Menu

Asset Tag: Variant detection

July 7, 2019  |  

Contributions of Zea mays subspecies mexicana haplotypes to modern maize.

Maize was domesticated from lowland teosinte (Zea mays ssp. parviglumis), but the contribution of highland teosinte (Zea mays ssp. mexicana, hereafter mexicana) to modern maize is not clear. Here, two genomes for Mo17 (a modern maize inbred) and mexicana are assembled using a meta-assembly strategy after sequencing of 10 lines derived from a maize-teosinte cross. Comparative analyses reveal a high level of diversity between Mo17, B73, and mexicana, including three Mb-size structural rearrangements. The maize spontaneous mutation rate is estimated to be 2.17?×?10-8 ~3.87?×?10-8 per site per generation with a nonrandom distribution across the genome. A higher deleterious mutation rate is observed in the pericentromeric regions, and might be caused by differences in recombination frequency. Over 10% of the maize genome shows evidence of introgression from the mexicana genome, suggesting that mexicana contributed to maize adaptation and improvement. Our data offer a rich resource for constructing the pan-genome of Zea mays and genetic improvement of modern maize varieties.

Read more
July 7, 2019  |  

Hidden genetic variation shapes the structure of functional elements in Drosophila.

Mutations that add, subtract, rearrange, or otherwise refashion genome structure often affect phenotypes, although the fragmented nature of most contemporary assemblies obscures them. To discover such mutations, we assembled the first new reference-quality genome of Drosophila melanogaster since its initial sequencing. By comparing this new genome to the existing D. melanogaster assembly, we created a structural variant map of unprecedented resolution and identified extensive genetic variation that has remained hidden until now. Many of these variants constitute candidates underlying phenotypic variation, including tandem duplications and a transposable element insertion that amplifies the expression of detoxification-related genes associated with nicotine resistance. The abundance of important genetic variation that still evades discovery highlights how crucial high-quality reference genomes are to deciphering phenotypes.

Read more
July 7, 2019  |  

SV2: Accurate structural variation genotyping and de novo mutation detection from whole genomes.

Structural Variation (SV) detection from short-read whole genome sequencing is error prone, presenting significant challenges for population or family-based studies of disease.Here we describe SV2, a machine-learning algorithm for genotyping deletions and duplications from paired-end sequencing data. SV2 can rapidly integrate variant calls from multiple structural variant discovery algorithms into a unified call set with high genotyping accuracy and capability to detect de novo mutations. SV2 is freely available on GitHub (https://github.com/dantaki/SV2).Supplementary data are available at Bioinformatics online.© The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Read more
July 7, 2019  |  

Copy number variation and expression analysis reveals a nonorthologous pinta gene family member involved in butterfly vision.

Vertebrate (cellular retinaldehyde-binding protein) and Drosophila (prolonged depolarization afterpotential is not apparent [PINTA]) proteins with a CRAL-TRIO domain transport retinal-based chromophores that bind to opsin proteins and are necessary for phototransduction. The CRAL-TRIO domain gene family is composed of genes that encode proteins with a common N-terminal structural domain. Although there is an expansion of this gene family in Lepidoptera, there is no lepidopteran ortholog of pinta. Further, the function of these genes in lepidopterans has not yet been established. Here, we explored the molecular evolution and expression of CRAL-TRIO domain genes in the butterfly Heliconius melpomene in order to identify a member of this gene family as a candidate chromophore transporter. We generated and searched a four tissue transcriptome and searched a reference genome for CRAL-TRIO domain genes. We expanded an insect CRAL-TRIO domain gene phylogeny to include H. melpomene and used 18 genomes from 4 subspecies to assess copy number variation. A transcriptome-wide differential expression analysis comparing four tissue types identified a CRAL-TRIO domain gene, Hme CTD31, upregulated in heads suggesting a potential role in vision for this CRAL-TRIO domain gene. RT-PCR and immunohistochemistry confirmed that Hme CTD31 and its protein product are expressed in the retina, specifically in primary and secondary pigment cells and in tracheal cells. Sequencing of eye protein extracts that fluoresce in the ultraviolet identified Hme CTD31 as a possible chromophore binding protein. Although we found several recent duplications and numerous copy number variants in CRAL-TRIO domain genes, we identified a single copy pinta paralog that likely binds the chromophore in butterflies.© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Read more
July 7, 2019  |  

A recurrence-based approach for validating structural variation using long-read sequencing technology.

Although numerous algorithms have been developed to identify structural variations (SVs) in genomic sequences, there is a dearth of approaches that can be used to evaluate their results. This is significant as the accurate identification of structural variation is still an outstanding but important problem in genomics. The emergence of new sequencing technologies that generate longer sequence reads can, in theory, provide direct evidence for all types of SVs regardless of the length of the region through which it spans. However, current efforts to use these data in this manner require the use of large computational resources to assemble these sequences as well as visual inspection of each region. Here we present VaPoR, a highly efficient algorithm that autonomously validates large SV sets using long-read sequencing data. We assessed the performance of VaPoR on SVs in both simulated and real genomes and report a high-fidelity rate for overall accuracy across different levels of sequence depths. We show that VaPoR can interrogate a much larger range of SVs while still matching existing methods in terms of false positive validations and providing additional features considering breakpoint precision and predicted genotype. We further show that VaPoR can run quickly and efficiency without requiring a large processing or assembly pipeline. VaPoR provides a long read-based validation approach for genomic SVs that requires relatively low read depth and computing resources and thus will provide utility with targeted or low-pass sequencing coverage for accurate SV assessment. The VaPoR Software is available at: https://github.com/mills-lab/vapor.© The Authors 2017. Published by Oxford University Press.

Read more
July 7, 2019  |  

The state of whole-genome sequencing

Over the last decade, a technological paradigm shift has slashed the cost of DNA sequencing by over five orders of magnitude. Today, the cost of sequencing a human genome is a few thousand dollars, and it continues to fall. Here, we review the most cost-effective platforms for whole-genome sequencing (WGS) as well as emerging technologies that may displace or complement these. We also discuss the practical challenges of generating and analyzing WGS data, and how WGS has unlocked new strategies for discovering genes and variants underlying both rare and common human diseases.

Read more
July 7, 2019  |  

Genomic resources and their influence on the detection of the signal of positive selection in genome scans.

Genome scans represent powerful approaches to investigate the action of natural selection on the genetic variation of natural populations and to better understand local adaptation. This is very useful, for example, in the field of conservation biology and evolutionary biology. Thanks to Next Generation Sequencing, genomic resources are growing exponentially, improving genome scan analyses in non-model species. Thousands of SNPs called using Reduced Representation Sequencing are increasingly used in genome scans. Besides, genome sequences are also becoming increasingly available, allowing better processing of short-read data, offering physical localization of variants, and improving haplotype reconstruction and data imputation. Ultimately, genome sequences are also becoming the raw material for selection inferences. Here, we discuss how the increasing availability of such genomic resources, notably genome sequences, influences the detection of signals of selection. Mainly, increasing data density and having the information of physical linkage data expand genome scans by (i) improving the overall quality of the data, (ii) helping the reconstruction of demographic history for the population studied to decrease false-positive rates and (iii) improving the statistical power of methods to detect the signal of selection. Of particular importance, the availability of a high-quality reference genome can improve the detection of the signal of selection by (i) allowing matching the potential candidate loci to linked coding regions under selection, (ii) rapidly moving the investigation to the gene and function and (iii) ensuring that the highly variable regions of the genomes that include functional genes are also investigated. For all those reasons, using reference genomes in genome scan analyses is highly recommended. © 2015 John Wiley & Sons Ltd.

Read more
July 7, 2019  |  

Novel FANCI mutations in Fanconi anemia with VACTERL association.

Fanconi anemia (FA) is an inherited bone marrow failure syndrome caused by mutations in DNA repair genes; some of these patients may have features of the VACTERL association. Autosomal recessive mutations in FANCI are a rare cause of FA. We identified FANCI mutations by next generation sequencing in three patients in our FA cohort among several whose mutated gene was unknown. Four of the six mutations are novel and all mutations are likely deleterious to protein function. There are now 16 reported cases of FA due to FANCI of whom 7 have at least 3 features of the VACTERL association (44%). This suggests that the VACTERL association in patients with FA may be seen in patients with FANCI mutations more often than previously recognized. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Read more
July 7, 2019  |  

Timing, rates and spectra of human germline mutation.

Germline mutations are a driving force behind genome evolution and genetic disease. We investigated genome-wide mutation rates and spectra in multi-sibling families. The mutation rate increased with paternal age in all families, but the number of additional mutations per year differed by more than twofold between families. Meta-analysis of 6,570 mutations showed that germline methylation influences mutation rates. In contrast to somatic mutations, we found remarkable consistency in germline mutation spectra between the sexes and at different paternal ages. In parental germ line, 3.8% of mutations were mosaic, resulting in 1.3% of mutations being shared by siblings. The number of these shared mutations varied significantly between families. Our data suggest that the mutation rate per cell division is higher during both early embryogenesis and differentiation of primordial germ cells but is reduced substantially during post-pubertal spermatogenesis. These findings have important consequences for the recurrence risks of disorders caused by de novo mutations.

Read more
July 7, 2019  |  

In planta comparative transcriptomics of host-adapted strains of Ralstonia solanacearum.

Background. Ralstonia solanacearum is an economically important plant pathogen with an unusually large host range. The Moko (banana) and NPB (not pathogenic to banana) strain groups are closely related but are adapted to distinct hosts. Previous comparative genomics studies uncovered very few differences that could account for the host range difference between these pathotypes. To better understand the basis of this host specificity, we used RNAseq to profile the transcriptomes of an R. solanacearum Moko strain and an NPB strain under in vitro and in planta conditions. Results. RNAs were sequenced from bacteria grown in rich and minimal media, and from bacteria extracted from mid-stage infected tomato, banana and melon plants. We computed differential expression between each pair of conditions to identify constitutive and host-specific gene expression differences between Moko and NPB. We found that type III secreted effectors were globally up-regulated upon plant cell contact in the NPB strain compared with the Moko strain. Genes encoding siderophore biosynthesis and nitrogen assimilation genes were highly up-regulated in the NPB strain during melon pathogenesis, while denitrification genes were up-regulated in the Moko strain during banana pathogenesis. The relatively lower expression of oxidases and the denitrification pathway during banana pathogenesis suggests that R. solanacearum experiences higher oxygen levels in banana pseudostems than in tomato or melon xylem. Conclusions. This study provides the first report of differential gene expression associated with host range variation. Despite minimal genomic divergence, the pathogenesis of Moko and NPB strains is characterized by striking differences in expression of virulence- and metabolism-related genes.

Read more
July 7, 2019  |  

Resolving complex structural genomic rearrangements using a randomized approach.

Complex chromosomal rearrangements are structural genomic alterations involving multiple instances of deletions, duplications, inversions, or translocations that co-occur either on the same chromosome or represent different overlapping events on homologous chromosomes. We present SVelter, an algorithm that identifies regions of the genome suspected to harbor a complex event and then resolves the structure by iteratively rearranging the local genome structure, in a randomized fashion, with each structure scored against characteristics of the observed sequencing data. SVelter is able to accurately reconstruct complex chromosomal rearrangements when compared to well-characterized genomes that have been deeply sequenced with both short and long reads.

Read more
July 7, 2019  |  

Rapid evolution of citrate utilization by Escherichia coli by direct selection requires citT and dctA.

The isolation of aerobic citrate-utilizing Escherichia coli (Cit(+)) in long-term evolution experiments (LTEE) has been termed a rare, innovative, presumptive speciation event. We hypothesized that direct selection would rapidly yield the same class of E. coli Cit(+) mutants and follow the same genetic trajectory: potentiation, actualization, and refinement. This hypothesis was tested with wild-type E. coli strain B and with K-12 and three K-12 derivatives: an E. coli ?rpoS::kan mutant (impaired for stationary-phase survival), an E. coli ?citT::kan mutant (deleted for the anaerobic citrate/succinate antiporter), and an E. coli ?dctA::kan mutant (deleted for the aerobic succinate transporter). E. coli underwent adaptation to aerobic citrate metabolism that was readily and repeatedly achieved using minimal medium supplemented with citrate (M9C), M9C with 0.005% glycerol, or M9C with 0.0025% glucose. Forty-six independent E. coli Cit(+) mutants were isolated from all E. coli derivatives except the E. coli ?citT::kan mutant. Potentiation/actualization mutations occurred within as few as 12 generations, and refinement mutations occurred within 100 generations. Citrate utilization was confirmed using Simmons, Christensen, and LeMaster Richards citrate media and quantified by mass spectrometry. E. coli Cit(+) mutants grew in clumps and in long incompletely divided chains, a phenotype that was reversible in rich media. Genomic DNA sequencing of four E. coli Cit(+) mutants revealed the required sequence of mutational events leading to a refined Cit(+) mutant. These events showed amplified citT and dctA loci followed by DNA rearrangements consistent with promoter capture events for citT. These mutations were equivalent to the amplification and promoter capture CitT-activating mutations identified in the LTEE.IMPORTANCE E. coli cannot use citrate aerobically. Long-term evolution experiments (LTEE) performed by Blount et al. (Z. D. Blount, J. E. Barrick, C. J. Davidson, and R. E. Lenski, Nature 489:513-518, 2012, http://dx.doi.org/10.1038/nature11514 ) found a single aerobic, citrate-utilizing E. coli strain after 33,000 generations (15 years). This was interpreted as a speciation event. Here we show why it probably was not a speciation event. Using similar media, 46 independent citrate-utilizing mutants were isolated in as few as 12 to 100 generations. Genomic DNA sequencing revealed an amplification of the citT and dctA loci and DNA rearrangements to capture a promoter to express CitT, aerobically. These are members of the same class of mutations identified by the LTEE. We conclude that the rarity of the LTEE mutant was an artifact of the experimental conditions and not a unique evolutionary event. No new genetic information (novel gene function) evolved. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Read more
July 7, 2019  |  

Third-generation sequencing and the future of genomics

Third-generation long-range DNA sequencing and mapping technologies are creating a renaissance in high-quality genome sequencing. Unlike second-generation sequencing, which produces short reads a few hundred base-pairs long, third-generation single-molecule technologies generate over 10,000 bp reads or map over 100,000 bp molecules. We analyze how increased read lengths can be used to address long-standing problems in de novo genome assembly, structural variation analysis and haplotype phasing.

Read more
July 7, 2019  |  

Draft genome sequences of three European laboratory derivatives from enterohemorrhagic Escherichia coli O157:H7 strain EDL933, including two plasmids.

Escherichia coliO157:H7 EDL933, isolated in 1982 in the United States, was the first enterohemorrhagicE. coli(EHEC) strain sequenced. Unfortunately, European labs can no longer receive the original strain. We checked three European EDL933 derivatives and found major genetic deviations (deletions, inversions) in two strains. All EDL933 strains contain the cryptic EHEC-plasmid, not reported before. Copyright © 2016 Fellner et al.

Read more
July 7, 2019  |  

Exploring structural variants in environmentally sensitive gene families.

Environmentally sensitive plant gene families like NBS-LRRs, receptor kinases, defensins and others, are known to be highly variable. However, most existing strategies for discovering and describing structural variation in complex gene families provide incomplete and imperfect results. The move to de novo genome assemblies for multiple accessions or individuals within a species is enabling more comprehensive and accurate insights about gene family variation. Earlier array-based genome hybridization and sequence-based read mapping methods were limited by their reliance on a reference genome and by misplacement of paralogous sequences. Variant discovery based on de novo genome assemblies overcome the problems arising from a reference genome and reduce sequence misplacement. As de novo genome sequencing moves to the use of longer reads, artifacts will be minimized, intact tandem gene clusters will be constructed accurately, and insights into rapid evolution will become feasible. Copyright © 2016 Elsevier Ltd. All rights reserved.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.