Dataset Information

A Comparison of Mainstream Genotyping Platforms for the Evaluation and Use of Barley Genetic Resources.

ABSTRACT: We compared the performance of two commonly used genotyping platforms, genotyping-by-sequencing (GBS) and single nucleotide polymorphism-arrays (SNP), to investigate the extent and pattern of genetic variation within a collection of 1,000 diverse barley genotypes selected from the German Federal ex situ GenBank hosted at IPK Gatersleben. Each platform revealed equivalent numbers of robust bi-allelic SNPs (39,733 and 37,930 SNPs for the 50K SNP-array and GBS datasets respectively). A small overlap of 464 SNPs was common to both platforms, indicating that the methodologies we used selectively access informative polymorphism in different portions of the barley genome. Approximately half of the GBS dataset was comprised of SNPs with minor allele frequencies (MAFs) below 1%, illustrating the power of GBS to detect rare alleles in diverse germplasm collections. While desired for certain applications, the highly robust calling of alleles at the same SNPs across multiple populations is an advantage of the SNP-array, allowing direct comparisons of data from related or unrelated studies. Overall MAFs and diversity statistics (π) were higher for the SNP-array data, potentially reflecting the conscious removal of markers with a low MAF in the ascertainment population. A comparison of similarity matrices revealed a positive correlation between both approaches, supporting the validity of using either for entire GenBank characterization. To explore the potential of each dataset for focused genetic analyses we explored the outcomes of their use in genome-wide association scans for row type, growth habit and non-adhering hull, and discriminant analysis of principal components for the drivers of sub-population differentiation. Interpretation of the results from both types of analysis yielded broadly similar conclusions indicating that choice of platform used for such analyses should be determined by the research question being asked, group preferences and their capabilities to extract and interpret the different types of output data easily and quickly. Access to the requisite infrastructure for running, processing, analyzing, querying, storing, and displaying either datatype is an additional consideration. Our investigations reveal that for barley the cost per genotyping assay is less for SNP-arrays than GBS, which translates to a cost per informative datapoint being significantly lower for the SNP-array.

SUBMITTER: Darrier B

PROVIDER: S-EPMC6499090 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The rapid development of next-generation sequencing platforms has enabled the use of sequencing for routine genotyping across a range of genetics studies and breeding applications. Genotyping-by-sequencing (GBS), a low-cost, reduced representation sequencing method, is becoming a common approach for whole-genome marker profiling in many species. With quickly developing sequencing technologies, adapting current GBS methodologies to new platforms will leverage these advancements for future studies. To test new semiconductor sequencing platforms for GBS, we genotyped a barley recombinant inbred line (RIL) population. Based on a previous GBS approach, we designed bar code and adapter sets for the Ion Torrent platforms. Four sets of 24-plex libraries were constructed consisting of 94 RILs and the two parents and sequenced on two Ion platforms. In parallel, a 96-plex library of the same RILs was sequenced on the Illumina HiSeq 2000. We applied two different computational pipelines to analyze sequencing data; the reference-independent TASSEL pipeline and a reference-based pipeline using SAMtools. Sequence contigs positioned on the integrated physical and genetic map were used for read mapping and variant calling. We found high agreement in genotype calls between the different platforms and high concordance between genetic and reference-based marker order. There was, however, paucity in the number of SNP that were jointly discovered by the different pipelines indicating a strong effect of alignment and filtering parameters on SNP discovery. We show the utility of the current barley genome assembly as a framework for developing very low-cost genetic maps, facilitating high resolution genetic mapping and negating the need for developing de novo genetic maps for future studies in barley. Through demonstration of GBS on semiconductor sequencing platforms, we conclude that the GBS approach is amenable to a range of platforms and can easily be modified as new sequencing technologies, analysis tools and genomic resources develop.

Project description:ImportanceIncreasing BRCA1 and BRCA2 (collectively termed herein as BRCA) gene testing is required to improve cancer management and prevent BRCA-related cancers.ObjectiveTo evaluate mainstream genetic testing using cancer-based criteria in patients with cancer.Design, setting, and participantsA quality improvement study and cost-effectiveness analysis of different BRCA testing selection criteria and access procedures to evaluate feasibility, acceptability, and mutation detection performance was conducted at the Royal Marsden National Health Service Foundation Trust as part of the Mainstreaming Cancer Genetics (MCG) Programme. Participants included 1184 patients with cancer who were undergoing genetic testing between September 1, 2013, and February 28, 2017.Main outcomes and measuresMutation rates, quality-adjusted life-years (QALYs), and incremental cost-effectiveness ratios were the primary outcomes.ResultsOf the 1184 patients (1158 women [97.8%]) meeting simple cancer-based criteria, 117 had a BRCA mutation (9.9%). The mutation rate was similar in retrospective United Kingdom (10.2% [235 of 2294]) and prospective Malaysian (9.7% [103 of 1061]) breast cancer studies. If traditional family history criteria had been used, more than 50% of the mutation-positive individuals would have been missed. Of the 117 mutation-positive individuals, 115 people (98.3%) attended their genetics appointment and cascade to relatives is underway in all appropriate families (85 of 85). Combining with the equivalent ovarian cancer study provides 5 simple cancer-based criteria for BRCA testing with a 10% mutation rate: (1) ovarian cancer; (2) breast cancer diagnosed when patients are 45 years or younger; (3) 2 primary breast cancers, both diagnosed when patients are 60 years or younger; (4) triple-negative breast cancer; and (5) male breast cancer. A sixth criterion-breast cancer plus a parent, sibling, or child with any of the other criteria-can be added to address family history. Criteria 1 through 5 are considered the MCG criteria, and criteria 1 through 6 are considered the MCGplus criteria. Testing using MCG or MCGplus criteria is cost-effective with cost-effectiveness ratios of $1330 per discounted QALYs and $1225 per discounted QALYs, respectively, and appears to lead to cancer and mortality reductions (MCG: 804 cancers, 161 deaths; MCGplus: 1020 cancers, 204 deaths per year over 50 years). Use of MCG or MCGplus criteria might allow detection of all BRCA mutations in patients with breast cancer in the United Kingdom through testing one-third of patients. Feedback questionnaires from 259 patients and 23 cancer team members (12 oncologists, 8 surgeons, and 3 nurse specialists) showed acceptability of the process with 100% of patients pleased they had genetic testing and 100% of cancer team members confident to approve patients for genetic testing. Use of MCGplus criteria also appeared to be time and resource efficient, requiring 95% fewer genetic consultations than the traditional process.Conclusions and relevanceThis study suggests that mainstream testing using simple, cancer-based criteria might be able to efficiently deliver consistent, cost-effective, patient-centered BRCA testing.

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A Comparison of Mainstream Genotyping Platforms for the Evaluation and Use of Barley Genetic Resources.

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