Dataset Information

Shedding light on dark genes: enhanced targeted resequencing by optimizing the combination of enrichment technology and DNA fragment length.

ABSTRACT: The exome contains many obscure regions difficult to explore with current short-read sequencing methods. Repetitious genomic regions prevent the unique alignment of reads, which is essential for the identification of clinically-relevant genetic variants. Long-read technologies attempt to resolve multiple-mapping regions, but they still produce many sequencing errors. Thus, a new approach is required to enlighten the obscure regions of the genome and rescue variants that would be otherwise neglected. This work aims to improve the alignment of multiple-mapping reads through the extension of the standard DNA fragment size. As Illumina can sequence fragments up to 550?bp, we tested different DNA fragment lengths using four major commercial WES platforms and found that longer DNA fragments achieved a higher genotypability. This metric, which indicates base calling calculated by combining depth of coverage with the confidence of read alignment, increased from hundreds to thousands of genes, including several associated with clinical phenotypes. While depth of coverage has been considered crucial for the assessment of WES performance, we demonstrated that genotypability has a greater impact in revealing obscure regions, with ~1% increase in variant calling in respect to shorter DNA fragments. Results confirmed that this approach enlightened many regions previously not explored.

SUBMITTER: Iadarola B

PROVIDER: S-EPMC7287100 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Shedding light on dark genes: enhanced targeted resequencing by optimizing the combination of enrichment technology and DNA fragment length.

Iadarola Barbara B Xumerle Luciano L Lavezzari Denise D Paterno Marta M Marcolungo Luca L Beltrami Cristina C Fortunati Elisabetta E Mei Davide D Vetro Annalisa A Guerrini Renzo R Parrini Elena E Rossato Marzia M Delledonne Massimo M

Scientific reports 20200610 1

The exome contains many obscure regions difficult to explore with current short-read sequencing methods. Repetitious genomic regions prevent the unique alignment of reads, which is essential for the identification of clinically-relevant genetic variants. Long-read technologies attempt to resolve multiple-mapping regions, but they still produce many sequencing errors. Thus, a new approach is required to enlighten the obscure regions of the genome and rescue variants that would be otherwise neglec ...[more]

PMID: 32523024

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Project description:Background. Research addressing distinctions and similarities between people's malevolent character traits (i.e., the Dark Triad: Machiavellianism, narcissism, and psychopathy) has detected inconsistent linear associations to temperament traits. Additionally, these dark traits seem to have a common core expressed as uncooperativeness. Hence, some researchers suggest that the dark traits are best represented as one global construct (i.e., the unification argument) rather than as ternary construct (i.e., the uniqueness argument). We put forward the dark cube (cf. Cloninger's character cube) comprising eight dark profiles that can be used to compare individuals who differ in one dark character trait while holding the other two constant. Our aim was to investigate in which circumstances individuals who are high in each one of the dark character traits differ in Cloninger's "light" character traits: self-directedness, cooperativeness, and self-transcendence. We also investigated if people's dark character profiles were associated to their light character profiles. Method. A total of 997 participants recruited from Amazon's Mechanical Turk (MTurk) responded to the Short Dark Triad and the Short Character Inventory. Participants were allocated to eight different dark profiles and eight light profiles based on their scores in each of the traits and any possible combination of high and low scores. We used three-way interaction regression analyses and t-tests to investigate differences in light character traits between individuals with different dark profiles. As a second step, we compared the individuals' dark profile with her/his character profile using an exact cell-wise analysis conducted in the ROPstat software (http://www.ropstat.com). Results. Individuals who expressed high levels of Machiavellianism and those who expressed high levels of psychopathy also expressed low self-directedness and low cooperativeness. Individuals with high levels of narcissism, in contrast, scored high in self-directedness. Moreover, individuals with a profile low in the dark traits were more likely to end up with a profile high in cooperativeness. The opposite was true for those individuals with a profile high in the dark traits. The rest of the cross-comparisons revealed some of the characteristics of human personality as a non-linear complex dynamic system. Conclusions. Our study suggests that individuals who are high in Machiavellianism and psychopathy share a unified non-agentic and uncooperative character (i.e., irresponsible, low in self-control, unempathetic, unhelpful, untolerant), while individuals high in narcissism have a more unique character configuration expressed as high agency and, when the other dark traits are high, highly spiritual but uncooperative. In other words, based on differences in their associations to the light side of character, the Dark Triad seems to be a dyad rather than a triad.

Project description:The black caiman is one of the largest neotropical top predators, which means that it could play a structuring role within swamp ecosystems. However, because of the difficulties inherent to studying black caimans, data are sorely lacking on many aspects of their general biology, natural history, and ecology, especially in French Guiana. We conducted a detailed study of the Agami Pond black caiman population using a multidisciplinary approach. The aim was to better understand the species' dietary ecology and movements in the pond, and thus its functional role in pond system. We gathered natural history data, tracked caiman movements using satellite transmitters, and characterized feeding ecology via stable isotope analysis. Our study was carried out over three sampling periods and spanned both wet and dry seasons, which differ in their hydrological and ecological conditions. Our results show that black caiman abundance and age demographics differed between seasons in Agami Pond. In the dry season, Agami Pond is one of the only areas within the marsh to hold water. It thus contains large quantities of different fish species, which form the basis of the black caiman's diet. Caiman body size, a proxy for age class, was around 1.5 meters. During the wet season, which corresponds to the breeding period for migratory birds (e.g., Agami herons), adult black caimans are present in Agami Pond. Adults were most abundant in the inundated forest. There, most individuals measured up to 2 meters. They also exhibited a particular "predatory" behavior near bird nests, preying on fallen chicks and adults. Juveniles and subadults were present during both seasons in the pond's open waters. These behavioral observations were backed up by stable isotope analysis, which revealed ontogenetic variation in the caiman's isotopic values. This isotopic variation reflected variation in diet that likely reduced intraspecific competition between adults and young. The telemetry and microchip data show that different age classes had different movement patterns and that seasonal variation in the pond may influence caiman prey availability and reproductive behavior. The new information gathered should help predict this species' responses to potential ecosystem disturbance (e.g., water pollution, habitat destruction) and inform the development of an effective conservation plan that involves locals and wildlife officials.

Project description:Despite widespread interest in next-generation sequencing (NGS), the adoption of personalized clinical genomics and mutation profiling of cancer specimens is lagging, in part because of technical limitations. Tumors are genetically heterogeneous and often contain normal/stromal cells, features that lead to low-abundance somatic mutations that generate ambiguous results or reside below NGS detection limits, thus hindering the clinical sensitivity/specificity standards of mutation calling. We applied COLD-PCR (coamplification at lower denaturation temperature PCR), a PCR methodology that selectively enriches variants, to improve the detection of unknown mutations before NGS-based amplicon resequencing.We used both COLD-PCR and conventional PCR (for comparison) to amplify serially diluted mutation-containing cell-line DNA diluted into wild-type DNA, as well as DNA from lung adenocarcinoma and colorectal cancer samples. After amplification of TP53 (tumor protein p53), KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), IDH1 [isocitrate dehydrogenase 1 (NADP(+)), soluble], and EGFR (epidermal growth factor receptor) gene regions, PCR products were pooled for library preparation, bar-coded, and sequenced on the Illumina HiSeq 2000.In agreement with recent findings, sequencing errors by conventional targeted-amplicon approaches dictated a mutation-detection limit of approximately 1%-2%. Conversely, COLD-PCR amplicons enriched mutations above the error-related noise, enabling reliable identification of mutation abundances of approximately 0.04%. Sequencing depth was not a large factor in the identification of COLD-PCR-enriched mutations. For the clinical samples, several missense mutations were not called with conventional amplicons, yet they were clearly detectable with COLD-PCR amplicons. Tumor heterogeneity for the TP53 gene was apparent.As cancer care shifts toward personalized intervention based on each patient's unique genetic abnormalities and tumor genome, we anticipate that COLD-PCR combined with NGS will elucidate the role of mutations in tumor progression, enabling NGS-based analysis of diverse clinical specimens within clinical practice.

Project description:BackgroundExposure to light at night (LAN) can perturb the biological clock and affect sleep and health. Previous epidemiological studies have evaluated LAN levels measured by satellites, but the validity of this measure as a proxy for personal LAN exposure is unclear. In addition, outdoor satellite-measured LAN levels are higher in urban environments, which means that this measure could potentially represent a proxy for other, likely urban, environmental exposures.ObjectivesWe evaluated correlations of satellite-assessed LAN with measured bedroom light levels and explored correlations with other environmental exposures, in particular, air pollution, green space, and area-level socioeconomic position (SEP).MethodsWe compared satellite measurements with evening and nighttime bedroom measurements of illuminance (in units of lux) for 256 children, and we evaluated correlations between satellite-based measures and other urban exposures such as air pollution, area-level SEP, and surrounding green space for 3,021 children.ResultsSatellite-measured LAN levels (nanowatts per centimeter squared per steradian) were not correlated with measured evening or nighttime lux levels [Spearman correlation coefficients ([Formula: see text]) [Formula: see text] to 0.04]. There was a weak correlation with measurements during the darkest time period if parents and their children reported that outdoor light sometimes or usually influenced indoor light levels ([Formula: see text], [Formula: see text]). In contrast, satellite-measured LAN levels were correlated with air pollution ([Formula: see text] with [Formula: see text], [Formula: see text] with [Formula: see text]), and surrounding green space ([Formula: see text] for green space within [Formula: see text] of the home). A weak correlation with area-level SEP was also observed ([Formula: see text]).ConclusionsOutdoor satellite-assessed outdoor LAN exposure levels were correlated with urban environmental exposures, but they were not a good proxy for indoor evening or nighttime personal exposure as measured in our study population of 12-y-old children. Studies planning to evaluate potential risks from LAN should consider such modifying factors as curtains and indoor lighting and the use of electronic devices and should include performing indoor or personal measurements to validate any exposure proxies. The moderate-to-strong correlation of outdoor LAN with other environmental exposures should be accounted for in epidemiological investigations. https://doi.org/10.1289/EHP3431.

Dataset Information

Shedding light on dark genes: enhanced targeted resequencing by optimizing the combination of enrichment technology and DNA fragment length.

Publications

Shedding light on dark genes: enhanced targeted resequencing by optimizing the combination of enrichment technology and DNA fragment length.

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