High-throughput Genotyping Platforms Research Articles

Targeted resequencing of coding DNA sequences for SNP discovery in nonmodel species.

Targeted capture coupled with high-throughput sequencing can be used to gain information about nuclear sequence variation at hundreds to thousands of loci. Divergent reference capture makes use of molecular data of one species to enrich target loci in other (related) species. This is particularly valuable for nonmodel organisms, for which often no a priori knowledge exists regarding these loci. Here, we have used targeted capture to obtain data for 809 nuclear coding DNA sequences (CDS) in a nonmodel organism, the Eurasian lynx Lynx lynx, using baits designed with the help of the published genome of a related model organism (the domestic cat Felis catus). Using this approach, we were able to survey intraspecific variation at hundreds of nuclear loci in L.lynx across the species' European range. A large set of biallelic candidate SNPs was then evaluated using a high-throughput SNP genotyping platform (Fluidigm), which we then reduced to a final 96 SNP-panel based on assay performance and reliability; validation was carried out with 100 additional Eurasian lynx samples not included in the SNP discovery phase. The 96 SNP-panel developed from CDS performed very successfully in the identification of individuals and in population genetic structure inference (including the assignment of individuals to their source population). In keeping with recent studies, our results show that genic SNPs can be valuable for genetic monitoring of wildlife species.

CHIIMP: An automated high-throughput microsatellite genotyping platform reveals greater allelic diversity in wild chimpanzees.

Short tandem repeats (STRs), also known as microsatellites, are commonly used to noninvasively genotype wild‐living endangered species, including African apes. Until recently, capillary electrophoresis has been the method of choice to determine the length of polymorphic STR loci. However, this technique is labor intensive, difficult to compare across platforms, and notoriously imprecise. Here we developed a MiSeq‐based approach and tested its performance using previously genotyped fecal samples from long‐term studied chimpanzees in Gombe National Park, Tanzania. Using data from eight microsatellite loci as a reference, we designed a bioinformatics platform that converts raw MiSeq reads into locus‐specific files and automatically calls alleles after filtering stutter sequences and other PCR artifacts. Applying this method to the entire Gombe population, we confirmed previously reported genotypes, but also identified 31 new alleles that had been missed due to sequence differences and size homoplasy. The new genotypes, which increased the allelic diversity and heterozygosity in Gombe by 61% and 8%, respectively, were validated by replicate amplification and pedigree analyses. This demonstrated inheritance and resolved one case of an ambiguous paternity. Using both singleplex and multiplex locus amplification, we also genotyped fecal samples from chimpanzees in the Greater Mahale Ecosystem in Tanzania, demonstrating the utility of the MiSeq‐based approach for genotyping nonhabituated populations and performing comparative analyses across field sites. The new automated high‐throughput analysis platform (available at https://github.com/ShawHahnLab/chiimp) will allow biologists to more accurately and effectively determine wildlife population size and structure, and thus obtain information critical for conservation efforts.

Multi-level genomic analyses suggest new genetic variants involved in human memory.

Development of high-throughput genotyping platforms provides an opportunity to identify new genetic elements related to complex cognitive functions. Taking advantage of multi-level genomic analysis, here we studied the genetic basis of human short-term (STM, n = 1623) and long-term (LTM, n = 1522) memory functions. Heritability estimation based on single nucleotide polymorphism showed moderate (61%, standard error 35%) heritability of short-term memory but almost zero heritability of long-term memory. We further performed a two-step genome-wide association study, but failed to find any SNPs that could pass genome-wide significance and survive replication at the same time. However, suggestive significance for rs7011450 was found in the shared component of the two STM tasks. Further inspections on its nearby gene zinc finger and at-hook domain containing and SNPs around this gene showed suggestive association with STM. In LTM, a polymorphism within branched chain amino acid transaminase 2 showed suggestive significance in the discovery cohort and has been replicated in another independent population of 1862. Furthermore, we performed a pathway analysis based on the current genomic data and found pathways including mTOR signaling and axon guidance significantly associated with STM capacity. These findings warrant further replication in other larger populations.

Clinical Impact and Prognostic Role of KRAS/BRAF/PIK3CA Mutations in Stage I Colorectal Cancer.

Stage I colorectal carcinoma has excellent prognosis, with 5-year survival rate up to 95%. The occurrence of lymphovascular invasion, tumor budding, high number of PDC, or lymph node micrometastases is associated with tumor progression. The aim of this study was to evaluate the mutational status of 62 stage I colorectal carcinomas (CRC) (taken from 37 patients surviving more than five years since the initial diagnosis and from 25 patients who died of disease) and to correlate it with histopathological features and the clinical outcome. Mutations of KRAS, NRAS, BRAF, and PIK3CA genes were analyzed through Myriapod Colon Status Kit, using the high-throughput genotyping platform Sequenom MassARRAY System. Mutations in those genes were found in 31 cases (50%) and mainly in those with poor prognosis. The most frequent mutations occurred at codons 12 and 13 of the KRAS gene (40% of cases). We found concomitant PIK3CA mutations in 5 cases (8%). The presence of PIK3CA mutations was mainly observed in tumors with poor prognosis and with unfavorable histopathological prognostic features. High PDC grade (P = 0.0112), the presence of tumor budding (P = 0.0334), LVI (P < 0.0001), KRAS mutations (P = 0.0228), PIK3CA mutations (P = 0.0214), multiple genetic mutations in KRAS and PIK3CA genes (P = 0.039), and nodal micrometastases (P < 0.0001) were significant prognostic variables for CSS. The presence of LVI was the only independent and statistically significant prognostic variable for CSS in our cohort of pTNM stage I CRCs. The analysis of KRAS/PIK3CA mutational status may be used to identify patients with stage I CRC at high risk of bad outcome and who may need additional treatments, including biological therapies.

Genome Wide Association Mapping of Seedling and Adult Plant Resistance to Barley Stripe Rust (Puccinia striiformis f. sp. hordei) in India.

Barley stripe rust is caused by Puccinia striiformis f.sp. hordei, (Psh), occurs worldwide, and is a major disease in South Asia. The aim of this work was to identify and estimate effects of loci underlying quantitative resistance to rust at seedling and adult plant stages. HI-AM panel of 261 barley genotypes consisting of released cultivars from North and South America, Europe, Australia, advanced breeding lines, and local landraces from ICARDA barley program were screened at seedling and adult plant stages for resistance to Psh. Seedling resistance was evaluated with the five prevalent Psh races in India. Screening for the adult plant stage resistance was also performed in two different locations by inoculating with a mixture of the five races used for seedling screeing. The panel was genotyped using DaRT-Seq high-throughput genotyping platform. The genome-wide association mapping (GWAM) showed a total of 45 QTL located across the seven barley chromosomes for seedling resistance to the five races and 18 QTL for adult plant stage resistance. Common QTL for different races at seedling stage were found on all chromosomes except on chromosome 1H. Four common QTL associated with seedling and adult plant stage resistance were found on chromosomes 2, 5, and 6H. Moreover, one of the QTL located on the long arm of chromosome 5H showed stable effects across environments for adult plant stage resistance. Several QTL identified in this study were also reported before in bi-parental and association mapping populations studies validating current GWAM. However 15 new QTL were found at adult plant stage on all chromosomes except the 4H, explaining up to 36.79% of the variance. The promising QTL detected at both stages, once validated, can be used for MAS in Psh resistance breeding program globally.

Molecular Markers Improve Breeding Efficiency in Apomictic Poa Pratensis L.

Kentucky bluegrass (Poa pratensis L.) is a highly adapted and important turfgrass species in cool-season climates. It has high and variable polyploidy, small and metacentric chromosomes, and a facultative apomictic breeding system. As a result of the polyploidy and apomixis, identifying hybrids for Mendelian selection, identifying fixed apomictic progeny of desirable hybridizations for cultivar development, or differentiating among cultivars with subtle phenotypic differences is challenging without the assistance of molecular markers. Herein, we show data and review previous research showing the uses and limitations of using molecular markers for hybrid detection, apomixis assessment, and cultivar discrimination. In order to differentiate among different apomictic offtypes, both molecular markers and flow cytometry are necessary. For assessing similarity among progeny of hybridizations, as well as discriminating among cultivars, sets of markers are necessary and cryptic molecular variation must be considered. High throughput genotyping platforms are critical for increased genotyping efficiency.

Genotyping of the ABCG2 gene using Matrix-Associated Laser Desorption/Ionisation, Time-of-Flight Mass Spectrometry.

The aim of this study was to evaluate the applicability of genotyping of the ABCG2 gene using MALDI-TOF MS and to estimate the allele frequency in the Japanese population. Jr (a-) phenotype has a prevalence of approximately 0·05% among Japanese blood donors; DNA-based genotyping was conducted to investigate the molecular basis of the Jr (a-) phenotype along with serological typing. To detect all SNPs of the ABCG2 gene, a high-throughput SNP genotyping platform is needed. Overall, 1004 Jr (a-) blood samples were collected from blood donors in Japan and pre-genotyped. To detect the SNPs of the ABCG2 gene using MALDI-TOF MS, polymerase chain reaction and unextend primer were designed. In total, 205 Jr (a-) samples were genotyped using MALDI-TOF MS analysis. The SNPs of 1004 Jr (a-) samples were identified using the HRM analysis and DNA sequencing, and 799 of 1004 (80%) Jr (a-) samples had the homozygous for c.376 T. The designed primers for MALDI-TOF MS perfectly detected the SNPs of the ABCG2 gene. A total of 205 Jr (a-) samples were genotyped using MALDI-TOF MS. Calling failures occurred in only two samples with the mutations c.736CT to c.376C and c.421C to c.421CA. The concordance rate between the pre-genotyped and MALDI-TOF MS-based genotyping results was very high (99·02%) for all ABCG2 alleles. Jr (a-) Japanese donors had almost the homozygous for c.376 T. However, detections of more than 20 SNPs of the ABCG2 gene for the JR blood group genotyping are needed. MALDI-TOF MS-based genotyping was highly concordant with the pre-genotyped results for all ABCG2 alleles.

Role of Genomic tools for Mungbean [Vigna radiata (L.) Wilczek] improvement

Molecular markers are routinely utilized worldwide in all major crops as a component of breeding. The pace of development of molecular markers, establishment of marker–trait associations for important agronomic traits and other genomic sources has been accelerated in other pulses than the mungbean. The efforts are underway to use high-throughput genotyping platforms besides developing more genomic resources. So far, progress in the use of marker-assisted selection as a part of mungbean breeding programmes has been very limited. In this article, we have reviewed the progress made, limitations encountered and future possibilities for the application of marker-assisted selection in the genetic improvement of mungbean crops.

Multi-Locus Mixed Model Analysis Of Stem Rust Resistance In Winter Wheat.

Genome-wide association mapping is a powerful tool for dissecting the relationship between phenotypes and genetic variants in diverse populations. With the improved cost efficiency of high-throughput genotyping platforms, association mapping is a desirable method of mining populations for favorable alleles that hold value for crop improvement. Stem rust, caused by the fungus f. sp. is a devastating disease that threatens wheat ( L.) production worldwide. Here, we explored the genetic basis of stem rust resistance in a global collection of 1411 hexaploid winter wheat accessions genotyped with 5390 single nucleotide polymorphism markers. To facilitate the development of resistant varieties, we characterized marker-trait associations underlying field resistance to North American races and seedling resistance to the races TTKSK (Ug99), TRTTF, TTTTF, and BCCBC. After evaluating several commonly used linear models, a multi-locus mixed model provided the maximum statistical power and improved the identification of loci with direct breeding application. Ten high-confidence resistance loci were identified, including SNP markers linked to and and at least three newly discovered resistance loci that are strong candidates for introgression into modern cultivars. In the present study, we assessed the power of multi-locus association mapping while providing an in-depth analysis for its practical ability to assist breeders with the introgression of rare alleles into elite varieties.

Introduction of High Throughput and Cost Effective SNP Genotyping Platforms in Soybean

We presented here the application of two in-plate SNP (single nucleotide polymorphism) genotyping platforms for soybean plants [Glycine max (L.) Merr.], KASP® (Kompetitive Allele Specific PCR genotyping, LGC Genomics) and TaqMan® (Life Technologies) respectively. These two systems offer us an ability to determine the genotypes of 384 individual samples accurately and efficiently by allele specific PCR in a single plate using typical PCR conditions. Both of the systems require small quantity of genomic DNA obtained from a simple DNA extraction. The genomic sequences containing target SNPs can easily be used as a basic blueprint to design the probes and primers of KASP® and TaqMan® assays whether the sequences are obtained from the genome sequence of soybean William 82 (Wm82.a2.v1), Illumina Soy50k SNPs, or parallel resequencing. Moreover, we listed the pros and cons of the two systems and explained the principles behind the platforms. The high call rate and clear clustering separation of the SNPs can be readily obtained from these platforms without conducting any assay optimization processes. These platforms can routinely be performed on 96/384-well plate format with or without an automation procedure. Therefore, these platforms are especially suitable for the SNP genotyping on a particular trait with a large sample size, gene fine mapping, and marker assisted selection. Further, they require little hands-on experience and achieve per-site and per-individual costs below that of current SSR, AFLP, RFLP, and SNP chip technologies. The platforms can be used for genotyping on a wide range of organisms due to their simplicity and flexibility of handling. Meanwhile, we also especially presented some of the advantages using KASP® SNP genotyping pipeline, which was cost effective in the selection of allele specific assay and therefore, efficiently facilitated the soybean genotyping across large numbers (thousands or more) of individual lines for a great range of markers (hundreds to thousands) in our laboratory.

BAYESIAN CLUSTERING OF INDONESIAN RICE GERMPLASM

Model-based clustering where the inference on the parameters follow the Bayesian principle has been used to cluster 467 accessions of Indonesian rice germplasm which consist of released varieties, landraces, introduction lines, improved lines and wild species. A model-based Bayesian cluster analysis of genotype data can be used to evaluate the genetic backgrounds of rice populations of interest. Such analyses can be used to infer population structure, assign individuals to sub populations, and to study hybrid populations. Thus, the goal of this research was to examine the genotype data of numerous accession of rice germplasm using the model bayesian cluster analysis. The 1536 SNP-chip design was performed for genome scanning of the accession using the high throughput genotyping platform, the data of which were used for clustering. The result indicated that the germplasm can be clustered into five cluster based on similarities on genetic profile, i.e. similarities in gene frequencies across genome among individuals. Each cluster can be identified by reference lines, i.e. the lines or varieties that their genetic profile uniquely belong to one cluster and do not have or very rare introgression from lines or varieties of other clusters. Many introgressions have been identified among lines in all clusters which indicated that most of Indonesia rice germplasm, including local and introduced varieties were the results of crosses that occurred either in naturally fixation or breeding program activities that crossed one line/varieties to the others. There is also cluster in which no reference line and almost all lines/varieties in that cluster are known to have same common specific phenotype, e.g. aromatic.

Genetic diversity and genomic strategies for improving drought and waterlogging tolerance in soybeans.

Drought and its interaction with high temperature are the major abiotic stress factors affecting soybean yield and production stability. Ongoing climate changes are anticipated to intensify drought events, which will further impact crop production and food security. However, excessive water also limits soybean production. The success of soybean breeding programmes for crop improvement is dependent on the extent of genetic variation present in the germplasm base. Screening for natural genetic variation in drought- and flooding tolerance-related traits, including root system architecture, water and nitrogen-fixation efficiency, and yield performance indices, has helped to identify the best resources for genetic studies in soybean. Genomic resources, including whole-genome sequences of diverse germplasms, millions of single-nucleotide polymorphisms, and high-throughput marker genotyping platforms, have expedited gene and marker discovery for translational genomics in soybean. This review highlights the current knowledge of the genetic diversity and quantitative trait loci associated with root system architecture, canopy wilting, nitrogen-fixation ability, and flooding tolerance that contributes to the understanding of drought- and flooding-tolerance mechanisms in soybean. Next-generation mapping approaches and high-throughput phenotyping will facilitate a better understanding of phenotype-genotype associations and help to formulate genomic-assisted breeding strategies, including genomic selection, in soybean for tolerance to drought and flooding stress.

Application of Genomic Technologies to the Breeding of Trees

The recent introduction of next generation sequencing (NGS) technologies represents a major revolution in providing new tools for identifying the genes and/or genomic intervals controlling important traits for selection in breeding programs. In perennial fruit trees with long generation times and large sizes of adult plants, the impact of these techniques is even more important. High-throughput DNA sequencing technologies have provided complete annotated sequences in many important tree species. Most of the high-throughput genotyping platforms described are being used for studies of genetic diversity and population structure. Dissection of complex traits became possible through the availability of genome sequences along with phenotypic variation data, which allow to elucidate the causative genetic differences that give rise to observed phenotypic variation. Association mapping facilitates the association between genetic markers and phenotype in unstructured and complex populations, identifying molecular markers for assisted selection and breeding. Also, genomic data provide in silico identification and characterization of genes and gene families related to important traits, enabling new tools for molecular marker assisted selection in tree breeding. Deep sequencing of transcriptomes is also a powerful tool for the analysis of precise expression levels of each gene in a sample. It consists in quantifying short cDNA reads, obtained by NGS technologies, in order to compare the entire transcriptomes between genotypes and environmental conditions. The miRNAs are non-coding short RNAs involved in the regulation of different physiological processes, which can be identified by high-throughput sequencing of RNA libraries obtained by reverse transcription of purified short RNAs, and by in silico comparison with known miRNAs from other species. All together, NGS techniques and their applications have increased the resources for plant breeding in tree species, closing the former gap of genetic tools between trees and annual species.

Genetics in chronic venous disease.

Chronic venous disease is highly prevalent in the Western world, with varicose veins being the most common form of clinical manifestation. With recent developments in sequencing technology, clinicians and geneticists alike are embarking on a journey to identify and unravel the genetic candidates of chronic venous disease. There is now currently substantial evidence to suggest the presence of genetic influences in the aetiology and pathology of venous disease. Despite this, the precise nature and profile of the genes involved in chronic venous disease remain a poorly understood entity. Moreover, it is strikingly apparent that the majority of venous genetic studies conducted over the past decade do not adhere to fundamental research principles. The emergence of high-throughput genotyping platforms permits a more systematic search for inherited components of venous disease. Pursuing a genome-wide frontier has the potential to reveal novel critical metabolic pathways and explain the genetic susceptibility of chronic venous disease. An expedited knowledge of the genetic factors in the aetiology of venous disease may translate into better prevention or treatment, which would benefit patients suffering from its clinical sequelae. Researchers should be urged to foster collaborative links and design a genome-wide case-control association study as an international consortium to provide a statistically robust paradigm in the field of chronic venous disease genetics. This will carry promise for clinically relevant progress and represent a first step towards better understanding of the genetics of chronic venous disease aetiology.

Identification of TRPCs genetic variants that modify risk for lung cancer based on the pathway and two-stage study

ObjectiveStore operated calcium channels (SOCCs) and Receptor-operated calcium channels (ROCCs) are important pathways participating in regulation of intracellular Ca2+ concentration in various cell types. The purpose of our study is to determine whether genetic variations in key components of SOCCs and ROCCs are associated with lung cancer risk. MethodsWe identified 236 tagSNPs in 9 key genes related to SOCCs and ROCCs (TRPC1, TRPC3, TRPC4, TRPC6, TRPC7, ORAI1, ORAI2, STIM1, and STIM2) and evaluated their association with lung cancer risk in a two-stage case-control study with a total of 2433 lung cancer cases and 2433 cancer-free controls using Illumina high throughput genotyping platform. ResultsWe found consistently significant associations of TRPC4 rs9547991 and rs978156, and TRPC7 rs11748198 with increased risk of lung cancer among the three kinds of sources of populations (additive model in combined population: adjusted OR=1.33, 95% CI=1.11–1.59 for rs9547991; adjusted OR=1.21, 95% CI=1.08–1.35 for rs978156; and adjusted OR=1.28, 95% CI=1.10–1.47 for rs11748198). When combining the effects of TRPC7 rs11748198, and TRPC4 rs9547991 and rs978156, subjects carrying “≥1” variant alleles had a 1.29-fold increased risk of lung cancer (95% CI=1.15–1.46), compared with those carrying “0” variant allele. Lung cancer risk significantly increased with the increasing number of variant alleles of the three SNPs in a dose-dependent manner (P for trend=7.2×10−7). ConclusionThese findings suggested that TRPC4 rs9547991 and rs978156, and TRPC7 rs11748198 were candidate susceptibility markers for lung cancer in Chinese population. Our study provides the epidemiological evidence supporting a connection between TRPC members and lung cancer risks.

Paired Primary and Metastatic Tumor Analysis of Somatic Mutations in Synchronous and Metachronous Colorectal Cancer.

PurposeAlthough the mutation status of KRAS is highly concordant in primary and metastatic lesions, it has not been generalized to other major pathway genes.Materials and MethodsIn this study, 41 genes were evaluated and the mutational profiles were compared in 46 colorectal cancer patients with paired surgical specimens of primary and metastatic lesions: synchronous (n=27) and metachronous (n=19) lesions. A high-throughput mass spectrometry-based genotyping platform validated by orthogonal chemistry, OncoMap v.4.4, was used to evaluate the formalin-fixed, paraffin-embedded surgical specimens. The patients’ demographics, tumor characteristics, and microsatellite instability status were analyzed by a retrospective chart review.ResultsIn this study,with OncoMap, mutationswere identified in 80.4% of patientswith the following frequency: KRAS (39.1%), TP53 (28.3%), APC (28.3%), PIK3CA (6.5%), BRAF (6.5%), and NRAS (4.3%). Although 19.6% (9/46) of the patients showed no gene mutations, 43.5% (20/46) and 37.0% (17/46) had mutations in one and two or more genes, respectively. The synchronous and metachronous lesions showed similar mutational profiles. Paired samples between primary and metastatic tumors differed in 7.4% (2/27) and 10.5% (2/19) for synchronous and metachronous according to OncoMap.ConclusionThese findings indicate the major pathway genes, including KRAS, TP53, APC, PIK3CA, BRAF, and NRAS, are often concordant between the primary and metastatic lesions regardless of the temporal relationship of metastasis.

High-throughput 18K SNP array to assess genetic variability of the main grapevine cultivars from Sicily

The viticulture of Sicily, for its vocation, is one of the most important and ancient forms in Italy. Autochthonous grapevine cultivars, many of which known throughout the world, have always been cultivated in the island from many centuries. With the aim to preserve this large grapevine diversity, previous studies have already started to assess the genetic variability among the Sicilian cultivars by using morphological and microsatellite markers. In this study, simple sequence repeat (SSR) were utilized to verify the true-to-typeness of a large clone collection (101) belonging to 21 biotypes of the most 10 cultivated Sicilian cultivars. Afterwards, 42 Organization Internationale de la Vigne et du Vin (OIV) descriptors and a high-throughput single nucleotide polymorphism (SNP) genotyping array (Vitis18kSNP) were applied to assess genetic variability among cultivars and biotypes of the same cultivar. Ampelographic traits and high-throughput SNP genotyping platforms provided an accuracy estimation of genetic diversity in the Sicilian germplasm, showing the relationships among cultivars by cluster and multivariate analyses. The large SNP panel defined sub-clusters unable to discern among biotypes, previously classified by ampelographic analysis, belonging to each cultivar. These results suggested that a very large number of SNP did not cover the genome regions harboring few morphological traits. Genetic structure of the collection revealed a clear optimum number of groups for K = 3, clustering in the same group a significant portion of family-related genotypes. Parentage analysis highlighted significant relationships among Sicilian grape cultivars and Sangiovese, as already reported, but also the first evidences of the relationships between Nero d’Avola and both Inzolia and Catarratto. Finally, a small panel of highly informative markers (12 SNPs) allowed us to isolate a private profile for each Sicilian cultivar, providing a new tool for cultivar identification.

Use of a High-Throughput Genotyping Platform (OncoMap) for RAS Mutational Analysis to Predict Cetuximab Efficacy in Patients with Metastatic Colorectal Cancer.

PurposeCetuximab demonstrates improved efficacy outcomes in patients with metastatic colorectal cancer (mCRC) harboring wild-type KRAS exon 2. Resistance to cetuximab is mediated by activating less frequent mutations in the RAS genes beyond KRAS exon 2. We performed extended RAS Mutational analysis using a high-throughput genotyping platform (OncoMap) and evaluated extended RAS analysis for predicting cetuximab efficacy in patients harboring wild-type KRAS exon 2 tumors following Sanger sequencing.Materials and MethodsExtended RAS analysis was performed on 227 wild-type KRAS exon 2 mCRC patients who received cetuximab as salvage treatment using OncoMap ver. 4.0. Targeted genes included exon 2, exon 3, and exon 4, both in KRAS and NRAS, and included BRAF exon 15. We assessed efficacy by the new RAS mutation status.ResultsThe OncoMap detected 57 additional mutations (25.1%): 25 (11%) in KRAS exon 2 and 32 (14.1%) beyond KRAS exon 2. Survival differences were observed after dividing patients into the wild-type RAS group (n=170) and mutant RAS group (n=57) using OncoMap. Progression-free survival was 4.8 months versus 1.8 months (hazard ratio [HR], 0.44; 95% confidence interval [CI], 0.32 to 0.61), and overall survival was 11.9 months versus 8.4 months (HR, 0.65; 95% CI, 0.47 to 0.88).ConclusionSanger sequencing is not sufficient for selecting candidates for cetuximab treatment. High-throughput extended RAS genotyping is a feasible approach for this purpose and identifies patients who might benefit from cetuximab treatment.

Development and evaluation of a high-throughput single nucleotide polymorphism multiplex assay for assigning pedigrees in common carp

Accurate pedigree information is critical when managing animal breeding programmes and ensure the highest rate of genetic gain. The abundance of available genomic data and the development of high-throughput genotyping platforms have facilitated the use of single nucleotide polymorphisms (SNPs) as the best DNA markers for genomic selection studies. Furthermore, the superior qualities of SNPs compared with those of microsatellite markers allow standardization between laboratories, which is crucial for developing an international set of markers for use in traceability studies. The objective of this study was to develop a high-throughput SNP array to assign common carp pedigrees accurately. A 48-SNP array was developed based on the Fluidigm genotyping platform, and a phylogenetic analysis was performed to distinguish different pedigrees. A likelihood-based approach was used to infer parental pairs, and the pair with the highest LOD score (log of the ratio of the likelihood given parentage to likelihood given non-parentage) was assigned. The SNP genotypes of the offspring and candidate parents tested were collected, and 94% of the offspring were assigned to the most-likely parent pair, which was consistent with the actual pedigree records. Using this SNP assay will allow implementation of offspring testing at large commercial farms where improved accuracy of pedigree assignments and genetic evaluations will increase genetic gains in the common carp aquaculture industry.

Identification of Novel Oncogenic Mutations in Thyroid Cancer

Thyroid cancer patients frequently have favorable outcomes. However, a small subset develops aggressive disease refractory to traditional treatments. Therefore, we sought to characterize oncogenic mutations in thyroid cancers to identify novel therapeutic targets that may benefit patients with advanced, refractory disease. Data on 239 thyroid cancer specimens collected between January 2009 and September 2014 were obtained from the Dana Farber/Brigham and Women's Cancer Center. The tumors were analyzed with the OncoMap-4 or OncoPanel high-throughput genotyping platforms that survey up to 275 cancer genes and 91 introns for DNA rearrangement. Of the 239 thyroid cancer specimens, 128 (54%) had oncogenic mutations detected. These 128 tumors had 351 different mutations detected in 129 oncogenes or tumor suppressors. Examination of the 128 specimens demonstrated that 55% (n= 70) had 1 oncogenic mutation, and 45% (n= 48) had more than 1 mutation. The 351 oncogenic mutations were in papillary (85%), follicular (4%), medullary (7%), and anaplastic (4%) thyroid cancers. Analysis revealed that 2.3% (n= 3 genes) of the somatic gene mutations were novel. These included AR (n= 1), MPL (n= 2), and EXT2 (n= 1), which were present in 4 different papillary thyroid cancer specimens. New mutations were found in an additional 13 genes known to have altered protein expression in thyroid cancer: BLM, CBL, CIITA, EP300, GSTM5, LMO2, PRAME, SBDS, SF1, TET2, TNFAIP3, XPO1, and ZRSR2. This analysis revealed that several previously unreported oncogenic gene mutations exist in thyroid cancers and may be targets for the development of future therapies. Further investigation into the role of these genes is warranted.