Evaluation of 96 SNPs in 14 populations for worldwide individual identification

Great advances have been made recently in searching for individual identification single-nucleotide polymorphisms (IISNPs or IDSNPs). Such SNPs as suggested by SNPforID scientists and by Pakstis et al., are promising, although they were selected from older or smaller databases rather than the most recent database. Here, we describe a new computational strategy for developing IDSNPs based on HapMap. We searched through HapMap r27 for SNPs having minor allele frequencies ≥0.30 in all its 11 populations and found more than 1881 qualified SNPs. We examined 96 of them with 183 DNA samples from three Chinese populations using Illumina arrays. The average allele frequency for these 96 SNPs among the three populations was 0.495/0.505, the average number of identical SNP genotypes shared by two individuals among the 14 populations (three Chinese and 11 HapMap) was 37.9, and the random matching probability for two unrelated Hans to match in all 96 genotypes was 9.793 × 10(-39). Thus, most of these 96 SNPs are universally applicable.     

个体识别SNPs位点组合筛选与法医学应用价值初探

目的筛选用于包括中国主要民族在内的多个群体个体识别的SNPs位点组合体系。方法以Kidd实验室筛选的86个SNPs位点、欧洲SNPforID组织构建的52-plex SNPs复合检测体系为基础,收集和整理这些位点在HapMap数据库中11个人群的分型数据,计算各位点杂合度和Fst值,筛选杂合度〉0.4,Fst值〈0.06,并在研究人群中处于Hardy-Weinberg和连锁平衡的位点组合。针对这些位点,采用MassARRAY分子阵列技术对自行收集的8个人群（尼日利亚人、坦桑尼亚查加人、印度人、丹麦人、俄罗斯汉特人、中国汉族、藏族、维吾尔族）308份样本进行分型,统计群体遗传学参数。结果按本文标准共筛选出66个SNPs位点,均符合Hardy-Weinberg平衡,之间互不连锁,平均杂合度和Fst值分别为0.475、0.014。在本文收集的8个人群中的随机匹配概率在1.45E-24~4.72E-27之间,累积非父排除率为0.999 995 608~0.999 997 876之间。结论本文筛选的SNPs组合系统具有较强的个体识别能力,可用于本文调查的HapMap数据库中11个人群和本文收集的8个人群的个体识别鉴定。     

X染色体上高信息量SNP位点及其法医学价值

人类X染色体长154.8Mb,其上密布SNP位点,蕴涵着大量的信息。用于法医学鉴定的X-SNP标记多态性好、突变率低。本研究从Hapmap、NCBI的数据库中筛选了167个高信息量SNP位点,这些位点的等位基因在北京汉族人群中的分布频率均高于0.3,通过高通量、高灵敏度的检测方法可对各个X-SNP位点进行分型验证,通过正确的统计学分析可得到其法医学多态性参数。X-SNP位点具有一些常染色体遗传标记无法比拟的优点,作为常规STR基因座的补充,能用于解决特殊的亲子鉴定案,性别鉴定和混合斑鉴定。     

Genome‐wide Screen for Individual Identification SNPs (IISNPs) and the Confirmation of Six of Them in Han Chinese with Pyrosequencing Technology*

Abstract: Single nucleotide polymorphisms (SNPs) offer promise to forensic DNA analysts, but it remains uncertain whether a panel of individual identification SNPs can be as informative as the Combined DNA Index System short tandem repeats. Based on the highly accurate and publicly available HapMap SNP database (r21a) and a minor allele frequency cutoff of ≥0.45, we completed a genome‐wide screen through 3,905,819 SNPs with internally modified computer programs and identified 1439 SNPs with high heterozygosity and low F_st values among four populations (Utah Caucasian, Han Chinese, Tokyo Japanese, and Nigerian Yoruba). Using pyrosequencing technology, we studied six loci in a relatively large group of samples to determine whether these loci were as informative as the HapMap data suggest. These SNPs performed as expected in the Han Chinese in terms of heterozygosity and F_st. The 1439 identified SNPs should provide a comprehensive and reliable set of loci for identity and relationship testing.     

Mass spectrometry-based SNP genotyping as a potential tool for ancestry inference and human identification in Chinese Han and Uygur populations

Ancestry informative SNPs (AISNPs) are genetic variants that exhibit substantially different frequencies between populations from different geographical regions; thus, they can provide some valuable information regarding samples and be used in predicting an individual's ancestry origin. In this study, we selected the potentially best SNPs from our previous study with genome-wide high-density SNP data in mainland Chinese Uygur and Han populations and investigated the allele distribution patterns and genetic information of AISNPs with a mass spectrometry-based SNP genotyping panel. Mass spectrometry-based detection technology offers the opportunity to analyze forensic DNA samples and obtain SNP variants with accuracy and ease. The panel can distinguish and cluster Han and Uygur populations and is suitable for human identification and parentage testing in the two populations. Heatmap, PCA, and Structure analyses indicated that the ideal 64 AISNPs can collectively provide additional information on differences among populations from East Asia, South Asia, Europe and Africa. Additionally, the results proved that the Uygur population is the admixture of East Asia and Europe.     

Inferring ancestral origin using a single multiplex assay of ancestry-informative marker SNPs

Tests that infer the ancestral origin of a DNA sample have considerable potential in the development of forensic tools that can help to guide crime investigation. We have developed a single-tube 34-plex SNP assay for the assignment of ancestral origin by choosing ancestry-informative markers (AIMs) exhibiting highly contrasting allele frequency distributions between the three major population-groups. To predict ancestral origin from the profiles obtained, a classification algorithm was developed based on maximum likelihood. Sampling of two populations each from African, European and East Asian groups provided training sets for the algorithm and this was tested using the CEPH Human Genome Diversity Panel. We detected negligible theoretical and practical error for assignments to one of the three groups analyzed with consistently high classification probabilities, even when using reduced subsets of SNPs. This study shows that by choosing SNPs exhibiting marked allele frequency differences between population-groups a practical forensic test for assigning the most likely ancestry can be achieved from a single multiplexed assay.     

法医SNP复合检测体系的构建及应用

目的构建48-SNP位点复合检测体系,用于个体识别、性别鉴定、ABO基因分型。方法采集225份无关个体样本(血斑及口腔拭子),18份案例样本(不同组织及体液斑),选择43个常染色体位点、4个ABO基因位点和1个性别鉴定位点,根据单碱基延伸技术通过GenomeLabTMSNPstream基因分型系统进行SNP分型;并检测体系灵敏度、同一个体不同组织同一性及模拟腐败检材。结果 48-SNP体系分型结果与测序结果的一致性为100%,最小DNA检出量为0.25ng,不同组织来源样本检测同一性很好;利用该体系检测225名无关汉族个体,所有位点均符合Hardy-Weinberg平衡,整个系统的随机匹配概率为9.4×10-18,累积非父排除率(CEP)为0.999 788,累积个体识别率大于0.999 999 999 999 999 99。结论本文48-SNP体系能同时进行个体识别、ABO基因分型和性别鉴定,可以作为现有STR检验体系的补充。     

Tri-allelic SNP markers enable analysis of mixed and degraded DNA samples

For the analysis of degraded DNA in disaster victim identification (DVI) and criminal investigations, single nucleotide polymorphisms (SNPs) have been recognized as promising markers mainly because they can be analyzed in short sized amplicons. Most SNPs are bi-allelic and are thereby ineffective to detect mixtures, which may lead to incorrect genotyping. We developed an algorithm to find non-binary (i.e. tri-allelic or tetra-allelic) SNPs in the NCBI dbSNP database. We selected 31 potential tri-allelic SNPs with a minor allele frequency of at least 10%. The tri-allelic nature was confirmed for 15 SNPs residing on 14 different chromosomes. Multiplex SNaPshot™ assays were developed, and the allele frequencies of 16 SNPs were determined among 153 Dutch and 111 Netherlands Antilles reference samples. Using these multiplex SNP assays, the presence of a mixture of two DNA samples in a ratio up to 1:8 could be recognized reliably. Furthermore, we compared the genotyping efficiency of the tri-allelic SNP markers and short tandem repeat (STR) markers by analyzing artificially degraded DNA and DNA from 30 approximately 500-year-old bone and molar samples. In both types of degraded DNA samples, the larger sized STR amplicons failed to amplify whereas the tri-allelic SNP markers still provided valuable information. In conclusion, tri-allelic SNP markers are suited for the analysis of degraded DNA and enable the detection of a second DNA source in a sample.     

Characterization of human control region sequences of the African American SWGDAM forensic mtDNA data set

The scientific working group on DNA analysis Methods (SWGDAM) mitochondrial DNA (mtDNA) population data set is used to infer the relative rarity of control region mtDNA profiles obtained from evidence samples and of profiles used for identification of missing persons. In this study, the African American haplogroup patterns in the SWGDAM data were analyzed in a phylogenetic context to determine relevant single nucleotide polymorphisms (SNPs) and to describe haplogroup distributions for Africans observed in these data sets. Over 200 SNPs (n=217) were observed in the African American data set (n=1148). These SNPs ranged from having 1-39 changes in the phylogenetic tree, with sites 152 and 16519 being the most variable. On average there were 5.8 changes for a character on the tree. The most variable sites (with 19 or more changes each) observed included 16093, 16129, 16189, 16311, 16362, 16519, 146, 150, 152, 189, and 195. These rapidly changing sites are consistent with other published analyses. Only 34 SNPs are needed to identify all clusters containing 10 or more individuals in the African American data set. The results show that the African American SWGDAM mtDNA data set contains variation consistent with that described in continental African populations. Thirteen of the 18 haplogroups previously observed in African populations were observed and include: L1a, L1b, L1c, L2a, L2b, L2c, L3b, L3d, L3e1, L3e2, L3e3, L3e4 and L3f. Haplogroup L2a is the most commonly observed cluster (18.8%) in the African American data set. The next most common haplogroups in the African American data set include the clusters L1c (11.0%), L1b (9.1%), L3e2 (9.0%) and L3b (8.1%). Approximately 8% of the haplogroups observed within African Americans were common in European Caucasians or East Asians; these were H (n=32), J (n=4), K (n=5), T (n=2), U5 (n=6), U6 (n=9 also known from North Africa), A (n=12), B (n=7), C (n=4), and M (n=16), respectively. The European Caucasian and East Asian haplogroups are expected due to admixture between individuals with recent ancestry in Western Eurasia and sub-Saharan Africa. The genetic characterization of these relevant data sets is fully consistent with other published mtDNA genetic variation. The sequence diversity observed in this data set makes it a valuable tool for forensic applications.     

SNP Miniplexes for Individual Identification of Random‐Bred Domestic Cats

Phenotypic and genotypic characteristics of the cat can be obtained from single nucleotide polymorphisms (SNPs) analyses of fur. This study developed miniplexes using SNPs with high discriminating power for random‐bred domestic cats, focusing on individual and phenotypic identification. Seventy‐eight SNPs were investigated using a multiplex PCR followed by a fluorescently labeled single base extension (SBE) technique (SNaPshot^®). The SNP miniplexes were evaluated for reliability, reproducibility, sensitivity, species specificity, detection limitations, and assignment accuracy. Six SNPplexes were developed containing 39 intergenic SNPs and 26 phenotypic SNPs, including a sex identification marker, ZFXY. The combined random match probability (cRMP) was 6.58 × 10^?19 across all Western cat populations and the likelihood ratio was 1.52 × 10¹⁸. These SNPplexes can distinguish individual cats and their phenotypic traits, which could provide insight into crime reconstructions. A SNP database of 237 cats from 13 worldwide populations is now available for forensic applications.     

World population data for the HLA-DQA1, PM and D1S80 loci with least and most common profile frequencies for combinations of loci estimated following NRC II guidelines

All published and unpublished gene frequency data for the PCR-based loci HLA-DQA1, LDLR, GYPA, HBGG, D7S8, GC, and D1S80 that could be located are presented in summary tables. These gene frequencies provide the data necessary for estimating probabilities of chance match according to NRC II guidelines for any DNA profile that includes any combination of these loci for any of the populations. To illustrate the range of polymorphism for combined locus profiles, least and most common profile frequencies were estimated following NRC II guidelines for: the PM loci for all populations for which PM data were available; and for combinations of HLA-DQA1/PM, HLA-DQA1/D1S80, PM/D1S80, and HLA-DQA1/ PM/D1S80 for populations for which data were available for the relevant combinations. The profile frequencies were calculated at theta values of zero and 0.01. Minimum allele frequencies (MAF) were calculated, and are shown, for each data set for which the MAF was greater than the lowest observed allele frequency. Least common profile frequencies were calculated using MAF in those cases to illustrate a conservative estimate. The effect of using MAF versus lowest observed allele frequency in estimating least common profile frequencies is briefly illustrated as well. We finally show that aggregate U.S. gene frequency data for the classical MN and GC polymorphisms for both Caucasian and African-American populations is fully in accord with the DNA-based gene frequency data obtained from PM reverse dot-blot strips for GYPA and GC, respectively.     

Identification in blood stains through DNA typing with C4 and HLA-DR probes

A restriction fragment length polymorphism analysis using double digestion of DNA preparations with XbaI and BglII restriction enzymes and hybridization with C4 and HLA-DR probes is described. The typing conditions selected reveal extensive individual variation in both C4 and DR gene regions. In our panel of 46 unrelated individuals, 37 different phenotypic patterns were recognized when both probes were used, and preliminary discriminative power values of 0.865 and 0.914 were calculated for C4 and DR beta, respectively. The probability of a chance match using both systems is probably about 1.5.10(-2). The potential of this method for individual identification of blood stains was demonstrated on DNA prepared from 6-month-old dried blood stains from seven panel individuals. The seven individuals were all identified when comparing stain DNA patterns with panel control patterns. No RFLP pattern changes were observed following storage of blood stains. Based on these experiments with C4 and DR beta DNA typing under laboratory conditions, it is concluded that DNA typing with such probes may become a powerful tool in future stain identification analyses.     

Investigation of 74 microhaplotypes for kinship testing in US populations

Microhaplotypes are markers that consist of haplotype blocks of SNPs, which can be analyzed by massively parallel sequencing technologies. These allow determining the haplotype phase at every locus by clonal sequencing each DNA strand. MHs are polymorphic loci with same size alleles, no stutter, and lower mutation rate than STRs. They can provide the same power of discrimination of STR-kits, thus useful for mixture deconvolution, but more accurate ancestry prediction than STRs. In this study we investigated the potential of a recently developed 74plex-MH panel for kinship testing using the Familias software.Samples from families of four major US population groups were collected and genotyped using the 74plex-MH panel. MH allele frequency data from 347 individuals were imported into Familias software along with STR allele frequency data of 29 loci (NIST dataset) from 1036 individuals. Different family scenarios were tested and these included unrelated vs parent-child, unrelated vs full siblings, unrelated vs half siblings, unrelated vs cousin pairs. The prediction of the kinship relation for the four populations of interest was reported as Log10 of the likelihood ratio (LR).Overall, the panel of 74MHs and 29STRs showed similar performance in predicting the correct kinship scenarios tested. Correct prediction was reported for parent-child, full siblings, and half sibling scenarios, but not for the cousin pairs scenario. The panel of 74 MHs showed larger Log10LR values than the 29 STR-assay, thus demonstrating the effectiveness of this biomarker as a tool for kinship testing in addition to mixture deconvolution and ancestry prediction.     

Twelve short tandem repeat loci Y chromosome haplotypes: genetic analysis on populations residing in North America

A total of 2443 male individuals, previously typed for the 13 CODIS STR loci, distributed across the five North American population groups African American, Asian, Caucasian, Hispanic, and Native American were typed for the Y-STR loci DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439 using the PowerPlex Y System. All population samples were highly polymorphic for the 12 Y-STR loci with the marker DYS385a/b being the most polymorphic across all sample populations. The Native American population groups demonstrated the lowest genetic diversity, most notably at the DYS393 and DYS437 loci. Almost all of the 12-locus haplotypes observed in the sample populations were represented only once in the database. Haplotype diversities were greater than 99.6% for the African Americans, Caucasians, Hispanics, and Asians. The Native Americans had the lowest haplotype diversities (Apaches, 97.0%; Navajo, 98.1%). Population substructure effects were greater for Y-haplotypes, compared with that for the autosomal loci. For the apportionment of variance for the 12 Y-STRs, the within sample population variation was the largest component (>98% for each major population group and approximately 97% in Native Americans), and the variance component contributed by the major population groups was less than the individual component, but much greater than among sample populations within a major group (11.79% versus 1.02% for African Americans/Caucasians/Hispanics and 15.35% versus 1.25% for all five major populations). When each major population is analyzed individually, the R(ST) values were low but showed significant among group heterogeneity. In 692 confirmed father-son pairs, 14 mutation events were observed with the average rate of 1.57x10(-3)/locus/generation (a 95% confidence bound of 0.83x10(-3) to 2.69x10(-3)). Since the Y-STR loci reside on the non-recombining region of the Y chromosome, the counting method is one approach suggested for conveying an estimate of the rarity of the Y-haplotype. Because the Y-STR loci are not all in disequilibrium to the same extent, the counting method is a very conservative approach. The data also support that autosomal STR frequencies can be multiplied by the upper bound frequency estimate of a Y-haplotype in the individual population group or those pooled into major population groups (i.e., Caucasian, African American, Hispanic, and Asian). These analyses support use of the haplotype population data for estimating Y-STR profile frequencies for populations residing in North America.     

30个 InDels在中国三个民族的遗传学调查及法医学应用

目的调查Qiagen Investigator@ DIPplex试剂盒30个InDels多态性位点在中国汉族、藏族、维吾尔族人群中的群体遗传学数据,评估其法医学应用价值。方法采集汉、藏、维吾尔族各90名无关个体静脉血,提取DNA。使用3130xL毛细管电泳对该270份样品进行分型,通过统计计算相关的群体遗传学参数。结果实验得到270份样品的分型及基因型频率,30个InDels未明显偏离Hardy-Weinberg平衡及连锁平衡,在汉族、藏族、维吾尔族三个人群中的随机匹配概率分别为1.42×10（-11）、7.19×10（-12）、4.74×10（-13）,累积非父排除率（CPE）均大于0．9951。结论该组插入缺失位点在中国的汉族、藏族、维吾尔族人群中具有较高的多态性,能达到较高的个体识别能力,可以作为现有STR检验体系的补充。     

Analysis of genotype frequencies and interlocus association for the PM, DQA1, and D1S80 loci in four populations

Allele frequencies of the LDLR, HBGG, GYPA, D7S8, GC, DQA1, and D1S80 loci are presented and genotypes are analyzed for each of four ethnic groups: African Americans (n = 200), US Caucasians (n = 200), US Hispanics (n = 200), and Japanese (n = 89). Hardy-Weinberg genotypic proportions were observed in all but two of the 28 population-locus tests undertaken. Those two instances are attributable to type I statistical error. Gametic equilibrium among loci is an assumption invoked for application of the product rule to utilize the discriminatory power from two or more loci simultaneously. Two statistical methods, a genotype matching statistic and log-linear modeling, were used to evaluate gametic disequilibrium. The match statistic, comparing observed to expected likelihood of genotypic identity for seven loci among pairs of individuals within the database, revealed only one statistically significant deviation among 20 tests. As expected, the probability of match was generally lowest in the test on all ethnic groups combined, indicating that allele frequencies differ among ethnic groups for some of the loci. This was confirmed with the statistic theta to measure ethnic stratification, in which about 0.10 of the genetic variation is apportioned among the four ethnic groups for four of the structural loci (LDLR, HBGG, GC, and DQA1), while for GYPA, D7S8, and D1S80, variation is more uniformly distributed among ethnic groups. Log-linear modeling was also applied to the five PM loci. The most parsimonious log-linear model included only three higher order terms: the two-way interactions of three of the PM loci with ethnic group. These three instances (LDLR, HBGG, and GC) indicated differences in allele frequencies between ethnic groups. No two or higher way interaction (disequilibrium) was observed among loci. In summary, the assumptions of Hardy-Weinberg and gametic equilibrium that facilitate the use of the five PM loci, DQA1 and D1S80 in forensic applications are consistent with the allele and genotype frequencies observed in these populations.     

血小板同种抗原基因中9个单核苷酸多态性在广西地区壮族和汉族人群中的差异分析

目的检测血小板同种抗原基因中9个单核苷酸多态性在广西地区壮族和汉族人群中的差异。方法利用基于单碱基延伸的单核苷酸多态性（SNP）分型芯片,对广西壮族地区99例壮族个体和107例汉族个体的染色体基因组上10个SNP位点进行了分型,其中9个位于6个血小板同种抗原基因中,1个位于基因间区。此外,结合Hapmap计划第二期公布的四个人群的SNP分型数据,分析这六个人群的遗传结构。结果广西原住汉族人在等位基因频率上未检测到与当地壮族有显著性的差异位点,但在基因型频率上,rs630014和rs9441951两位点是显著差异的。广西壮族人与广西汉族、北京汉族人及日本东京人的遗传结构相近,但与祖先来自欧洲西部和北部的犹他州居民以及尼日利亚伊巴丹的约鲁巴人有显著差异的遗传成分存在。结论壮汉两族由于历史上的多次基因交流可能导致其遗传信息在很大程度上是相近的。     

Characterization of a novel dimorphism in the 5' flanking region of the short tandem repeat (STR) locus,c-fes/fps (FES)

The FES short tandem repeat (STR) locus contains seven to 14 repeats of the tetranucleotide sequence ATTT. A novel 10 base pair dimorphism in the 5' flanking region of the FES locus was characterized in four broad populations: African-American, Hispanic, Caucasian, and Asian. The absence of the 10 base pair sequence, or (-) allele, was closely linked to FES STR alleles with 10 or fewer repeats. The presence of the 10 base pair sequence, or (+) allele, was closely linked to FES STR alleles with 12 or more repeats. The (-) and (+) alleles occurred equally often in FES STR allele 11. The nucleotide sequence (5'-GGCTGTTTTG-3') of the (+) allele, located 179 base pairs upstream of the FES STR, was determined to be consistent within and among the four populations. Statistical and sequence analysis confirmed the linkage between the two polymorphic sites. The results indicate that the exclusion rate of the FES locus is increased, above that for the STR alone, when both polymorphic characteristics are considered.     

Forensic Identification Using a Multiplex Assay of 47 SNPs*

Abstract: As a powerful alternative to short tandem repeat (STR) profiling, we have developed a novel panel of 47 single nucleotide polymorphisms (SNPs) for DNA profiling and ABO genotyping. We selected 42 of the 47 SNPs from a panel of 86 markers that were previously validated as universal individual identification markers and identified five additional SNPs including one gender marker and four ABO loci. Match probability of the 42 validated SNPs was found to be 9.5 × 10^?18 in Han Chinese. SNP analysis correctly assessed a panel of historical cases, including both paternity identifications in trios and individual identifications. In addition, while STR profiling of degraded DNA provided information for 11 loci of 16 potential markers with low peak intensities, SNPstream^® genotyping was sufficient to identify all 47 SNPs. In summary, SNP analysis is equally effective as STR profiling, but appears more suited for individual identification than STR profiling in cases where DNA may be degraded.     

Analysis of 50 SNPs in CYP2D6, CYP2C19, CYP2C9, CYP3A4 and CYP1A2 by MALDI-TOF mass spectrometry in Chinese Han population

One of the major challenges in the near future is the identification of genes that affect the metabolism of different drugs. Large scale association studies that utilise single nucleotide polymorphisms (SNPs) have been considered a valuable tool for this purpose. CYP2D6, CYP2C19, CYP2C9, CYP3A4 and CYP1A2 were found to be involved in the majority of hepatically cleared drugs. To determine the allele frequencies of some SNPs that may have great potential value in forensic science, we screened 50 SNPs in these 5 CYP genes in Chinese Han people using an accurate, high-throughput, cost-effective method. Primers were designed using the MassARRAY Assay Design software. Genomic DNA was prepared from blood samples obtained from individuals of Chinese Han origin. Multiplex PCR was performed to amplify the relevant gene fragments, and the polymorphisms were analysed by allele-specific primer extension followed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). A panel of genomic DNA samples previously genotyped by other methods were analysed simultaneously for quality control, and the results demonstrated that this assay was 100% accurate. A total of 17 of the analysed SNPs were polymorphic. Of these 17 SNPs, 8 (rs16947, rs28371725, rs1800754, rs4244285, rs4986893, rs12248560, rs3758580, rs2242480) had an allele frequency that was significantly different between this Chinese Han population and Caucasians (p<0.01). In addition, the frequencies of two of these SNPs (rs1800754, rs3758581) in our Chinese Han population differed significantly from the existing Chinese frequency data (p<0.01). The described method thus provides reliable results and enables the genotyping of up to thousands of samples by taking advantage of the high-throughput MALDI-TOF technology. The results herein are now included as a supplement to the P450 database.