The statistics of bulk segregant analysis using next generation sequencing.
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ABSTRACT:
SUBMITTER: Magwene PM
PROVIDER: S-EPMC3207950 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundBulk segregant analysis (BSA) combined with next generation sequencing is a powerful tool to identify quantitative trait loci (QTL). The impact of the size of the study population and the percentage of extreme genotypes analysed have already been assessed. But a good comparison of statistical approaches designed to identify QTL regions using next generation sequencing (NGS) technologies for BSA is still lacking.ResultsWe developed an R code to simulate QTLs in bulks of F2 contrasted lines. We simulated a range of recombination rates based on estimations using different crop species. The simulations were used to benchmark the ability of statistical methods identify the exact location of true QTLs. A single QTL led to a shift in allele frequency across a large fraction of the chromosome for plant species with low recombination rate. The smoothed version of all statistics performed best notably the smoothed Euclidean distance-based statistics was always found to be more accurate in identifying the location of QTLs. We propose a simulation approach to build confidence interval statistics for the detection of QTLs.ConclusionWe highlight the statistical methods best suited for BSA studies using NGS technologies in crops even when recombination rate is low. We also provide simulation codes to build confidence intervals and to assess the impact of recombination for application to other studies. This computational study will help select NGS-based BSA statistics that are useful to the broad scientific community.
| S-EPMC9258084 | biostudies-literature
Project description:Dwarfism is one of the most valuable traits in watermelon breeding mainly because of its contribution to yield as well as the decreased labor required to cultivate and harvest smaller plants. However, the underlying genetic mechanism is unknown. In this study, a candidate dwarfism gene was identified by applying next-generation sequencing technology to analyze watermelon plants. We completed a whole-genome re-sequencing of two DNA bulks (dwarf pool and vine pool) generated from plants in an F2 population. A genome-wide analysis of single nucleotide polymorphisms resulted in the detection of a genomic region harboring the candidate dwarfism gene Cla010726. The encoded protein was predicted to be a gibberellin 20-oxidase-like protein, which is a well-known "green revolution" protein in other crops. A quantitative real-time PCR investigation revealed that the Cla010726 expression level was significantly lower in the dwarf plants than in the normal-sized plants. The SNP analysis resulted in two SNP locating in the Cla010726 gene promoter of dsh F2 individuals. The results presented herein provide preliminary evidence that Cla010726 is a possible dwarfism gene.
| S-EPMC5811605 | biostudies-literature
Project description:Phenotype-driven forward genetic experiments are among the most powerful approaches for linking biology and disease to genomic elements. Although widely used in a range of model organisms, positional cloning of causal variants is still a very laborious process. Here, we describe a novel universal approach, named fast forward genetics that combines traditional bulk segregant techniques with next-generation sequencing technology and targeted genomic enrichment, to dramatically improve the process of mapping and cloning multiple mutants in a single experiment. In a two-step procedure the mutation is first roughly mapped by ‘light’ sequencing of the bulk segregant pool, followed by genomic enrichment and deep-sequencing of the mutant pool for the linked genomic region. The latter step allows for simultaneous fine-mapping and mutation discovery. We successfully applied this approach to three Arabidopsis mutants, but the method can in principle be applied to any model organism of interest and is largely independent of the genome size. Moreover, we show that both steps can be performed in multiplex using barcoded samples, thereby increasing efficiency enormously. Inducible overexpression of the RETINOBLASTOMA-RELATED (RBR-OE) gene in Arabidopsis roots causes the complete differentiation of stem cells and premature differentiation of daughter cells, leading to a full exhaustion of the primary root meristem. In order to identify regulators of RBR function in cell differentiation, RBR-OE plants in the Columbia background (Col0) were treated with EMS mutagenesis and a set of genetic suppressors of RBR-OE, which restores root growth capacity, were isolated. In this study, we used one the identified suppressor lines, which segregated as a recessive mutation. Mapping populations were generated by outcrossing to Ler ecotype. Seedlings from the F2 population were grown for 15 days post germination (dpg). A pool of 60 seedlings each with a clear suppressor phenotype (homozygous for suppressor mutation) and of 60 seedlings showing RBOE phenotype (Heterozygous for the suppressor mutation) were prepared and genomic DNA was isolated with the RNeasy Plant Mini Kit from QIAGEN according to manufacturer's protocol. The other two, mutants 136 and 193 were obtained in fluorescence based mutant screen and a QCmarker based mutagenesis, respectively. Mutants were generated by chemical mutagenesis (EMS) in Colombia (Col) genetic background. Mutants were subsequently crossed to the Landsberg (Ler) ecotype to create the mapping populations. Bulk-segregant pools of about 200 mutant as well as wild-type plants were generated for every mutant line.
2010-12-31 | E-GEOD-24511 | biostudies-arrayexpress
Project description:Foxtail millet has gradually become a model gramineous C4 crop owing to its short growth period and small genome. Research on the development of its spikelets is not only directly related to the yield and economic value of foxtail millet but also can provide a reference for studying the fertility of other C4 crops. In this study, a hybrid population containing 200 offspring was constructed from the Xinong8852 and An15 parental lines, and two extreme trait populations were constructed from the F2 generation for analysis of the spikelet sterility. The F2 population conformed to a 3:1 Mendelian segregation ratio, and it was thus concluded that this trait is likely controlled by a single recessive gene. Bulk segregant analysis sequencing (BSA-Seq) was used to determine the candidate regions and candidate genes related to the development of foxtail millet spikelets. Additionally, the functional analysis of differentially expressed genes in populations with different traits was conducted by bulk segregant RNA sequencing (BSR-Seq). Finally, conjunctive analysis of BSA-Seq and BSR-Seq results, combined with biological information analysis, revealed six genes on chromosome VII that were ultimately identified as candidate genes controlling foxtail millet spikelet development. This study provides a new reference for research on foxtail millet sterility and lays a solid foundation for the examination of fertility in other gramineous crops.
| S-EPMC9047506 | biostudies-literature
Project description:Bulk segregant analysis (BSA) using microarrays, and extreme array mapping (XAM) have recently been used to rapidly identify genomic regions associated with phenotypes in multiple species. These experiments, however, require the identification of single feature polymorphisms (SFP) between the cross parents for each new combination of genotypes, which raises the cost of experiments. The availability of the genomic polymorphism data in Arabidopsis thaliana, coupled with the efficient designs of Single Nucleotide Polymorphism (SNP) genotyping arrays removes the requirement for SFP detection and lowers the per array cost, thereby lowering the overall cost per experiment. To demonstrate that these approaches would be functional on SNP arrays and determine confidence intervals, we analyzed hybridizations of natural accessions to the Arabidopsis ATSNPTILE array and simulated BSA or XAM given a variety of gene models, populations, and bulk selection parameters. Our results show a striking degree of correlation between the genotyping output of both methods, which suggests that the benefit of SFP genotyping in context of BSA can be had with the cheaper, more efficient SNP arrays. As a final proof of concept, we hybridized the DNA from bulks of an F2 mapping population of a Sulfur and Selenium ionomics mutant to both the Arabidopsis ATTILE1R and ATSNPTILE arrays, which produced almost identical results. We have produced R scripts that prompt the user for the required parameters and perform the BSA analysis using the ATSNPTILE1 array and have provided them as supplemental data files.
| S-EPMC3029305 | biostudies-literature
Project description:BackgroundExome sequencing has transformed human genetic analysis and may do the same for other vertebrate model systems. However, a major challenge is sifting through the large number of sequence variants to identify the causative mutation for a given phenotype. In models like Xenopus tropicalis, an incomplete and occasionally incorrect genome assembly compounds this problem. To facilitate cloning of X. tropicalis mutants identified in forward genetic screens, we sought to combine bulk segregant analysis and exome sequencing into a single step.ResultsHere we report the first use of exon capture sequencing to identify mutations in a non-mammalian, vertebrate model. We demonstrate that bulk segregant analysis coupled with exon capture sequencing is not only able to identify causative mutations but can also generate linkage information, facilitate the assembly of scaffolds, identify misassembles, and discover thousands of SNPs for fine mapping.ConclusionExon capture sequencing and bulk segregant analysis is a rapid, inexpensive method to clone mutants identified in forward genetic screens. With sufficient meioses, this method can be generalized to any model system with a genome assembly, polished or unpolished, and in the latter case, it also provides many critical genomic resources.
| S-EPMC3526394 | biostudies-literature
Project description:Phenotype-driven forward genetic experiments are among the most powerful approaches for linking biology and disease to genomic elements. Although widely used in a range of model organisms, positional cloning of causal variants is still a very laborious process. Here, we describe a novel universal approach, named fast forward genetics that combines traditional bulk segregant techniques with next-generation sequencing technology and targeted genomic enrichment, to dramatically improve the process of mapping and cloning multiple mutants in a single experiment. In a two-step procedure the mutation is first roughly mapped by ‘light’ sequencing of the bulk segregant pool, followed by genomic enrichment and deep-sequencing of the mutant pool for the linked genomic region. The latter step allows for simultaneous fine-mapping and mutation discovery. We successfully applied this approach to three Arabidopsis mutants, but the method can in principle be applied to any model organism of interest and is largely independent of the genome size. Moreover, we show that both steps can be performed in multiplex using barcoded samples, thereby increasing efficiency enormously.
2010-12-31 | GSE24511 | GEO
Project description:BACKGROUND:Cold stress can cause serious abiotic damage that limits the growth, development and yield of rice. Cold tolerance during the booting stage of rice is a key factor that can guarantee a high and stable yield under cold stress. The cold tolerance of rice is controlled by quantitative trait loci (QTLs). Based on the complex genetic basis of cold tolerance in rice, additional efforts are needed to detect reliable QTLs and identify candidate genes. In this study, recombinant inbred lines (RILs) derived from a cross between a cold sensitive variety, Dongnong422, and strongly cold-tolerant variety, Kongyu131, were used to screen for cold-tolerant loci at the booting stage of rice. RESULTS:A novel major QTL, qPSST6, controlling the percent seed set under cold water treatment (PSST) under the field conditions of 17 °C cold water irrigation was located on the 28.4 cM interval on chromosome 6. Using the combination of bulked-segregant analysis (BSA) and next-generation sequencing (NGS) technology (Seq-BSA), a 1.81 Mb region that contains 269 predicted genes on chromosome 6 was identified as the candidate region of qPSST6. Two genes, LOC_Os06g39740 and LOC_Os06g39750, were annotated as "response to cold" by gene ontology (GO) analysis. qRT-PCR analysis revealed that LOC_Os06g39750 was strongly induced by cold stress. Haplotype analysis also demonstrate a key role of LOC_Os06g39750 in regulating the PSST of rice, suggesting that it was the candidate gene of qPSST6. CONCLUSIONS:The information obtained in this study is useful for gene cloning of qPSST6 and for breeding cold-tolerant varieties of rice using marker assisted selection (MAS).
| S-EPMC5906412 | biostudies-literature
Project description:Next-generation sequencing (NGS)-based bulked-segregant analysis (BSA) approaches have been proven successful for rapidly mapping genes in plant species. However, most such methods are based on mutants and usually only one gene controlling the mutant phenotype is identified. In this study, NGS-based BSA was employed to map simultaneously two qualitative genes controlling cotyledon color of seed in soybean. Yellow-cotyledon (YC) and green-cotyledon (GC) bulks from progenies of a biparental population (Zhonghuang 30 × Jiyu 102) were sequenced. The SNP-index of each SNP locus in YC and GC bulks was calculated and two genomic regions on chromosomes 1 and 11 harboring, respectively, loci qCC1 and qCC2 were identified by ?(SNP-index) analysis. These two BSA-seq-derived loci were further validated with SSR markers and fine-mapped. qCC1 was mapped to a 30.7-kb region containing four annotated genes and qCC2 was mapped to a 67.7-kb region with nine genes. These two regions contained, respectively, genes D1 and D2, which had previously been identified by homology-based cloning as being associated with cotyledon color. Sequence analysis of the NGS data also identified a frameshift deletion in the coding region of D1. These results suggested that BSA-seq could accelerate the mapping of loci controlling qualitative traits, even if a trait is controlled by more than one locus.
| S-EPMC5449466 | biostudies-literature
Project description:NGPS is a method for de-novo, full-length protein sequencing in high throughput. The method is based on cleavage of the protein at semi-random sites by microwave-assisted acid hydrolysis (MAAH), enrichment of LC-MS/MS amenable peptides from the hydrolysate by solid-phase-extraction, LC-MS/MS analysis, de-novo long peptide tag sequencing of resulting peptides and assembly of peptide tags into consensus contigs.
2017-04-03 | PXD003804 | Pride