Melt analysis of mismatch amplification mutation assays (melt-MAMA): a functional study of a cost-effective SNP genotyping assay in bacterial models.

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA), is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg). Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs) and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of Melt-MAMA, which should prove useful to the wider scientific community

La sismologie citoyenne, un révélateur des crises en Haïti

On January 12, 2010, Haiti was hit by one of the largest seismic disasters known to date. This disaster is part of a long series of crises that affect Haitians, whether natural (hurricanes, earthquakes...) or related to political and economic factors (demonstrations, impoverishment...).Using a citizen seismology approach, a multidisciplinary team of seismo-sociologists used low-cost seismic sensors (Raspberry Shake, or RS) placed in the homes of Haitian citizens in order to complete the national seismic network and improve the risk perception, the level of preparedness and the scientific knowledge of the population. Through these objectives, it is in fact a paradigm shift that is aimed at, in which seismic networks are not only composed of sensors, but also of citizens and scientists who gather around these tools and their information.Presented here are the results of a qualitative survey of 15 RS seismometer hosts in Haiti. Semi-structured interviews were conducted to learn more about their experience, to assess their motivations, their difficulties and expectations in this citizen seismology process. It was found that RS hosts expressed pride and interest in being part of the network and actively contributing to risk reduction in their community. Some of them reported a form of empowerment in that they could compensate for the deficiency of the state, which they generally distrust. Thus, the combination of the seismologists' approach and the sociologists' analysis based on the citizen science approach allows us to understand issues that go beyond those solely related to earthquakes. We conclude that a citizen seismology approach is a very relevant way to understand the general environment of a crisis in order to better respond to it and to contribute to risk reduction: RS become indicators of major issues in Haitian society, beyond earthquakes

Lectures et \ue9critures ha\ueftiennes. n. 49 "Francofonia"

Des sp\ue9cialistes de la litt\ue9rature ha\ueftienne interrogent les oeuvres d'auteurs particuli\ue8rement repr\ue9sentatifs de cette litt\ue9rature (Jacques Roumain, Louis-Joseph Janvier, Lyonel Trouillot, Marie Chauvet), font le point sur les tendances contemporaines de ces \ue9critures francophones, mais situent aussi la litt\ue9rature ha\ueftienne au centre des grandes mutations et transformations qui concernent le XXIe si\ue8cle (mondialisation et globalisation)

A socio-seismology experiment in Haiti

Earthquake risk reduction approaches classically apply a top-down model where scientific information is processed to deliver risk mitigation measures and policies understandable by all, while shielding end-users from the initial, possibly complex, information. Alternative community-based models exist but are rarely applied at a large scale and rely on valuable, but non-scientific, observations and experiences of local populations. In spite of risk reduction efforts based on both approaches, changes in behaviour or policies to reduce earthquake risk are slow or even non-existent, in particular in developing countries. Here we report on the initial stage of a project that aims at testing, through a participatory seismology experiment in Haiti-a country struck by a devastating earthquake in January 2010-whether public or community involvement through the production and usage of seismic information can improve earthquake awareness and, perhaps, induce grassroots protection initiatives. This experiment is made possible by the recent launch of very low-cost, plug-and-play,Raspberry Shakeseismological stations, the relative ease of access to the internet even in developing countries such as Haiti, and the familiarity of all with social networks as a way to disseminate information. Our early findings indicate that 1) the seismic data collected is of sufficient quality for real-time detection and characterization of the regional seismicity, 2) citizens are in demand of earthquake information and trust scientists, even though they appear to see earthquakes through the double lens of tectonics and magic/religion, 3) the motivation of seismic station hosts has allowed data to flow without interruption for more than a year, including through a major political crisis in the Fall of 2019 and the current COVID19 situation. At this early stage of the project, our observations indicate that citizen-seismology in a development context has potential to engage the public while collecting scientifically-relevant seismological information

Genotyping over a broad range of DNA amounts.

<p>Melt-MAMA genotyping accuracy is not diminished at lower amounts of DNA, even at near-single copy for some assays. The sensitivity of individual melt-MAMAs varies greatly. This <i>B. anthracis</i> melt-MAMA (A.Br.003 clade) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032866#pone.0032866-VanErt1" target="_blank">[4]</a> accurately genotyped DNA regardless of starting amounts as long as it was sufficient to support amplification. (A & B) The respective amplification plots of genomic DNA of ‘G’ allele and ‘A’ SNP allele templates show the amplification curves of templates titrated in ten-fold serial dilutions and in replicates of eight. The number assigned to each amplification curve (1–8) denotes the DNA amount for the starting template. (C) The temperature-dissociation (melt) curve derivatives for all initial template amounts are shown (numbers denote DNA amount shown). This panel illustrates that genotyping accuracy was not affected by DNA amounts, even at near-single copy levels. Similar to TaqMan assays, the detection of low levels of DNA template by Melt-MAMA is also subject to stochastic sampling effects (<i>B. anthracis</i> single copy ∼6 fg), which is predictable using a Poisson distribution <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032866#pone.0032866-VanErt1" target="_blank">[4]</a>.</p

Real-time PCR amplification and dissociation (melt) curve plots.

<p><i>B. anthracis</i> Melt-MAMA SYBR® Green assay targeting the A.Br.004 genetic clade. (A & C) The amplification of two alleles are illustrated for haploid template (<i>Bacillus anthracis</i>) possessing an ‘A’ polymorphic SNP-state or ‘G’ state. Each amplification plot represents a single PCR reaction containing a reverse “common” primer and two allele-specific MAMA primers. The AS-MAMA primers anneal to the same template target and then compete for extension across the SNP position. The polymerase-mediated extension rate of the 3′match AS-MAMA primer (perfect primer-template complex) exceeds that of the 3′mismatched MAMA primer (mismatched primer-template complex), thus the perfect match primer-template complex outcompetes the mismatched primer-template complex and dominates the PCR amplification. (B & D) Plots of the temperature-dissociation (melt) curve of the final PCR products for the two allele templates are shown next to their respective amplification plots (green arrows). Allele-specific PCR products are easily differentiated through temperature-dissociation (melt) curve analysis, which is conferred by the GC-clamp engineered on one of the AS-MAMA primer.</p