ORIGINAL ARTICLES
BIOPRESERVATION AND BIOBANKING Volume 12, Number 6, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/bio.2014.0036
DNA Storage under High Temperature Conditions Does Not Affect Performance in Human Leukocyte Antigen Genotyping via Next-Generation Sequencing (DNA Integrity Maintained in Extreme Conditions) Shana L McDevitt,1 Michael E Hogan,2 Derek J Pappas,1 Lily Y Wong,2 and Janelle A Noble1
Background: Stable dry-state storage of DNA is desirable to minimize required storage space and to reduce electrical and shipping costs. DNA purified from various commercially available dry-state stabilization matrices has been used successfully in downstream molecular applications (e.g., quantitative polymerase chain reaction [qPCR], microarray, and sequence-based genotyping). However, standard DNA storage conditions still include freezing of DNA eluted in aqueous buffers or nuclease-free water. Broad implementation of dry-state, long-term DNA storage requires enhancement of such dry-state DNA stabilization products to control for temperature fluctuations at specimen collection, transit, and storage. This study tested the integrity of genomic DNA subjected to long-term storage on GenTegraTM DNA stabilization matrices (GenTegra LLC, Pleasanton, CA) at extreme conditions, as defined by a 4-year storage period at ambient temperature with an initial incubation for 7 months at 37C, 56C, or ambient temperature. Subsequently, purified DNA performance and integrity were measured by qPCR and next-generation sequencing (NGS)-based human leokocyte antigen (HLA) genotyping. Results: High molecular weight genomic DNA samples were recovered from the GenTegra product matrix and exhibited integrity comparable to a highly characterized commercial standard under assessment by qPCR. Samples were genotyped for classical HLA loci using next generation sequencing-based methodolgy on the Roche 454 GS Junior instrument. Amplification efficiency, sequence coverage, and sequence quality were all comparable with those produced from a cell line DNA sequenced as a control. No significant differences were observed in the mean, median, or mode quality scores between samples and controls ( p ‡ 0.4). Conclusions: Next generation HLA genotyping was chosen to test the integrity of GenTegra-treated genomic DNA due to the requirment for long sequence reads to genotype the highly polymorphic classical HLA genes. Experimental results demonstrate the efficacy of the GenTegra product as a suitable genomic DNA preservation tool for collection and long-term biobanking of DNA at fluctuating and high temperatures.
Introduction
T
he preservation of purified DNA, during long-term biobanking or shipping, has become a major concern in the field of applied genetics and public health screening.1 For cryogenic preservation of high-value DNA samples, mechanical and liquid nitrogen storage for biobanking, or dry ice and chemical packs for shipping are proven technologies. However, cryogenic preservation methods introduce substantial cost and some measure of risk, such as chemical refrigerant failure during shipping or power loss. Refrigeration-free biosample preservation could dramatically reduce the cost and risk associated with cryogenic preservation. Consequently, refrigeration-free approaches to purified DNA preservation 1 2
have been developed that preserve purified DNA via desiccation in the presence of added chemical stabilizers. Successful dry-state DNA preservation at lab ambient temperatures for many months has been reported, resulting in DNA of sufficient quality to support routine laboratory analyses such as quantitative polymerase chain reaction (qPCR), sequence based typing (SBT), and microarrays.2–5 However, one compelling application of refrigeration-free DNA stabilization is for collection of samples in the field, where the conditions are likely to be far more harsh than in the laboratory setting. Here we assess the performance of GenTegraTM (GenTegra LLC, Pleasanton, CA) under conditions that emulate ‘‘worst-case’’ ambient temperature storage or shipping. Using high-quality DNA purified from freshly collected blood, DNA
Children’s Hospital Oakland Research Institute, Oakland, California. GenTegra LLC, Pleasanton, California.
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was subjected to room temperature storage for 4 years, which included a 7-month initial treatment at elevated temperatures (37C or 56C) to reflect conditions that could be encountered during sample collection, shipping, or long-term DNA banking. Integrity of the DNA stored under these adverse conditions was compared to that of DNA stored at 4C. Performance of the DNA stored under adverse conditions was tested by its use in 2.5 kb quantitative polymerase chain reaction (qPCR) and in next-generation sequencing (NGS) based human leukocyte antigen (HLA) genotyping. HLA genotyping is complicated by extreme HLA sequence polymorphism; 12,242 HLA alleles have been documented as of October, 2014 (http://hla.allelles.org).
Methods DNA storage and purification Genomic DNA was extracted from 12 mL fresh venous blood samples collected in EDTA tubes from two different donors (Memorial Blood Centers, St. Paul, MN). Samples were collected by 4 pm, shipped overnight at 4C, stored at 4C upon receipt, and purified within 24 hours thereafter. DNA was purified using QIAamp DNA Blood Midi Kit (Qiagen Corp, Hilden, Germany). DNA concentration was measured by both Nanodrop UV/VIS spectrospcopy (Thermo Scientific, Waltham, MA) and Qubit fluorometry (Life Technologies, Carlsbad, CA). A total of 18 aliquots of DNA, 17 from one blood sample and one from the second blood sample, were arbitrarily split into two groups (C1-C9 and D1-D9) and stored in three plates. 3 mg (C samples) or 5 mg (D samples) of purified DNA sample (in TE buffer) were applied per well of a microplate containing the GenTegraTM DNA stabilization matrix (GenTegra LLC, Pleasanton, CA). Each plate contained three samples from each group. Each DNA sample in GenTegra was then air-dried per the manufacturer’s recommendation; the wells were sealed and then stored as follows for 4 years. One plate was heated to 37C in an oven for 7 months, then transferred to a warehouse for ambient temperature storage (*25C) for 3 years, 5 months (Fig. 1A). A second plate was heated to 56C in an oven for 7 months, then transferred to a warehouse for ambient temperature storage (*25C) for 3 years, 5 months (Fig. 1A). A third plate was kept in a warehouse at *25C for the entire 4 year time period. Subsequent to the 4-year storage, the 3 mg (C group) and 5 mg (D group) samples were rehydrated with 20 mL and 33 mL water, respectively. 100 ng of each DNA sample (as assessed by NanoDrop) was then run on a 0.8% agarose gel with ethidium bromide for 20 min on an E-gel base at 75V (Life Technologies, Carlsbad, CA) (Fig. 1B).
qPCR assessment of DNA quality 25 ng of each stored DNA sample was subjected to qPCR targeting a 2.5 kb region of mitochondrial DNA. Each reaction also included 250 nM of forward primer mt2568-F (5¢-CGCACGGACTACAACCACGAC-3¢), reverse primer mt2568-R (5¢-CTGTGGGGGGTGTCTTTGGGG-3¢), and FastStart SYBR Green with ROX (Roche Applied Sciences, Indianapolis, IN). qPCR was performed on the Applied Biosystems 7500 Fast Real-time PCR System (Life Technologies, Foster City, CA). The thermal cycling conditions were: 1) 95C for 10 min; 2) 40 cycles of 95C for 30 sec, 68C for
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1 min, 70C for 2 min, 3) 77C for 30 sec, followed by a dissociation step post-cycling. qPCR was performed in triplicate and the average CT value for each sample was plotted in Figure 1C, relative to CTs obtained for 25 ng of a highly validated control DNA, which was stored in the laboratory at 4C (PN 11691112001; Roche Applied Sciences, Indianapolis, IN).
HLA genotyping using next-generation sequencing (Roche 454) Amplicons (PCR products) were generated from purified genomic DNA (gDNA) from all eighteen samples, six samples per condition, stored dry with the GenTegraTM product and from a pre-purified and commercially supplied cell line human gDNA sample stored at - 20C (HAR: International Histocompatibility Workshop #9149) as a positive control (PTC). Fourteen amplicons were generated from each DNA sample using commercially available Roche 454 HLA GType Medium Resolution (MR) and High Resolution (HR) HLA fusion primer assay plates (Roche 454, Branford, CT) (6). Roche 454 GType MR assay plates include fusion primers to amplify HLA-A, -B, and -C exons 2 and 3 (exon 2 amplicons lengths are 560 base pair (bp), 446 bp, and 490 bp, respectively; exon 3 amplicon lengths are 495 bp, 425 bp, and 673 bp, respectively), HLA-DQB1 exon 2 (389 bp), and HLADRBX (366 bp); where X includes HLA-DRB1 and all additional DRB genes. Roche 454 GType HR assay plates include fusion primers to amplify HLA-A, -B, and -C exons 4 (740 bp, 464 bp, and 443 bp, respectively), HLA-DQB1 exon 3 (469 bp), HLA-DQA1 exon 2 (488 bp), and HLADPB1 exon 2 (430 bp). Roche 454 fusion primers consist of a 10-base multiplex ID (MID) tag, flanked by a locus-specific primer on the 3¢ end, and an ‘‘A’’ or ‘‘B’’ 454-specific primer adaptor sequence on the 5¢ end.6,7 The MID tag serves as a barcode for sample identification by data analysis programs.7,8 PCRs were performed in a 25 mL volume. Each reaction contained 20 ng of genomic DNA, 2 units of AmpliTaq Gold Polymerase (Life Technologies, Carlsbad, CA), 1X AmpliTaq PCR Gold Buffer (Life Technologies, Carlsbad, CA), 1.5 mM AmpliTaq Gold MgCl2 Solution (Life Technologies, Carlsbad, CA), 0.3 mM of PCR Grade Nucleotide Mix (Roche Applied Sciences, Indianapolis, IN), and 10% Amresco brand glycerol (Amresco, Solan, OH). Genomic DNA and PCR mastermix were aliquoted directly into the Roche 454 GType primer assay plates and PCR was performed using the thermal cycler GenAmp PCR 9700 system (Life Technologies, Foster City, CA). Cycling conditions were: 1) 94C for 5 min, 2) 31 cycles of 94C for 15 sec, 62C for 15 sec, and 72C for 30 sec, 3) 72C for 8 min. PCR products were visualized with the Advanced Analytical 96-Capillary Gel Electrophoresis based Fragment Analyzer (Advanced Analytical Technical Instruments, Ames, IA). PCR products were pooled, based on Quant-iT PicoGreen dsDNA reagent concentration data (Life Technologies, Carlsbad, CA), into a single, eqimolar amplicon library, purified with Agencourt AMPure XP beads (Beckman Coulter, Pasadena, CA), re-quantified using Quant-iT PicoGreen dsDNA reagent, and mixed with 454 capture beads (Roche 454, Branford, CT) after dilution to 1 · 106 molecule per microliter concentration. Individual HLA amplicon molecules within the library were clonally amplified and bound to 454 capture beads in emulsion PCR.6,7 DNA bound beads were enriched and deposited onto a single region 454 Titanium
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FIG. 1. (A) DNA storage timecourse. Purified gDNA sample was treated with GenTegraTM DNA stabilization matrix and stored at one of three temperatures for 7 months, followed by an incubation at ambient temperature (*25C) for a total period of 4 years. (B) Agarose gel assessment of gDNA quality. Subsequent to storage, the samples were rehydrated and visualized using agarose gel electrophoresis. A (collapsed) DNA band with an apparent molecular weight of 40 kb indicates that the average duplex DNA strand length for such samples is in excess of approximately 40 kb. (C) PCR assessment of DNA quality. qPCR was performed on an aliquot of each stored DNA sample employing a 2.5 kb region of mitochondrial DNA as the target. The average qPCR CT values for each sample are plotted relative to CTs obtained for control DNA.
PicoTiter plate (Roche 454, Branford, CT).6,7 Molecules were pyrosequenced on the Roche 454 GS Junior system (Roche 454, Branford, CT).6,7
Data analysis and statistics FASTA formatted sequencing reads were filtered based on alignment to an International Immunogenetics Information System (IMGT) HLA reference database using the Roche 454 Amplicon Variant Analyzer (Roche 454, Branford, CT).9 Sequencing reads from a subsequent FASTA file were analyzed with SCORETM software and Conexio AssignTM ATF HLA genotyping softwares. 454 GS Junior data processing tools, SFFfile and SFFinfo, were used to parse files and to extract sequencing adaptor trimmed and untrimmed sample-specific base calling quality metric scores
using manufacturer protocols. Raw base quality scores for all sequencing reads, from the original SFF file, were then analyzed per sample in reference to controls in R (http:// www.r-project.org). Wilcoxon rank sum tests were used to test for significance between sample and control means; significance threshold was set at p £ 0.05.
Results and Discussion Stored genomic DNA maintained integrity The integrity of eighteen gDNA samples recovered from the GenTegra product matrix was confirmed. As seen in Figure 1B, a limiting, high molecular weight DNA band, with apparent size greater than 40 kilobases, was detected with 0.8% agarose electrophoresis for all samples, indicating that,
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upon recovery from the GenTegra product, DNA chain length remains in the high molecular weight range expected for genomic DNA samples.
qPCR assay validated mitochondrial DNA integrity The quality of DNA obtained after long-term dry-state storage was assessed by an in-house qPCR assay targeting a 2.5 kb region of human mitochondrial DNA. The target size is approximately 10 times longer than that typically employed for qPCR and, thus, is capable of assessing DNA integrity by PCR in the presence of lesions at a density as low as 0.5 per kilobase. A mitochondrial DNA target was chosen to measure DNA quality as a surrogate for all other gene quality, in the same way that ribosomal RNA is used routinely as a surrogate for total RNA quality (e.g., the well known rRNA Bioanalyzer ‘‘RIN’’ score for RNA). Detailed instructions for reproducing this assay are available as a white paper, which may be obtained on the GenTegra website, at no cost, ([email protected]). In this assay, CT values obtained by qPCR were compared to those obtained from a highly characterized commercial human gDNA standard (PN 11691112001; Roche Applied Sciences, Indianapolis, IN). CT values obtained for the gDNA samples stored in GenTegra were found to be in the range of 19–20 cycles, independent of storage condition. CT values for all stored samples (Fig. 1) were found to be approximately three cycles fewer than the un-stored, control DNA, which was kept at 4C. Since DNA damage, or the presence of contaminants would increase, rather than decrease CT values, the qPCR data in Figure 1C are consistent with the gel electrophoresis data of Figure 1 and demonstrate that the DNA samples stored in the GenTegra matrix are at least as intact as freshly-isolated (never dried) control DNA.
Stored genomic DNA amplifies for troublesome HLA loci All eighteen samples and the positive control cell-line DNA (Fig. 2: PTC) amplified successfully for each of the target HLA loci. Amplicons were visualized by 2% agarose gel electrophoresis and by capillary gel electrophoresis. Nonspecific amplification was not observed (Fig. 2). HLA-A exon 4 amplicons are the largest in the set of fourteen (740 bp). Figure 2 displays capillary gel electrophoresis trace overlays of HLA-A exon 4 amplicons from all 18 samples and postive control PCR products around the target size, 740 bp. Amplicon concentrations ranged from 10.5 nanograms per microliter (ng/mL) to 17.2 ng/mL with a standard deviation of – 2.23 ng/mL for the longest HLA amplicon (HLA-A exon 4) as compared to postive control HLA-A exon 4 amplicon concentrations, 13.2 ng/mL and 13.7 ng/mL (SD = 0.23 ng/mL). In general, amplicon concentrations ranged from 5.56 ng/mL to 28.3 ng/mL for the experimental amplicons, as compared to control amplicon concentration ranges of 6.64 ng/mL to 24.4 ng/mL. The lowest concentrations in both groups were observed for HLA-C exon 3 (673 bp), which was the most difficult locus to amplify, possibly due to inefficient primer annealing.
Stored genomic DNA effectively used to genotype HLA Three sequencing runs were performed in total. Each run included all HLA amplicons for six experimental samples
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and one control. The same control library was sequenced in each run to control for run-to-run variation. The runs showed some variability in the number of quality filter passed sequencing reads, 126,665 reads, 110,766 reads, and 134,317 reads, respectively (454 software manual). All three sequencing runs, however, far exceed the 70,000 quality passed filter read benchmark set by Roche 454.10 As expected, identical HLA genotypes were assigned for 17 out of 17 identically sourced samples. The single sample sourced from a different individual produced a different genotype. Control genotypes were identical for each of the HLA loci among the three sequencing runs, demonstrating the consistency of the sequencing and genotype calling processes. 111 of 126 total genotypes (18 samples sequenced for 7 genes) were assigned without the need for manual user editing in the Conexio AssignTM ATF HLA genotyping software. Minimal user edits to remove sequence containing obvious sequencing error were necessary to assign genotypes to 11 out of the initial unassigned 15 genotypes. The remaining 4 genotypes cannot be assigned due to mid HLAB exon 3 sequencing error or cannot be assigned without excluding HLA-A exon four data. However, these failed assignments are attributable to limitations in 454 sequencing chemistry rather than the initial DNA integrity. 454 sequencing read length limitations. Roche 454 pyrosequencing chemistry delivers the longest read lengths of all clonally based sequencing platforms,10,11 with the ability to generate high quality reads 400 bp in length on either the 454 GS FLX or 454 GS Junior platforms.10 HLA-A exon 4 and HLA-C exon 3 amplicons, 740 and 673 bp, respectively, exceed the size of exons and far exceed the length limitations of the sequencing chemistry. For both amplicons, the exon lies near the upstream end; thus, the quality of the exon sequence from the forward sequence reactions was higher than that from the reverse sequences. In the reverse sequence reactions, the exon sequence was at the end of the read, and DNA sequencing tends to decrease in quality with read length.10 Figure 3 shows that, within this data set, all untrimmed sequencing reads maintain a high level of base calling quality (Q), where Q0 and Q40 are the minimum and maximum quality score range, respectively, until read lengths approach 430 bases. At this point, the ability of the sequencing software to call bases accurately steadily decreases. Moreover, when comparing the quality scores from reads at nucleotide position 423 (Fig. 3D), distribution of quality scores was not different for the test samples as compared to the control samples (adjusted p = 1). HLA-A exon 4 and HLA-C exon 3 reverse reads for all samples in the second sequencing run showed evidence of increased sequencing error at the 3¢ end. Failure to generate reverse direction sequences long enough to capture full HLA-A exon 4 and full HLA-C exon 3 sequence was also observed in the control sample from the second sequencing run. Only HLA-A genotypes for samples C8 and D3 were left unassigned. Genotypes for these samples were determined, albeit with increased ambiguity, upon the exclusion of all HLA-A exon 4 sequences. 454 sequencing homopolymer detection limitations. Genotype assignments for the 17 identically sourced samples were used to distinguish sequencing error from genomic variation where failed genotype assignments were made. Five HLA-B genotypes showed evidence of a base calling discrepancy
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FIG. 2. HLA-A exon 4 amplicon fragment analyzer trace overlay. Capillary electrophoresis was performed to visualize the HLA-A exon 4 amplicon size range distribution among all samples and controls. Upper and lower markers used for size determination are visible at positions of 35 base pairs (bp) and 1500 bp, respectively. Samples represented within each frame were amplified together on a PCR assay plate with the cell-line control (PTC) noted in the frame.
between the forward and reverse sequence reads from the same chromosome near the center of exon 2 near a high G-C rich region in the template strands. Regardless of input DNA quality, homopolymers (i.e., multiple molecules of the same nucleotide) can be problematic for 454 chemistry.10 454 sequencing data are represented by a series of intensity values, or a flowgram, for iterative deoxynucleotide flows during which multiple nucleotide incorporations may occur in the absence of a 3¢ chain terminator which can lead to substitution or deletion error.12 Nucleotide sequences off-set by one base in either direction cannot be aligned with confidence to a HLA reference. Three of the five samples with noted sequencing error (C1, C2, and C3) were in the 56C storage group while the remaining two samples (D2 and D3) were from the ambient
temperature storage group. No other replicates showed evidence of a similar issue with HLA-B sequence. Each of the noted samples shows a deletion in the reverse reads for both alleles at base position 389, which directly follows the G-C rich region noted above. This region is found in all HLA-B alleles described to date. Notably, the forward reads for these alleles per sample had no evidence of sequencing error. However, D7, D8, and D9, also from the 56C worstcase scenario treatment group, all showed no evidence of sequencing error in either forward or reverse reads, suggesting that the errors reflected in samples C7–C9, D2, and D3 were not due to the storage conditions or the subsequent quality of genomic DNA but were rather attributable to reduced sequencing run quality. Vandenbroucke et al. suggested that every amplicon has its own error profile dependent upon its originating sequence.10,13 Because all
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FIG. 3. Quality score distributions. (A–C) Average quality scores across all reads at indicated nucleotide position for each sample. (A) from sequencing run 1; (B) from sequencing run 2; and (C) from sequencing run 3. (D) Box plots of the quality score distributions for all reads at nucleotide position 423. Replicates were averaged and median centered.
HLA-B alleles contain the problematic sequence motif, which has a high error profile, the reduced sequencing quality of run 2, compared to that of runs 1 and 3, led to the apparently high standard error for this data set.
High molecular weight genomic DNA purified from the GenTegra product matrix was successfully used to genotype the most polymorphic genes in the human genome. Next generation HLA genotyping was chosen to test the integrity of GenTegra product matrix treated genomic DNA due to
Stored sample sequencing quality scores mirrored controls Quality scores are assigned to each base for each sequencing read by the 454 sequencing software, representing the log-probability that a base was not an overcall.10,12,14 These scores are reported in a FASTA-like format. 454 base quality scores are represented by Phred equivalent quality scores (Q = - 10 * log10 (error rate)) ranging from Q0–Q40, where a Q30 score represents a base-call with a probablility of 1/1000 of being incorrect while a Q40 score represents a 1/104 chance of being incorrect, in other words, representing a 99.99% accuracy rating.10,15 No significant differences were observed ( p ‡ 0.4) in the mean, median, or mode quality scores between samples and control (Fig. 4). Quality score results suggest that gDNA purified from the GenTegra product stored under all treatment conditions behaves similarly to cell line gDNA stored in best-case conditions at - 20C.
Conclusions Taken together, consistency of HLA genotypes from identically sourced samples and lack of statistically significant difference in average quality score for stored samples compared to controls, validate the integrity of gDNA purified from the GenTegra product matrix after all tested treatment conditions, including the worst-case, 56C group.
FIG. 4. Quality score average comparisons. For each set of conditions, averages (mean, median, mode) were measured across all reads within a replicate and all sample replicates (n = 3).
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the requirment for high-quality, long, clonal sequencing reads. Results of this test demonstrate the efficacy of the product as a suitable genomic DNA preservation tool for collection and long-term biobanking of DNA at fluctuating and high temperatures. Thus, implementation of the GenTegra product matrix should greatly reduce sample storage cost without compromising data integrity. This 4-year pilot study was performed primarily on aliquots of a sample from a single individual, plus a single sample from a second individual included to control for sample-specific effects. All test samples were stored dry in the GenTegra product. Planned follow-up studies will include both samples from more individuals, as well as samples for direct comparison of dry storage with traditional liquid storage methods, both at 4C and at - 20C. Given the excellent quality of the DNA after long-term dry storage reported here, the expectation is that future studies will show that this method is at least comparable, and perhaps superior, to liquid storage in terms of quality of resulting DNA. The combination of the excellent DNA quality shown in this report with the substantial cost savings due to nonrefrigeration of samples creates a compelling argument for dry state storage, especially for projects involving large numbers of samples.
Author Disclosure Statement SLM, DJP, and JAN are all employees of Children’s Hospital Oakland Research Institute. MEH and LYW were employees of IntegenX during the duration of the project and are now employed by GenTegra, LLC. Funding for this work was provided by IntegenX Corp. (Pleasanton, CA, USA), and, in part, by NIH grant DK61722 (J.A.N.).
References 1. Vaught JB, Henderson MK. Biological sample collection, processing, storage and information management. IARC scientific publications. 2011;163:23–42. 2. Wan E, Akana M, Pons J, et al. Green technologies for room temperature nucleic acid storage. Curr Issues Mol Biol 2010;12:135–142. 3. Byrnes S, Fan A, Trueb J, et al. A portable, pressure driven, room temperature nucleic acid extraction and storage system for point of care molecular diagnostics. Anal Methods 2013;5:3177–3184.
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4. Lee SB, Clabaugh KC, Silva B, et al. Assessing a novel room temperature DNA storage medium for forensic biological samples. Forensic Sci Intl Genet 2012;6:31–40. 5. Frippiat C, Zorbo S, Leonard D, et al. Evaluation of novel forensic DNA storage methodologies. Forensic Sci Intl Genet 2011;5:386–392. 6. De Santis D, Dinauer D, Duke J, et al. 16(th) IHIW: review of HLA typing by NGS. Intl J Immunogenet 2013;40: 72–76. 7. Erlich HA, Valdes AM, McDevitt S, et al. Next generation sequencing reveals the association of DRB3*02:02 with type I diabetes. Diabetes 2013;62:2618–2622. 8. Erlich H. HLA DNA typing: Past, present, and future. Tissue Antigens 2012l;80:1–11. 9. Robinson J, Halliwell JA, McWilliam H, Lopez R, Parham P, Marsh SG. The IMGT/HLA database. Nucleic Acids Res 2013;41:D1222–1227. 10. Niklas N, Proll J, Danzer M, Stabentheiner S, Hofer K, Gabriel C. Routine performance and errors of 454 HLA exon sequencing in diagnostics. BMC Bioinformat 2013; 14:176. 11. Shokralla S, Spall JL, Gibson JF, Hajibabaei M. Nextgeneration sequencing technologies for environmental DNA research. Mol Ecol 2012;21:1794–1805. 12. Brockman W, Alvarez P, Young S, et al. Quality scores and SNP detection in sequencing-by-synthesis systems. Genome Res 2008;18:763–770. 13. Vandenbroucke I, Van Marck H, Verhasselt P, et al. Minor variant detection in amplicons using 454 massive parallel pyrosequencing: Experiences and considerations for successful applications. BioTechniques 2011;51: 167–177. 14. Ledergerber C, Dessimoz C. Base-calling for next-generation sequencing platforms. Briefings Bioinformatics 2011; 12:489–497. 15. Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 1998; 8:186–194.
Address correspondence to: Janelle A. Noble, PhD Children’s Hospital Oakland Research Institute 5700 Martin Luther King Jr. Way Oakland, CA 94609 E-mail: [email protected]
BIOPRESERVATION AND BIOBANKING Volume 12, Number 6, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/bio.2014.0036
DNA Storage under High Temperature Conditions Does Not Affect Performance in Human Leukocyte Antigen Genotyping via Next-Generation Sequencing (DNA Integrity Maintained in Extreme Conditions) Shana L McDevitt,1 Michael E Hogan,2 Derek J Pappas,1 Lily Y Wong,2 and Janelle A Noble1
Background: Stable dry-state storage of DNA is desirable to minimize required storage space and to reduce electrical and shipping costs. DNA purified from various commercially available dry-state stabilization matrices has been used successfully in downstream molecular applications (e.g., quantitative polymerase chain reaction [qPCR], microarray, and sequence-based genotyping). However, standard DNA storage conditions still include freezing of DNA eluted in aqueous buffers or nuclease-free water. Broad implementation of dry-state, long-term DNA storage requires enhancement of such dry-state DNA stabilization products to control for temperature fluctuations at specimen collection, transit, and storage. This study tested the integrity of genomic DNA subjected to long-term storage on GenTegraTM DNA stabilization matrices (GenTegra LLC, Pleasanton, CA) at extreme conditions, as defined by a 4-year storage period at ambient temperature with an initial incubation for 7 months at 37C, 56C, or ambient temperature. Subsequently, purified DNA performance and integrity were measured by qPCR and next-generation sequencing (NGS)-based human leokocyte antigen (HLA) genotyping. Results: High molecular weight genomic DNA samples were recovered from the GenTegra product matrix and exhibited integrity comparable to a highly characterized commercial standard under assessment by qPCR. Samples were genotyped for classical HLA loci using next generation sequencing-based methodolgy on the Roche 454 GS Junior instrument. Amplification efficiency, sequence coverage, and sequence quality were all comparable with those produced from a cell line DNA sequenced as a control. No significant differences were observed in the mean, median, or mode quality scores between samples and controls ( p ‡ 0.4). Conclusions: Next generation HLA genotyping was chosen to test the integrity of GenTegra-treated genomic DNA due to the requirment for long sequence reads to genotype the highly polymorphic classical HLA genes. Experimental results demonstrate the efficacy of the GenTegra product as a suitable genomic DNA preservation tool for collection and long-term biobanking of DNA at fluctuating and high temperatures.
Introduction
T
he preservation of purified DNA, during long-term biobanking or shipping, has become a major concern in the field of applied genetics and public health screening.1 For cryogenic preservation of high-value DNA samples, mechanical and liquid nitrogen storage for biobanking, or dry ice and chemical packs for shipping are proven technologies. However, cryogenic preservation methods introduce substantial cost and some measure of risk, such as chemical refrigerant failure during shipping or power loss. Refrigeration-free biosample preservation could dramatically reduce the cost and risk associated with cryogenic preservation. Consequently, refrigeration-free approaches to purified DNA preservation 1 2
have been developed that preserve purified DNA via desiccation in the presence of added chemical stabilizers. Successful dry-state DNA preservation at lab ambient temperatures for many months has been reported, resulting in DNA of sufficient quality to support routine laboratory analyses such as quantitative polymerase chain reaction (qPCR), sequence based typing (SBT), and microarrays.2–5 However, one compelling application of refrigeration-free DNA stabilization is for collection of samples in the field, where the conditions are likely to be far more harsh than in the laboratory setting. Here we assess the performance of GenTegraTM (GenTegra LLC, Pleasanton, CA) under conditions that emulate ‘‘worst-case’’ ambient temperature storage or shipping. Using high-quality DNA purified from freshly collected blood, DNA
Children’s Hospital Oakland Research Institute, Oakland, California. GenTegra LLC, Pleasanton, California.
402
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was subjected to room temperature storage for 4 years, which included a 7-month initial treatment at elevated temperatures (37C or 56C) to reflect conditions that could be encountered during sample collection, shipping, or long-term DNA banking. Integrity of the DNA stored under these adverse conditions was compared to that of DNA stored at 4C. Performance of the DNA stored under adverse conditions was tested by its use in 2.5 kb quantitative polymerase chain reaction (qPCR) and in next-generation sequencing (NGS) based human leukocyte antigen (HLA) genotyping. HLA genotyping is complicated by extreme HLA sequence polymorphism; 12,242 HLA alleles have been documented as of October, 2014 (http://hla.allelles.org).
Methods DNA storage and purification Genomic DNA was extracted from 12 mL fresh venous blood samples collected in EDTA tubes from two different donors (Memorial Blood Centers, St. Paul, MN). Samples were collected by 4 pm, shipped overnight at 4C, stored at 4C upon receipt, and purified within 24 hours thereafter. DNA was purified using QIAamp DNA Blood Midi Kit (Qiagen Corp, Hilden, Germany). DNA concentration was measured by both Nanodrop UV/VIS spectrospcopy (Thermo Scientific, Waltham, MA) and Qubit fluorometry (Life Technologies, Carlsbad, CA). A total of 18 aliquots of DNA, 17 from one blood sample and one from the second blood sample, were arbitrarily split into two groups (C1-C9 and D1-D9) and stored in three plates. 3 mg (C samples) or 5 mg (D samples) of purified DNA sample (in TE buffer) were applied per well of a microplate containing the GenTegraTM DNA stabilization matrix (GenTegra LLC, Pleasanton, CA). Each plate contained three samples from each group. Each DNA sample in GenTegra was then air-dried per the manufacturer’s recommendation; the wells were sealed and then stored as follows for 4 years. One plate was heated to 37C in an oven for 7 months, then transferred to a warehouse for ambient temperature storage (*25C) for 3 years, 5 months (Fig. 1A). A second plate was heated to 56C in an oven for 7 months, then transferred to a warehouse for ambient temperature storage (*25C) for 3 years, 5 months (Fig. 1A). A third plate was kept in a warehouse at *25C for the entire 4 year time period. Subsequent to the 4-year storage, the 3 mg (C group) and 5 mg (D group) samples were rehydrated with 20 mL and 33 mL water, respectively. 100 ng of each DNA sample (as assessed by NanoDrop) was then run on a 0.8% agarose gel with ethidium bromide for 20 min on an E-gel base at 75V (Life Technologies, Carlsbad, CA) (Fig. 1B).
qPCR assessment of DNA quality 25 ng of each stored DNA sample was subjected to qPCR targeting a 2.5 kb region of mitochondrial DNA. Each reaction also included 250 nM of forward primer mt2568-F (5¢-CGCACGGACTACAACCACGAC-3¢), reverse primer mt2568-R (5¢-CTGTGGGGGGTGTCTTTGGGG-3¢), and FastStart SYBR Green with ROX (Roche Applied Sciences, Indianapolis, IN). qPCR was performed on the Applied Biosystems 7500 Fast Real-time PCR System (Life Technologies, Foster City, CA). The thermal cycling conditions were: 1) 95C for 10 min; 2) 40 cycles of 95C for 30 sec, 68C for
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1 min, 70C for 2 min, 3) 77C for 30 sec, followed by a dissociation step post-cycling. qPCR was performed in triplicate and the average CT value for each sample was plotted in Figure 1C, relative to CTs obtained for 25 ng of a highly validated control DNA, which was stored in the laboratory at 4C (PN 11691112001; Roche Applied Sciences, Indianapolis, IN).
HLA genotyping using next-generation sequencing (Roche 454) Amplicons (PCR products) were generated from purified genomic DNA (gDNA) from all eighteen samples, six samples per condition, stored dry with the GenTegraTM product and from a pre-purified and commercially supplied cell line human gDNA sample stored at - 20C (HAR: International Histocompatibility Workshop #9149) as a positive control (PTC). Fourteen amplicons were generated from each DNA sample using commercially available Roche 454 HLA GType Medium Resolution (MR) and High Resolution (HR) HLA fusion primer assay plates (Roche 454, Branford, CT) (6). Roche 454 GType MR assay plates include fusion primers to amplify HLA-A, -B, and -C exons 2 and 3 (exon 2 amplicons lengths are 560 base pair (bp), 446 bp, and 490 bp, respectively; exon 3 amplicon lengths are 495 bp, 425 bp, and 673 bp, respectively), HLA-DQB1 exon 2 (389 bp), and HLADRBX (366 bp); where X includes HLA-DRB1 and all additional DRB genes. Roche 454 GType HR assay plates include fusion primers to amplify HLA-A, -B, and -C exons 4 (740 bp, 464 bp, and 443 bp, respectively), HLA-DQB1 exon 3 (469 bp), HLA-DQA1 exon 2 (488 bp), and HLADPB1 exon 2 (430 bp). Roche 454 fusion primers consist of a 10-base multiplex ID (MID) tag, flanked by a locus-specific primer on the 3¢ end, and an ‘‘A’’ or ‘‘B’’ 454-specific primer adaptor sequence on the 5¢ end.6,7 The MID tag serves as a barcode for sample identification by data analysis programs.7,8 PCRs were performed in a 25 mL volume. Each reaction contained 20 ng of genomic DNA, 2 units of AmpliTaq Gold Polymerase (Life Technologies, Carlsbad, CA), 1X AmpliTaq PCR Gold Buffer (Life Technologies, Carlsbad, CA), 1.5 mM AmpliTaq Gold MgCl2 Solution (Life Technologies, Carlsbad, CA), 0.3 mM of PCR Grade Nucleotide Mix (Roche Applied Sciences, Indianapolis, IN), and 10% Amresco brand glycerol (Amresco, Solan, OH). Genomic DNA and PCR mastermix were aliquoted directly into the Roche 454 GType primer assay plates and PCR was performed using the thermal cycler GenAmp PCR 9700 system (Life Technologies, Foster City, CA). Cycling conditions were: 1) 94C for 5 min, 2) 31 cycles of 94C for 15 sec, 62C for 15 sec, and 72C for 30 sec, 3) 72C for 8 min. PCR products were visualized with the Advanced Analytical 96-Capillary Gel Electrophoresis based Fragment Analyzer (Advanced Analytical Technical Instruments, Ames, IA). PCR products were pooled, based on Quant-iT PicoGreen dsDNA reagent concentration data (Life Technologies, Carlsbad, CA), into a single, eqimolar amplicon library, purified with Agencourt AMPure XP beads (Beckman Coulter, Pasadena, CA), re-quantified using Quant-iT PicoGreen dsDNA reagent, and mixed with 454 capture beads (Roche 454, Branford, CT) after dilution to 1 · 106 molecule per microliter concentration. Individual HLA amplicon molecules within the library were clonally amplified and bound to 454 capture beads in emulsion PCR.6,7 DNA bound beads were enriched and deposited onto a single region 454 Titanium
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FIG. 1. (A) DNA storage timecourse. Purified gDNA sample was treated with GenTegraTM DNA stabilization matrix and stored at one of three temperatures for 7 months, followed by an incubation at ambient temperature (*25C) for a total period of 4 years. (B) Agarose gel assessment of gDNA quality. Subsequent to storage, the samples were rehydrated and visualized using agarose gel electrophoresis. A (collapsed) DNA band with an apparent molecular weight of 40 kb indicates that the average duplex DNA strand length for such samples is in excess of approximately 40 kb. (C) PCR assessment of DNA quality. qPCR was performed on an aliquot of each stored DNA sample employing a 2.5 kb region of mitochondrial DNA as the target. The average qPCR CT values for each sample are plotted relative to CTs obtained for control DNA.
PicoTiter plate (Roche 454, Branford, CT).6,7 Molecules were pyrosequenced on the Roche 454 GS Junior system (Roche 454, Branford, CT).6,7
Data analysis and statistics FASTA formatted sequencing reads were filtered based on alignment to an International Immunogenetics Information System (IMGT) HLA reference database using the Roche 454 Amplicon Variant Analyzer (Roche 454, Branford, CT).9 Sequencing reads from a subsequent FASTA file were analyzed with SCORETM software and Conexio AssignTM ATF HLA genotyping softwares. 454 GS Junior data processing tools, SFFfile and SFFinfo, were used to parse files and to extract sequencing adaptor trimmed and untrimmed sample-specific base calling quality metric scores
using manufacturer protocols. Raw base quality scores for all sequencing reads, from the original SFF file, were then analyzed per sample in reference to controls in R (http:// www.r-project.org). Wilcoxon rank sum tests were used to test for significance between sample and control means; significance threshold was set at p £ 0.05.
Results and Discussion Stored genomic DNA maintained integrity The integrity of eighteen gDNA samples recovered from the GenTegra product matrix was confirmed. As seen in Figure 1B, a limiting, high molecular weight DNA band, with apparent size greater than 40 kilobases, was detected with 0.8% agarose electrophoresis for all samples, indicating that,
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upon recovery from the GenTegra product, DNA chain length remains in the high molecular weight range expected for genomic DNA samples.
qPCR assay validated mitochondrial DNA integrity The quality of DNA obtained after long-term dry-state storage was assessed by an in-house qPCR assay targeting a 2.5 kb region of human mitochondrial DNA. The target size is approximately 10 times longer than that typically employed for qPCR and, thus, is capable of assessing DNA integrity by PCR in the presence of lesions at a density as low as 0.5 per kilobase. A mitochondrial DNA target was chosen to measure DNA quality as a surrogate for all other gene quality, in the same way that ribosomal RNA is used routinely as a surrogate for total RNA quality (e.g., the well known rRNA Bioanalyzer ‘‘RIN’’ score for RNA). Detailed instructions for reproducing this assay are available as a white paper, which may be obtained on the GenTegra website, at no cost, ([email protected]). In this assay, CT values obtained by qPCR were compared to those obtained from a highly characterized commercial human gDNA standard (PN 11691112001; Roche Applied Sciences, Indianapolis, IN). CT values obtained for the gDNA samples stored in GenTegra were found to be in the range of 19–20 cycles, independent of storage condition. CT values for all stored samples (Fig. 1) were found to be approximately three cycles fewer than the un-stored, control DNA, which was kept at 4C. Since DNA damage, or the presence of contaminants would increase, rather than decrease CT values, the qPCR data in Figure 1C are consistent with the gel electrophoresis data of Figure 1 and demonstrate that the DNA samples stored in the GenTegra matrix are at least as intact as freshly-isolated (never dried) control DNA.
Stored genomic DNA amplifies for troublesome HLA loci All eighteen samples and the positive control cell-line DNA (Fig. 2: PTC) amplified successfully for each of the target HLA loci. Amplicons were visualized by 2% agarose gel electrophoresis and by capillary gel electrophoresis. Nonspecific amplification was not observed (Fig. 2). HLA-A exon 4 amplicons are the largest in the set of fourteen (740 bp). Figure 2 displays capillary gel electrophoresis trace overlays of HLA-A exon 4 amplicons from all 18 samples and postive control PCR products around the target size, 740 bp. Amplicon concentrations ranged from 10.5 nanograms per microliter (ng/mL) to 17.2 ng/mL with a standard deviation of – 2.23 ng/mL for the longest HLA amplicon (HLA-A exon 4) as compared to postive control HLA-A exon 4 amplicon concentrations, 13.2 ng/mL and 13.7 ng/mL (SD = 0.23 ng/mL). In general, amplicon concentrations ranged from 5.56 ng/mL to 28.3 ng/mL for the experimental amplicons, as compared to control amplicon concentration ranges of 6.64 ng/mL to 24.4 ng/mL. The lowest concentrations in both groups were observed for HLA-C exon 3 (673 bp), which was the most difficult locus to amplify, possibly due to inefficient primer annealing.
Stored genomic DNA effectively used to genotype HLA Three sequencing runs were performed in total. Each run included all HLA amplicons for six experimental samples
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and one control. The same control library was sequenced in each run to control for run-to-run variation. The runs showed some variability in the number of quality filter passed sequencing reads, 126,665 reads, 110,766 reads, and 134,317 reads, respectively (454 software manual). All three sequencing runs, however, far exceed the 70,000 quality passed filter read benchmark set by Roche 454.10 As expected, identical HLA genotypes were assigned for 17 out of 17 identically sourced samples. The single sample sourced from a different individual produced a different genotype. Control genotypes were identical for each of the HLA loci among the three sequencing runs, demonstrating the consistency of the sequencing and genotype calling processes. 111 of 126 total genotypes (18 samples sequenced for 7 genes) were assigned without the need for manual user editing in the Conexio AssignTM ATF HLA genotyping software. Minimal user edits to remove sequence containing obvious sequencing error were necessary to assign genotypes to 11 out of the initial unassigned 15 genotypes. The remaining 4 genotypes cannot be assigned due to mid HLAB exon 3 sequencing error or cannot be assigned without excluding HLA-A exon four data. However, these failed assignments are attributable to limitations in 454 sequencing chemistry rather than the initial DNA integrity. 454 sequencing read length limitations. Roche 454 pyrosequencing chemistry delivers the longest read lengths of all clonally based sequencing platforms,10,11 with the ability to generate high quality reads 400 bp in length on either the 454 GS FLX or 454 GS Junior platforms.10 HLA-A exon 4 and HLA-C exon 3 amplicons, 740 and 673 bp, respectively, exceed the size of exons and far exceed the length limitations of the sequencing chemistry. For both amplicons, the exon lies near the upstream end; thus, the quality of the exon sequence from the forward sequence reactions was higher than that from the reverse sequences. In the reverse sequence reactions, the exon sequence was at the end of the read, and DNA sequencing tends to decrease in quality with read length.10 Figure 3 shows that, within this data set, all untrimmed sequencing reads maintain a high level of base calling quality (Q), where Q0 and Q40 are the minimum and maximum quality score range, respectively, until read lengths approach 430 bases. At this point, the ability of the sequencing software to call bases accurately steadily decreases. Moreover, when comparing the quality scores from reads at nucleotide position 423 (Fig. 3D), distribution of quality scores was not different for the test samples as compared to the control samples (adjusted p = 1). HLA-A exon 4 and HLA-C exon 3 reverse reads for all samples in the second sequencing run showed evidence of increased sequencing error at the 3¢ end. Failure to generate reverse direction sequences long enough to capture full HLA-A exon 4 and full HLA-C exon 3 sequence was also observed in the control sample from the second sequencing run. Only HLA-A genotypes for samples C8 and D3 were left unassigned. Genotypes for these samples were determined, albeit with increased ambiguity, upon the exclusion of all HLA-A exon 4 sequences. 454 sequencing homopolymer detection limitations. Genotype assignments for the 17 identically sourced samples were used to distinguish sequencing error from genomic variation where failed genotype assignments were made. Five HLA-B genotypes showed evidence of a base calling discrepancy
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FIG. 2. HLA-A exon 4 amplicon fragment analyzer trace overlay. Capillary electrophoresis was performed to visualize the HLA-A exon 4 amplicon size range distribution among all samples and controls. Upper and lower markers used for size determination are visible at positions of 35 base pairs (bp) and 1500 bp, respectively. Samples represented within each frame were amplified together on a PCR assay plate with the cell-line control (PTC) noted in the frame.
between the forward and reverse sequence reads from the same chromosome near the center of exon 2 near a high G-C rich region in the template strands. Regardless of input DNA quality, homopolymers (i.e., multiple molecules of the same nucleotide) can be problematic for 454 chemistry.10 454 sequencing data are represented by a series of intensity values, or a flowgram, for iterative deoxynucleotide flows during which multiple nucleotide incorporations may occur in the absence of a 3¢ chain terminator which can lead to substitution or deletion error.12 Nucleotide sequences off-set by one base in either direction cannot be aligned with confidence to a HLA reference. Three of the five samples with noted sequencing error (C1, C2, and C3) were in the 56C storage group while the remaining two samples (D2 and D3) were from the ambient
temperature storage group. No other replicates showed evidence of a similar issue with HLA-B sequence. Each of the noted samples shows a deletion in the reverse reads for both alleles at base position 389, which directly follows the G-C rich region noted above. This region is found in all HLA-B alleles described to date. Notably, the forward reads for these alleles per sample had no evidence of sequencing error. However, D7, D8, and D9, also from the 56C worstcase scenario treatment group, all showed no evidence of sequencing error in either forward or reverse reads, suggesting that the errors reflected in samples C7–C9, D2, and D3 were not due to the storage conditions or the subsequent quality of genomic DNA but were rather attributable to reduced sequencing run quality. Vandenbroucke et al. suggested that every amplicon has its own error profile dependent upon its originating sequence.10,13 Because all
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FIG. 3. Quality score distributions. (A–C) Average quality scores across all reads at indicated nucleotide position for each sample. (A) from sequencing run 1; (B) from sequencing run 2; and (C) from sequencing run 3. (D) Box plots of the quality score distributions for all reads at nucleotide position 423. Replicates were averaged and median centered.
HLA-B alleles contain the problematic sequence motif, which has a high error profile, the reduced sequencing quality of run 2, compared to that of runs 1 and 3, led to the apparently high standard error for this data set.
High molecular weight genomic DNA purified from the GenTegra product matrix was successfully used to genotype the most polymorphic genes in the human genome. Next generation HLA genotyping was chosen to test the integrity of GenTegra product matrix treated genomic DNA due to
Stored sample sequencing quality scores mirrored controls Quality scores are assigned to each base for each sequencing read by the 454 sequencing software, representing the log-probability that a base was not an overcall.10,12,14 These scores are reported in a FASTA-like format. 454 base quality scores are represented by Phred equivalent quality scores (Q = - 10 * log10 (error rate)) ranging from Q0–Q40, where a Q30 score represents a base-call with a probablility of 1/1000 of being incorrect while a Q40 score represents a 1/104 chance of being incorrect, in other words, representing a 99.99% accuracy rating.10,15 No significant differences were observed ( p ‡ 0.4) in the mean, median, or mode quality scores between samples and control (Fig. 4). Quality score results suggest that gDNA purified from the GenTegra product stored under all treatment conditions behaves similarly to cell line gDNA stored in best-case conditions at - 20C.
Conclusions Taken together, consistency of HLA genotypes from identically sourced samples and lack of statistically significant difference in average quality score for stored samples compared to controls, validate the integrity of gDNA purified from the GenTegra product matrix after all tested treatment conditions, including the worst-case, 56C group.
FIG. 4. Quality score average comparisons. For each set of conditions, averages (mean, median, mode) were measured across all reads within a replicate and all sample replicates (n = 3).
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the requirment for high-quality, long, clonal sequencing reads. Results of this test demonstrate the efficacy of the product as a suitable genomic DNA preservation tool for collection and long-term biobanking of DNA at fluctuating and high temperatures. Thus, implementation of the GenTegra product matrix should greatly reduce sample storage cost without compromising data integrity. This 4-year pilot study was performed primarily on aliquots of a sample from a single individual, plus a single sample from a second individual included to control for sample-specific effects. All test samples were stored dry in the GenTegra product. Planned follow-up studies will include both samples from more individuals, as well as samples for direct comparison of dry storage with traditional liquid storage methods, both at 4C and at - 20C. Given the excellent quality of the DNA after long-term dry storage reported here, the expectation is that future studies will show that this method is at least comparable, and perhaps superior, to liquid storage in terms of quality of resulting DNA. The combination of the excellent DNA quality shown in this report with the substantial cost savings due to nonrefrigeration of samples creates a compelling argument for dry state storage, especially for projects involving large numbers of samples.
Author Disclosure Statement SLM, DJP, and JAN are all employees of Children’s Hospital Oakland Research Institute. MEH and LYW were employees of IntegenX during the duration of the project and are now employed by GenTegra, LLC. Funding for this work was provided by IntegenX Corp. (Pleasanton, CA, USA), and, in part, by NIH grant DK61722 (J.A.N.).
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Address correspondence to: Janelle A. Noble, PhD Children’s Hospital Oakland Research Institute 5700 Martin Luther King Jr. Way Oakland, CA 94609 E-mail: [email protected]
