A centralized model for creating shared,standardized, microsatellite data that simplifies inter-laboratory collaboration

We demonstrate an efficient model for standardizing microsatellite DNA data among laboratories studying Oncorhynchus mykiss. Eight laboratories standardized 13 microsatellite loci following allele nomenclature of a central laboratory (average inter-laboratory genotyping concordance >98%). Following this central model, we have currently standardized 298 alleles from throughout the species native range. Although we focus here on O. mykiss, our experiences and recommendation apply equally to other broadly distributed species that may benefit from multi-laboratory collaborative data collection.     

combi.pl: a computer program to combine data sets with inconsistent microsatellite marker allele size information

Combining two data sets with allele information from overlapping microsatellite markers is often desirable, particularly in population genetic studies where a substantial body of published data exists. When genotyping is performed in different laboratories, allele size calling may not be presumed to be consistent. Our approach solves this problem by assigning allele sizes across studies using maximum-likelihood theory. Using data overlaps in samples and markers, allele shifts between two studies are calculated for each overlapping marker and a single file containing allele frequencies of consistent alleles is produced. The program (combi.pl) is written in PERL and available at http://data40.uni-tz.gwdg.de/~htaeube.     

Comparison of three microsatellite analysis methods for detecting genetic diversity in <Emphasis Type="Italic">Phytophthora sojae</Emphasis> (Stramenopila: Oomycete)

Analysis of an organism’s genetic diversity requires a method that gives reliable, reproducible results. Microsatellites are robust markers, however, detection of allele sizes can be difficult with some systems as well as consistency among laboratories. In this study, our two laboratories used 219 isolates of Phytophthora sojae to compare three microsatellite methods. Two capillary electrophoresis methods, the Applied Biosystems 3730 Genetic Analyzer and the CEQ 8000 Genetic Analysis system, detected an average of 2.4-fold more alleles compared to gel electrophoresis with a mean of 8.8 and 3.6 alleles per locus using capillary and gel methods, respectively. The two capillary methods were comparable, although allele sizes differed consistently by an average of 3.2 bp across isolates. Differences between capillary methods could be overcome if reference standard DNA genotypes are shared between collaborating laboratories.     

人类短串联重复序列HUMTH01基因座的遗传多态性

作者用扩增片段长度多态技术分析了人类短串联重复序列ＨＵＭＴＨ０１基因型及等位基因频率在中国成都地区汉族群体和德国科隆地区白人中的分布。用同步电泳技术比较不同引物ＰＣＲ产物分型结果,评估不同实验室ＨＵＭＴＨ０１群体数据的可比性。计算了２５个群体间ＨＵＭＴＨ０１ＳＴＲ的遗传距离,并构建了系统树。ＨＵＭＴＨ０１ＳＴＲ系统树分析不仅与传统遗传标记结果一致,并且获得了群体遗传的新线索。     

Standardizing selection and its consequences for genetic population structure

Type of selection for total fitness, specific for domestic animals and termed by us standardizing selection, is discussed. Consequences of such selection for the population structure genetic are considered taking a population of domestic pigs as an example. Examples of effective and ineffective standardizing selection are presented. Specific features of genetic determination of ten quantitative traits detected in analysis of standardizing selection are discussed.     

Standardized selection and its consequences for genetic population structure (examplified by Sus scrofa L.)

Type of selection for total fitness, specific for domestic animals and termed by us standardizing selection, is discussed. Consequences of such selection for the population structure genetic are considered taking a population of domestic pigs as an example. Examples of effective and ineffective standardizing selection are presented. Specific features of genetic determination often quantitative traits detected in analysis of standardizing selection are discussed.     

Plant names in vegetation databases – a neglected source of bias

Problem: The increasing availability of large vegetation databases holds great potential in ecological research and biodiversity informatics, However, inconsistent application of plant names compromises the usefulness of these databases. This problem has been acknowledged in recent years, and solutions have been proposed, such as the concept of “potential taxa” or “taxon views”. Unfortunately, awareness of the problem remains low among vegetation scientists. Methods: We demonstrate how misleading interpretations caused by inconsistent use of plant names might occur through the course of vegetation analysis, from relevés upward through databases, and then to the final analyses. We discuss how these problems might be minimized. Results: We highlight the importance of taxonomic reference lists for standardizing plant names and outline standards they should fulfill to be useful for vegetation databases. Additionally, we present the R package vegdata, which is designed to solve name‐related problems that arise when analysing vegetation databases. Conclusions: We conclude that by giving more consideration to the appropriate application of plant names, vegetation scientists might enhance the reliability of analyses obtained from large vegetation databases.     

A standard panel of microsatellites for Asian seabass (Lates calcarifer)

Microsatellites are the most popular markers for parentage assignment and population genetic studies. To meet the demand for international comparability for genetic studies of Asian seabass, a standard panel of 28 microsatellites has been selected and characterized using the DNA of 24 individuals from Thailand, Malaysia, Indonesia and Australia. The average allele number of these markers was 10.82 ± 0.71 (range: 6–19), and the expected heterozygosity averaged 0.76 ± 0.02 (range: 0.63–1.00). All microsatellites showed Mendelian inheritance. In addition, eight standard size controls have been developed by cloning a set of microsatellite alleles into a pGEM‐T vector to calibrate allele sizes determined by different laboratories, and are available upon request. Seven multiplex PCRs, each amplifying 3–5 markers, were optimized to accurately and rapidly genotype microsatellites. Parentage assignment using 10 microsatellites in two crosses (10 × 10 and 20 × 20) demonstrated a high power of these markers for revealing parent‐sibling connections. This standard set of microsatellites will standardize genetic diversity studies of Asian seabass, and the multiplex PCR sets will facilitate parentage assignment.     

Naming CHO cells for bio-manufacturing: Genome plasticity and variant phenotypes of cell populations in bioreactors question the relevance of old names

Chinese Hamster Ovary [CHO] cells are the workhorse for production of modern biopharmaceuticals. They are however immortalized cells with a high propensity for genetic change. Judging from published culture records, CHO cell populations have undergone hundreds of population doublings since their origin in the late 1950s. Different cell populations were established and named from 1 to 3 decades after their generation, such as CHO-Pro–, CHO-K1, CHO-DG44, CHO-S, CHO-DUK, CHO-DXB-11 to indicate origin and certain phenotypic features. These names are commonly used in scientific publications still today. This article discusses the relevance of such names. We argue that they provide a false sense of identity. To substantiate this, we provide the long (and poorly recorded) history of CHO cells as well as their highly complex genetics. Finally, we suggest an alternative naming system for CHO cells which provides more relevant information. While the implementation of a new naming convention will require substantial discussions among members of the relevant community, it should improve interpretation and comparability between laboratories. This, in turn will help scientific communities and industrial users to attain and further the full potential of CHO cells.     

Validation of single nucleotide polymorphism quantification in pooled DNA samples with SNaPIT. A glycosylase-mediated methods for polymorphism detection method

Association studies using genome scans to identify quantitative trait loci for multifactorial disorders, with anything approaching reasonable power, have been compromised by the need for a very dense array of genetic markers and large numbers of affected individuals. These requirements impose enormous burdens on the genotyping capacity for most laboratories. DNA pooling has been proposed as a possible approach to reduce genotyping costs and effort. We report on the application of the SNaPIT™ technology to evaluate allele frequencies in pooled DNA samples and conclude that it offers a cost effective, efficient and accurate estimator and provides several advantages over competing technologies in this regard.     

A splicing defect due to an exon-intron junctional mutation results in abnormal beta-hexosaminidase alpha chain mRNAs in Ashkenazi Jewish patients with Tay-Sachs disease

Abnormal beta-hexosaminidase alpha chain mRNAs from an Ashkenazi Jewish patient with the classical infantile Tay-Sachs disease contained intact or truncated intron 12 sequences. Sequence analysis showed a single nucleotide transversion at the 5' donor site of intron 12 from the normal G to C. This provides the first evidence that this junctional mutation, also found independently in two other laboratories by analysis of genomic clones, results in functional abnormality. Analysis with normal and mutant oligonucleotides as probes indicated that our patient was a compound heterozygote with only one allele having the transversion. The patient studied in the other two laboratories was also a compound heterozygote. Another Ashkenazi Jewish patient was normal in this region in both alleles. Thus, the splicing defect is the underlying genetic cause in some but not all Ashkenazi Jewish patients with Tay-Sachs disease.     

Single nucleotide polymorphism genotyping by heteroduplex analysis in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) are increasingly used for cultivar identification, construction of genetic maps, genetic diversity assessment, association mapping and marker-assisted breeding. Although there are several highly sensitive methods for the detection of polymorphisms, most of them are often beyond the budget of medium-throughput academic laboratories or seed companies. Heteroduplex analysis by enzymatic cleavage (CEL1CH) or denaturing high-performance liquid chromatography (dHPLC) has been successfully used to examine genetic variation in several plant and animal species. In this work, we assess and compare the performance of both methods in sunflower by genotyping SNPs from a set of 24 selected polymorphic candidate genes. The CEL1CH method allowed us to accurately detect allele differences in 10 out of 24 regions using an in-house prepared CEL1 enzyme (celery single strand endonuclease 1, Apium graveolens L.). Similarly, a total of 11 regions were successfully optimized for dHPLC analysis. As a scaling-up approach, both strategies were tested to genotype either 42 SNPs/indels in 22 sunflower accessions from the local germplasm bank or 33 SNPs/indels in 90 recombinant inbred lines (RILs) for genetic mapping purposes. Summarizing, a total of 601 genotypes were efficiently analyzed either with CEL1CH (110) or dHPCL (491). In conclusion, CEL1CH and dHPLC proved to be robust, complementary methods, allowing medium-scale laboratories to scale up the number of both SNPs and individuals to be included in genetic studies and targeted germplasm diversity characterization (EcoTILLING).     

Characterization of 12 microsatellite loci of the human MHC in a panel of reference cell lines

 The human genome contains a large number of interspersed microsatellite repeats which exhibit a high degree of polymorphism and are inherited in a Mendelian fashion, making them extremely useful genetic markers. Several microsatellites have been described in the HLA region, but allele nomenclature, a set of broadly distributed controls, and typing methods have not been standardized, which has resulted in discrepant microsatellite data between laboratories. In this report we present a detailed protocol for genotyping microsatellites using a semi-automated fluorescence-based method. Twelve microsatellites within or near the major histocompatibility complex (MHC) were typed in the 10th International Histocompatibility Workshop homozygous typing cell lines (HTCs) and alleles were designated based on size. All loci were sequenced in two HTCs providing some information on the level of complexity of the repeat sequence. A comparison of allele size obtained by genotyping versus that obtained by direct sequencing showed minor discrepancies in some cases, but these were not unexpected given the technical differences in the methodologies. Fluorescence-based typing of microsatellites in the MHC described herein is highly efficient, accurate, and reproducible, and will allow comparison of results between laboratories. Received: 10 May 1997 / Revised: 1 August 1997     

Microsatellite allele ladders in two species of Pacific salmon: preparation and field‐test results

To support microsatellite data communication, we have developed a convenient method for creating locus‐specific microsatellite allele ladders used to align data from different laboratories. The ladders were constructed by pooling polymerase chain reaction (PCR) products to create a template for amplification. Four ladders were field‐tested in six different laboratories using different genotyping platforms. Despite substantial differences in absolute size estimates of DNA fragments, each laboratory correctly scored unknown sample genotypes according to the ladder designations. The results indicate that our simple preparation method provides reliable allele ladders in a time‐efficient manner for verifying microsatellite genotypes across platforms.     

Technical reproducibility of genotyping SNP arrays used in genome-wide association studies

During the last several years, high-density genotyping SNP arrays have facilitated genome-wide association studies (GWAS) that successfully identified common genetic variants associated with a variety of phenotypes. However, each of the identified genetic variants only explains a very small fraction of the underlying genetic contribution to the studied phenotypic trait. Moreover, discordance observed in results between independent GWAS indicates the potential for Type I and II errors. High reliability of genotyping technology is needed to have confidence in using SNP data and interpreting GWAS results. Therefore, reproducibility of two widely genotyping technology platforms from Affymetrix and Illumina was assessed by analyzing four technical replicates from each of the six individuals in five laboratories. Genotype concordance of 99.40% to 99.87% within a laboratory for the sample platform, 98.59% to 99.86% across laboratories for the same platform, and 98.80% across genotyping platforms was observed. Moreover, arrays with low quality data were detected when comparing genotyping data from technical replicates, but they could not be detected according to venders' quality control (QC) suggestions. Our results demonstrated the technical reliability of currently available genotyping platforms but also indicated the importance of incorporating some technical replicates for genotyping QC in order to improve the reliability of GWAS results. The impact of discordant genotypes on association analysis results was simulated and could explain, at least in part, the irreproducibility of some GWAS findings when the effect size (i.e. the odds ratio) and the minor allele frequencies are low.     

Genetic and cultural transmission in Sicily as revealed by names and surnames

The study of names as cultural characters and of surnames, which behave like genetic markers, is useful for comparing cultural and genetic transmission. Genetic transmission has a unique vertical component, which also can be present in the transmission of cultural traits associated with a horizontal (or epidemic) component resulting from local customs or fashion. Our aims in this study are to infer genetic patterns in Sicily from surnames and names and to evaluate and compare the consequences of vertical versus horizontal transmission of cultural markers. Names and surnames of 88,383 consanguineous spouses collected in 16 dioceses of Sicily were analyzed by multivariate analysis to reveal and compare the geographic clusters obtained from both sets of data. As a result, both data sets indicate a major separation between the eastern and the western region of Sicily. Also, distance matrices obtained from names are highly correlated with those from surnames. But names seem to form fewer and larger geographic clusters, whereas surnames are more greatly subdivided into smaller clusters. The most common male names present a different pattern from surnames. Vertical transmission is the cause of the similarity of the main geographic patterns of names and surnames and their correspondence with findings from geography of genes, and horizontal cultural transmission explains the major differences. Furthermore, the genetic and cultural affinities can be correlated with the historical background of Sicily.     

SNPselector: a web tool for selecting SNPs for genetic association studies

SUMMARY: Single nucleotide polymorphisms (SNPs) are commonly used for association studies to find genes responsible for complex genetic diseases. With the recent advance of SNP technology, researchers are able to assay thousands of SNPs in a single experiment. But the process of manually choosing thousands of genotyping SNPs for tens or hundreds of genes is time consuming. We have developed a web-based program, SNPselector, to automate the process. SNPselector takes a list of gene names or a list of genomic regions as input and searches the Ensembl genes or genomic regions for available SNPs. It prioritizes these SNPs on their tagging for linkage disequilibrium, SNP allele frequencies and source, function, regulatory potential and repeat status. SNPselector outputs result in compressed Excel spreadsheet files for review by the user. AVAILABILITY: SNPselector is freely available at http://primer.duhs.duke.edu/