Y-chromosomal Short Tandem Repeat Research Articles

Technical note: multi-alleles at the DYS385ab locus with high frequency in a Han Chinese population from southwestern China.

Y-chromosome short tandem repeat (Y-STR) markers have been widely used in forensic applications and usually show monoallelic or diallelic genotypic patterns at certain double-copied loci. In this study, we have found 13 samples among 703 males with multi-alleles at the DYS385ab locus, including one with five mutant alleles, nine with four, and three with three. The frequency of abnormal DYS385ab genotypes was 1.85% (13/703), which is very high in the Han Chinese population. The percentage of samples with diallelic patterns at DYS385ab was higher than that of monoallelic patterns (80.23% vs. 17.92%). Additionally, the percentage of samples with tetra-allelic patterns at DYS385ab was higher than that of tri-allelic patterns (1.28% vs. 0.43%), suggesting that there are possibly two copies with duplicated events happening frequently on the Y chromosome. Interestingly, the peak height of allele 13 was two to three-folds higher than that of other alleles. The allele 18 peak height was also two-fold higher than others, which could potentially be explained by a duplication event mechanism. We also found that tri-allelic genotypes for alleles 13, 17, and 20, tetra-allelic genotypes for alleles 13, 14, 19, and 20, and tetra-allelic genotypes for alleles 12, 13, 19 and 21 were more common than others. Furthermore, all 13 samples had multi-alleles containing allele 13, implying a founder effect in this particular Chinese-specific ethnic group. Taken together, this study provides new information for this population and will be useful for paternal lineage identification, kinship analysis, and family relationship reconstruction using Y-STR forensic DNA analysis methods.

Forensic features and genetic legacy of the Baloch population of Pakistan and the Hazara population across Durand line revealed by Y-chromosomal STRs

The Hazara population across Durand line has experienced extensive interaction with Central Asian and East Asian populations. Hazara individuals have typical Mongolian facial appearances and they called themselves descendants of Genghis Khan's army. The people who speak the Balochi language are called Baloch. Previously, a worldwide analysis of Y-chromosomal haplotype diversity for rapidly mutating (RM) Y-STRs and with PowerPlex Y23 System (Promega Corporation Madison, USA) kit was created with collaborative efforts, but Baloch and Hazara population from Pakistan and Hazara population from Afghanistan were missing. In the current study, Yfiler Plus PCR Amplification Kit loci were examined in 260 unrelated Hazara individuals from Afghanistan, 153 Hazara individuals, and 111 Balochi individuals from Baluchistan Pakistan. For the Hazara population from Afghanistan and Pakistan overall, 380 different haplotypes were observed on these 27 Y-STR loci, gene diversities ranged from 0.51288 (DYS389I) to 0.9257 (DYF387S1), and haplotype diversity was 0.9992. For the Baloch population, every individual was unique at 27 Y-STR loci; gene diversity ranged from 0.5718 (DYS460) to 0.9371(DYF387S1). Twelve haplotypes were shared between 178 individuals, while only two haplotypes among these twelve were shared between 87 individuals in Hazara populations. Rst and Fst pairwise genetic distance analyses, multidimensional scaling plot, neighbor-joining tree, linear discriminatory analysis, and median-joining network were performed, which shed light on the history of Hazara and Baloch populations. The results of our study showed that the Yfiler Plus PCR Amplification Kit marker set provided substantially stronger discriminatory power in the Baloch population of Pakistan and the Hazara population across the Durand line.

The surname structure of Trentino (Italy) and its relationship with dialects and genes

Background Thanks to the availability of rich surname, linguistic and genetic information, together with its geographic and cultural complexity, Trentino (North-Eastern Italy) is an ideal place to test the relationships between genetic and cultural traits. Aim We provide a comprehensive study of population structures based on surname and dialect variability and evaluate their relationships with genetic diversity in Trentino. Subjects and methods Surname data were collected for 363 parishes, linguistic data for 57 dialects and genetic data for different sets of molecular markers (Y-chromosome, mtDNA, autosomal) in 10 populations. Analyses relied on different multivariate methods and correlation tests. Results Besides the expected isolation-by-distance-like patterns (with few local exceptions, likely related to sociocultural instances), we detected a significant and geography-independent association between dialects and surnames. As for molecular markers, only Y-chromosomal STRs seem to be associated with the dialects, although no significant result was obtained. No evidence for correlation between molecular markers and surnames was observed. Conclusion Surnames act as cultural markers as do other words, although in this context they cannot be used as reliable proxies for genetic variability at a local scale.

Genetic Polymorphism of 17 Y-Chromosomal STR Loci in the “On To’rt Urıw” Tribes of Karakalpak Population

Genetic Polymorphism of 17 Y-Chromosomal STR Loci in the “On To’rt Urıw” Tribes of Karakalpak Population

Ancestry and identity in Bronze Age Catacomb culture burials: A meta-tale of graves, skeletons, and DNA

After discovering the first kurgans in the steppes, the archaeologists were faced with the need to determine the social status of buried persons and the relationship between people buried within the same necropolis. Archaeology has developed its methods and criteria for assessing the social status of buried persons, such as the size of the burial kurgans, the location of burials in the center or on the periphery of the kurgan, the wealth of implements, etc. With the introduction of paleogenetic methods into archeology, new opportunities for research in this direction are opening up. The analysis of ancient DNA is a tool that allows you not to assume but to establish consanguinity.This study presents the archaeological and molecular analysis of human remains from the East-Manych variant of the Catacomb culture. Catacomb culture dominated eastern Ukraine and southern Russia in the 3rd millennium BCE. The skeletons were recovered from kurgans of the Ergeninskii kurgan group in Kalmykia (Russia) that were radiocarbon dated the Bronze Age (25th–23rd century BCE). Y-chromosome STR analysis revealed that both individuals belonged to haplogroup R1b. This paternal lineage appears at high frequency in central, western, and northern Europe, and commonly appears among the Yamnaya. Analysis of mitochondrial DNA variation revealed the Catacomb males to belong to haplogroups H and N, respectively, both of which also appeared in the Yamnaya. These genetic data suggest a possible relationship between the Catacomb and Yamnaya cultures and contribute to our understanding of the cultural and historical processes occurring in the steppes of Eastern Europe during the Bronze Age.

Haplotype diversity and phylogenetic relationship analysis of Chinese Yulin Han population using 59 Y-STR loci of two novel Y-STR typing systems

To investigate the genetic polymorphisms of 59 Y-chromosomal short tandem repeat (Y-STR) loci in the Yulin Han population, 229 unrelated healthy male individuals were analyzed using AGCU Y37 kit and AGCU Y-SUPP Plus kit. A total of 227 different haplotypes were obtained at the 59 Y-STR loci. Among them, 225 haplotypes were unique and 2 haplotypes occurred twice. The overall haplotypic diversity and discrimination capacity were 0.9999 and 0.9913, respectively. The phylogenetic relationships between the studied Yulin Han population and 17 previously reported reference populations were evaluated via multidimensional scaling and Neighbor-Joining analyses based on the haplotypic frequencies of ‘YHRD Maximal Loci’. Phylogenetic analysis revealed that Yulin Han population was closely related to Chinese Han and Hunan Yao populations. These results demonstrated that the 59 Y-STR loci were useful for forensic applications and population genetic studies.

Population data and genetic structure analysis based on 29 Y-STR loci among the ethnolinguistic groups in Burkina Faso.

Burkina Faso (BF) is a landlocked Sahelian country located in the middle of West Africa. Sixty-three local languages are spoken in BF. Despite this high diversity, the BF population remains poorly investigated, and updated forensic parameters with a large number of Y chromosome short tandem repeats (Y-STRs) are still missing. Herein, 447 DNA samples were typed for a cocktail of 29 Y-STR loci. None of these 447 individuals in total shared a common haplotype. The overall Y-STR haplotypes were successfully uploaded online on the Y-STR Haplotype Reference Database (YHRD) with the accession numbers YA004690 and YA004691. The main haplotype diversity was 0.9999999965, which is much higher than that obtained with 12 Y-STRs in a previous study. Haploid Match Probability for the whole dataset was 0.002237. The phylogenetic analysis of 24 ethnic groups of BF shows that the ethnic group named BISSA is closer to Gur speakers than Mande speakers, where they belong. In addition, genetic structure analysis of 49 African subpopulations sheds light on the fact that geographic proximity turns out to be one of the best predictors of genetic affinity between populations.

Population data of 27 Y-chromosome STRS in Aymara population from Peru

ABSTRACT In the present study, 90 samples from unrelated Aymaran males from the province of Puno were characterized with 27 Y-chromosome short tandem repeat (STR) loci using the Yfiler ™ Plus PCR Amplification Kit. Out of a total of the 90 haplotypes there were 89 distinct haplotypes. The statistical parameters applied to the data were haplotype diversity (0.9998), discrimination capacity (0.9889) and match probability (0.0114). Provisional Y haplogroups (major clade) were assigned with the majority being Q. The Aymara population was more closely related to a Bolivian population (Bolivia) and Iquitos (Peru) and was further away from the Shipibo-Conibo population of Ucayali, (Peru) and Aboriginal (Australia). The data were deposited in the YHRD database (accession number: YA004703). The analyses carried out show that the Y-STR loci from the Aymarans can be used for human identity casework and could be useful to study the history of the human populations in Latin America.

Next generation sequencing of Y-STRs in father-son pairs and comparison with traditional capillary electrophoresis

To evaluate the promising advantages of massively parallel sequencing (MPS) in our casework, we analysed a total of 33 Y-chromosomal short tandem repeats (Y-STRs) with traditional capillary electrophoresis (CE) and 25 Y-STRs using the newer MPS technology. We studied the outcome of both technologies in 64 father-son pairs using stock and custom-designed kits. Current MPS technology confirmed the 13 mutational events observed with CE and improved our understanding of the complex nature of STR mutations. By detecting isometric sequence variants between unrelated males, we show that sequencing Y-STRs using MPS can boost discrimination power.

Mutation analysis for 25 Y-STR markers in Japanese population.

In this study, we analyzed DNA samples from 213 Japanese father son pairs with 25 Y-chromosome short tandem repeat (Y-STR) (DYS576, DYS389I, DYS635, DYS389II, DYS627, DYS460, DYS458, DYS19, YGATAH4, DYS448, DYS391, DYS456, DYS390, DYS438, DYS392, DYS518, DYS570, DYS437, DYS385, DYS449, DYS393, DYS439, DYS481, DYF387S1, and DYS533) markers using the Yfiler™ Plus PCR amplification kit. We calculated Y-STR mutation rates for each locus to evaluate the efficacy of the 25 Y-STR markers for paternity testing and forensic identification using samples from male relatives. Six rapidly mutating Y-STR markers (DYS576, DYS627, DYS518, DYS570, DYS449 and DYF387S1), previously reported to have high mutation rates (>1.0×10-2), are included in the 25 Y-STR markers, but our findings revealed that the mutation rates for all Y-STR markers except for DYS576 and DYS458 were lower than 1.0×10-2. Therefore, the use of these 25 Y-STR markers may be useful for forensic identification in the Japanese population.

Developmental validation of the MGIEasy Signature Identification Library Prep Kit, an all-in-one multiplex system for forensic applications.

Analyzing genetic markers in nuclear and mitochondrial genomes is helpful in various forensic applications, such as individual identifications and kinship analyses. However, most commercial kits detect these markers separately, which is time-consuming, laborious, and more error-prone (mislabelling, contamination, ...). The MGIEasy Signature Identification Library Prep Kit (hereinafter "MGIEasy identification system"; MGI Tech, Shenzhen, China) has been designed to provide a simple, fast, and robust way to detect appropriate markers in one multiplex PCR reaction: 52 autosomal STRs, 27 X-chromosomal STRs, 48 Y-chromosomal STRs, 145 identity-informative SNPs, 53 ancestry-informative SNPs, 29 phenotype-informative SNPs, and the hypervariable regions of mitochondrial DNA (mtDNA). Here, we validated the performance of MGIEasy identification system following the guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM), assessing species specificity, sensitivity, mixture identification, stability under non-optimal conditions (degraded samples, inhibitor contamination, and various substrates), repeatability, and concordance. Libraries prepared using MGIEasy identification system were sequenced on a MGISEQ-2000 instrument (MGI Tech). MGIEasy-derived STR, SNP, and mtDNA genotypes were highly concordant with CE-based STR genotypes (99.79%), MiSeq FGx-based SNP genotypes (99.78%), and Sanger-based mtDNA genotypes (100%), respectively. This system was strongly human-specific, resistant to four common PCR inhibitors, and reliably amplified both low quantities of DNA (as low as 0.125ng) and degraded DNA (~ 150nt). Most of the unique alleles from the minor contributor were detected in 1:10 male-female and male-male mixtures; some minor Y-STR alleles were even detected in 1:1000 male-female mixtures. MGIEasy also successfully directly amplified markers from blood stains on FTA cards, filter papers, and swabs. Thus, our results demonstrated that MGIEasy identification system was suitable for use in forensic analyses due to its robust and reliable performance on samples of varying quality and quantity.

Exploitation of a novel slowly mutating Y-STRs set and evaluation of slowly mutating Y-STRs plus Y-SNPs typing strategy in forensic genetics and evolutionary research.

The Y-chromosome short tandem repeats (Y-STRs) loci with different mutation rates existing in the Y chromosome non-recombination region (NRY) allow to be applied in human forensics, genealogical researches, historical investigations and evolutionary studies. Currently, there is a high demand for pedigree search to narrow the scope of crime investigations. However, the commonly used Y-STRs kits generally contain Y-STRs with high mutation rates that could cause individuals from the same pedigree to display different haplotypes. Herein, we put forward a new strategy of Slowly Mutating (SM) Y-STRs plus Y-SNPs typing, which could not only improve the resolution and accuracy of pedigree search, but also be applicable to evolutionary research. First, we developed a nine SM Y-STRs assay by evaluating their mutation rates in 210 pedigrees. Then the gene diversity and efficiency of the SM Y-STRs and 172 Y-SNPs sets were investigated by 2304 unrelated males from 24 populations. Furthermore, network and time estimation analyses were performed to evaluate the new strategy's capability to reconstruct phylogenetic tree and reliability to infer the time to the most recent common ancestor (TMRCA). The nine SM Y-STRs assay even had a higher resolution and a comparable capacity of revealing population genetic differentiation compared to 172 Y-SNPs system. This new strategy could optimize the phylogenetic tree generated by commonly used Y-STR panels and obtain a quite consistent time estimations with the published dating.

Genetic structure and forensic characterization of 36 Y-chromosomal STR loci in Tibeto-Burman-speaking Yi population.

BackgroundMale‐specifically inherited Y‐STRs have been widely used in population genetics and forensic investigations.MethodsWe genotyped and analyzed Y chromosome haplotypes of 408 unrelated Tibeto‐Burman‐speaking Yi male individuals from Guizhou using Goldeneye® Y‐PLUS kit. Population comparisons between the Guizhou Yi and 67 reference groups were performed via the AMOVA, MDS, and phylogenetic relationship reconstruction.ResultsA total of 389 alleles and 396 haplotypes could be detected, and the allelic frequencies ranged from 0.0025 to 0.9875. The haplotype diversity, random match probability, and discrimination capacity values were 0.9999, 0.0026, and 0.9900, respectively. The gene diversity (GD) of 36 Y‐STR loci in the studied group ranged from 0.0248 (DYS645) to 0.9601 (DYS385a/b). Our newly genotyped Yi samples show a close affinity with other Tibeto‐Burman speaking groups in China and Southeast Asia.ConclusionsThe population stratification was almost consistent with the geographic distribution and language‐family, both among Chinese and worldwide ethnic groups. Our data may provide useful information for paternal lineage in the forensic application and population genetics, as well as evidence for archaeological and historical research.

Autosomal genetics and Y-chromosome haplogroup L1b-M317 reveal Mount Lebanon Maronites as a persistently non-emigrating population.

Currently, there are 18 different religious communities living in Lebanon. While evolving primarily within Lebanon, these communities show a level of local isolation as demonstrated previously from their Y-haplogroup distributions. In order to trace the origins and migratory patterns that may have led to the genetic isolation and autosomal clustering in some of these communities we analyzed Y-chromosome STR and SNP sample data from 6327 individuals, in addition to whole genome autosomal sample data from 609 individuals, from Mount Lebanon and other surrounding communities. We observed Y chromosome L1b Levantine STR branching that occurred around 5000 years ago. Autosomal DNA analyses suggest that the North Lebanese Mountain Maronite community possesses an ancestral Fertile Crescent genetic component distinct from other populations in the region. We suggest that the Levantine L1b group split from the Caucasus ancestral group around 7300 years ago and migrated to the Levant. This event was distinct from the earlier expansions from the Caucasus region that contributed to the wider Levantine populations. Differential cultural adaption by populations from the North Lebanese Mountains are clearly aligned with the L1b haplotype STR haplogroup clusters, indicating pre-existing and persistent cultural barriers marked by the transmission of L1b lineages. Our findings highlight the value of uniparental haplogroups and STR haplotype data for elucidating biosocial events among these populations.

Genetic polymorphisms of 44 Y chromosomal genetic markers in the Inner Mongolia Han population and its genetic relationship analysis with other reference populations

Y chromosomal genetic markers in the non-recombining region are commonly used for human evolution research, familial searching, and forensic male differentiation since they strictly follow paternal inheritance. Y chromosomal short tandem repeats (Y-STRs) possess extraordinarily advantages in forensic applications because of their high polymorphisms and special genetic pattern. Here, we assessed the genetic diversities of 41 Y-STRs and three Y chromosomal insertion/deletion (Y-InDels) loci in the Chinese Inner Mongolia Han population; besides, genetic differentiation analyses among the studied Han population and other previously reported populations were conducted based on 27 same Y-STRs. Totally, 425 alleles were observed in 324 Inner Mongolia Han individuals for these Y-markers. Gene diversities of these Y-markers distributed from 0.0306 to 0.9634. The haplotype diversity and discriminatory capacity of these Y-markers in the Inner Mongolia Han population were 0.9999 and 0.98457, respectively. Haplotype resolution comparisons of different Y-marker groups in the studied Han population revealed that higher haplotype resolution could be achieved for these 44 Y-markers. Population genetic analyses of the Inner Mongolia Han population and other reference populations demonstrated that the studied Han population had relatively closer genetic affinities with Northern Han Chinese populations than Southern Han and other minority groups. To sum up, these 44 Y-markers can be utilized as a valuable tool for male differentiation in the Inner Mongolia Han population.

27 Y-chromosomal STR haplotypic structure for the Chinese Han population from Changchun, Northeastern China.

Y- Changchun is the capital and largest city of Jilin Province in the northeast China. In this study, we genotyped and investigated haplotypes of 27 Y-STR loci in 1037 Changchun Han male individuals using commercially available AmpFlSTR Yfiler® Plus kit. We calculated the Gene diversity (GD) values and haplotype diversity (HD) as important forensic parameters. Furthermore, we observed genetic affinities between Changchun Han with other Northern Han Chinese populations and also Korans in Yanbian in the Multidimensional scaling and phylogenetic tree analysis.

A Y-chromosomal survey of Ecuador’s multi-ethnic population reveals new insights into the tri-partite population structure and supports an early Holocene age of the rare Native American founder lineage C3-MPB373

Ecuador is a multiethnic and pluricultural country with a complex history defined by migration and admixture processes. The present study aims to increase our knowledge on the Ecuadorian Native Amerindian groups and the unique South American Y-chromosome haplogroup C3-MPB373 through the analysis of up to 23 Y-chromosome STRs (Y-STRs) and several Y-SNPs in a sample of 527 Ecuadorians from 7 distinct populations and geographic areas, including Kichwa and non-Kichwa Native Amerindians, Mestizos and Afro-Ecuadorians. Our results reveal the presence of C3-MPB373 both in the Amazonian lowland Kichwa with frequencies up to 28 % and, for the first time, in notable proportions in Kichwa populations from the Ecuadorian highlands. The substantially higher frequencies of C3-MPB373 in the Amazonian lowlands found in Kichwa and Waorani individuals suggest a founder effect in that area. Notably, estimates for the time to the most recent common ancestor (TMRCA) in the range of 7.2–9.0 kya point to an ancient origin of the haplogroup and suggest an early Holocene expansion of C3-MPB373 into South America. Finally, the pairwise genetic distances (RST) separate the Kichwa Salasaka from all the other Native Amerindian and Ecuadorian groups, indicating a so far hidden diversity among the Kichwa-speaking populations and suggesting a more southern origin of this population. In sum, our study provides a more in-depth knowledge of the male genetic structure of the multiethnic Ecuadorian population, as well as a valuable reference dataset for forensic use.

Haplotype data and forensic evaluation of 23 Y-STR and 12 X-STR loci in eight ethnic groups from Eritrea

Eritrea is a multi-ethnic country of over 3 million of people consisting of different ethnic groups, having each its own language and cultural tradition. Due to the lack of population genetic data for markers of forensic interest, in this study, we analyzed the genetic polymorphisms of 23 Y-chromosome STR loci and of 12 X-chromosome STR loci in a sample of 255 unrelated individuals from 8 Eritrean ethnic groups, with the aim to generate a reference haplotype database for anthropological and forensic applications. X- and Y-chromosomes markers may indeed offer information especially in personal identification and kinship testing, when relying on the availability of large local population data to derive sufficiently accurate frequency estimates. The population genetic analyses in the Eritrean sample for both the two set of Y- and X-STR markers showed high power of discrimination both at country-based and population levels. Comparison population results highlight the importance of considering the ethnic composition within the analyzed country and the necessity of increasing available data especially when referring to heterogeneous populations such as the African ones.

Assessing 23 Y-STR loci mutation rates in Chinese Han father–son pairs from southwestern China

In this study, we have analyzed 23 Y-chromosomal short tandem repeats (Y-STRs) (DYS576, DYS389I, DYS389II, DYS448, DYS19, DYS391, DYS481, DYS549, DYS533, DYS438, DYS437, DYS570, DYS635, DYS390, DYS439, DYS392, DYS643, DYS393, DYS458, DYS460, DYS385ab, DYS456 and Y-GATA-H4) in 175 father-son sample pairs using a Microreader™ 24Y Direct ID system. Sixteen repeat mutations of father-son pairs at 10 loci, including three mutations at DYS570, 2 mutations at DYS549, DYS460, DYS458, and DYS576, and 1 mutation at other five loci, were revealed. Furthermore, all of the observed repeat mutations were single repeat changes with 5 (31.25%) repeat insertions and 11 (68.75%) repeat deletions. The deletion rate is more than two fold higher than of insertions (11:5 = 2.2-fold). Locus-specific mutation rates estimated varied between 5.71 × 10-3 (CI from 0.1 × 10-3 to 31.4 × 10-3) and 1.71 × 10-2 (CI from 3.6 × 10-3 to 49.3 × 10-3) for the 23 Y-STRs. An average mutation rate across all 23 Y-STR markers was estimated as 3.97 × 10-3 (CI 2.3 × 10-3 to 6.4 × 10-3). Thus, locus-specific mutation rates in DYS460, DYS458, and DYS438, estimated are much higher than previously published comprehensive data, but an average mutation rate across all 23 Y-STR markers is similar to previous reports (3.97 × 10-3 vs 4.34 × 10-3). These results by characterizing Y-STR mutations will not only provided new information for Y-STR mutations but also might be important for paternal lineage identification, kinship analysis, and family relationship reconstruction in our forensic Y-STR analysis.

Next generation sequencing of STR artifacts produced from historical bone samples

STR artifacts are commonly observed in electrophoretic data and can complicate interpretation of the profiles produced. Even when a consensus approach is applied, reproducible artifacts have the potential to convolute the analysis. DNA obtained from historical bone samples is often heavily degraded and damaged, requiring the use of more sensitive procedures to increase allele recovery. Additionally, skeletal remains exposed to environmental conditions may be afflicted with microbial DNA contamination that cross-reacts with the primers during short tandem repeat (STR) multiplex amplification. STR artifacts manifested as a result of these circumstances can be sourced and characterized using new sequencing technologies to potentially ease the analysis burden. For this study, PCR product from 17 low-quality bone samples exhibiting reproducible autosomal and Y-chromosomal STR (Y-STR) artifacts in capillary electrophoresis (CE) data were sequenced with next generation sequencing (NGS). Sequenced reads were bioinformatically sorted using STRait Razor to determine the authenticity of alleles and confirm the profile generated by CE. Sequence data from the PCR products and a subset of the associated extracts were further analyzed with Kaiju to classify the microbial species present and identify potential sources of artifact peaks. A suspected Y-STR artifact was similar in sequence to Pseudomonas sp. BAY1663, a species ubiquitously found in soil. Regions of homology were observed between the Pseudomonas genome and the presumed primer binding locations for Y-STRs included in the AmpFlSTR Y-Filer STR kit. Characterization of such supposed artifact peaks may aid in interpretation of CE data and ultimately lead to increased confidence in the reported results.