 21) % Count (of 21) % Count (of 21) % Count (of 63) % Positive species identifications Round 1 Klentaq Platinum Taq Pwo Round 2 Klentaq Herc II Vent Round 3 Klentaq Phusion rtth Effects of inhibition Round 1 Klentaq Platinum Taq Pwo Round 2 Klentaq Herc II Vent Round 3 Klentaq Phusion rtth Across these extracts and their dilutions, Klentaq (31/63 successes) outperformed Phusion (13/63) (p = ) and rtth (3/63) (p = ) in its use for species identification among these 21 vertebrae Ability to overcome inhibition The ability of the polymerases to overcome inhibition across all of the experiments are found in Table 1. These data are summarized in Table 3 as percent inhibited, and discussed below as such Round 1: Klentaq, Platinum Taq, Pwo Klentaq and Platinum Taq were inhibited by all 21 full concentration extracts, whereas Pwo was inhibited by 18/21 (85.7%) of the extracts. We note also that the extraction negative control (1/13/12-A) that accompanied the second batch of extractions was inhibited to all three polymerases. This observation likely stems from the unintentional carry-over of some chemical through the extraction process (e.g. alcohol and/or phenol). This was the only extraction negative control that behaved in this manner. After diluting the samples to 1:10, Klentaq was inhibited by 42.9% (9/21) of the extracts and showed to be slightly inhibited by extraction negative control 1/13/12-A, which did not occur with the other two polymerases. Platinum Taq was inhibited by 85.7% (18/21) and Pwo was inhibited by 38.1% of the 1:10 diluted extracts. At 1:50 dilutions Klentaq was the least inhibited (4.8%), followed by Pwo (14.3%) and Platinum Taq (42.9%). Across all extracts and dilution series, Klentaq was significantly less inhibited (49.2%) in this round of experiments than Platinum Taq (76.2%) (p = 0.003). Pwo, was less inhibited (46%) than Platinum Taq (p = ). While Pwo performed no differently than Klentaq (p = ), Klentaq out performed Pwo in its return of species identifications (as described under Section 3.1.1) Round 2: Klentaq, Herc II, Vent All full concentration extracts were deemed to be inhibited for all polymerases. Klentaq and Herc II were inhibited by 95.2% of extracts diluted 1:10, while Vent polymerase was 100% inhibited. At 1:50 dilutions, Klentaq was inhibited by 28.6% of extracts. The performance of Herc II did not improve with 95.2% of PCR reactions were inhibited, albeit the single 1:50 diluted extract (#155) not inhibited was different than the extract (#37) that was slightly inhibited at a 1:10 dilution. The performance of Vent improved with 71.4% of the 1:50 extractions determined to be inhibited. Across all of the experiments, Klentaq was less inhibited (74.6%) than Herc II (96.8%) (p = ) and Vent 90.5% (p = ) Round 3: Klentaq, Phusion, rtth Full concentration extracts caused PCR inhibition for all reactions for all polymerases. In addition, rtth was inhibited by one of the extraction negative controls (EC 2/10/12-F). Klentaq was inhibited by 38.1% of the 1:10 diluted extracts. Both Phusion and rtth were 100% inhibited by these same extracts. At 1:50 dilutions, Klentaq was inhibited by 4.8% of the samples. In contrast, 76.2% of the Phusion and 90.5% of rtth reactions failed to amplify the 1:50 diluted extracts. Overall Klentaq proved to be less susceptible to inhibition (47.6%) compared with Phusion (92.1%, p = ) and rtth (96.8%, p = ). Considering the whole of the results reported here, it is important to note that our experimental approach to test for inhibition produced results that require further assessment to account for the following. First, in round one of experiments, 1:10 dilute extracts that amplified successfully for salmonid (i.e., sample #420 with Klentaq and samples #401 and #420 with Platinum Taq) demonstrated to be inhibited against amplifying the Goose Collective ancient positive control. Across the extractions that ultimately yielded 75 positive species identifications, 19 of them (25.3%) were also determined to be inhibited. This was

11 152 C. Monroe et al. / Forensic Science International 228 (2013) observed in 8/13 (61.5%) successes using Phusion, 6/7 (85.7%) using Herc II, 2/3 (66.6%) using Platinum Taq, 2/49 (4.1%) using Klentaq, and 1/2 (50%) using rtth. While the outcomes of our experimental design suggest further tests are warranted, the results are still instructive to the extent that it was designed for evaluating the efficacies of the polymerase tested here. With one exception (i.e., sample #337 extract with Pwo) all of the full concentration extracts were determined to be inhibitory to all of the polymerases tested in this study. That 8/63 (12.7%) of these extracts were visibly colorless demonstrates again that a pigment free extract does not guarantee that an extract is free of inhibitors. Conversely, all 55 full concentration extracts exhibiting coloration, ranging from being very lightly tinged to dark brown in color, were inhibited, demonstrating that any degree of pigmentation in an extract is a good predictor of co-extracted PCR inhibitors. 4. Conclusions Overall, Omni Klentaq LA outperformed the other 8 polymerases in two respects: (1) its successful use in genetic species identification of these vertebrae, and (2) its ability to amplify an ancient DNA positive control when spiked with a volume of potentially inhibited extract from the vertebrae. Therefore, Klentaq appears particularly well suited to situations were DNA is coextracted with PCR inhibitors from bone samples. This is consistent with previous studies using Klentaq, which emphasized its ability to overcome inhibitors naturally occurring from blood, soil, and feces [31,32] as well as from added concentrations of fulvic and humic acids [40]. We temper this recommendation with the following observations that: (1) Klentaq did not statistically outperform either Platinum Taq or Herc II in positive species identifications (see Section 3.1.2), and (2) in one instance Platinum Taq was used to make a species identification that was not achieved with Klentaq (see Section 3.1.1). However, we did not test the additive buffers that the manufacturers of Klentaq (DNA Polymerases Inc.) suggest will further enhance PCR amplification [62]. Additionally at the time of writing this manuscript, Omni Klentaq LA is the most cost effective choice at $0.43/U (when 625U are purchased) compared to Platinum Taq ($0.86/U, when 500U are purchased) and Herc II ($0.46, when 500U are purchased). Acknowledgments This project was supported by Award No DN-BX-K008 awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. 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