DiNAMO: Exact method for degenerate IUPAC motifs discovery, characterization of sequence-specific errors

National audienceNext generation sequencing technologies are still associated with relatively high error rates, about 1%, which correspond to thousands of errors in the scale of a complete genome. Each region needs therefore to be sequenced several times and variants are usually filtered based on depth criteria. The significant number of artifacts, in spite of those filters, shows the limit of conventional approaches and indicates that some sequencing artifacts are recurrent. This recurrence underlines that sequencing errors can depend on the upstream nucleotide sequence context. Our goal is to search for overrepresented motifs that tend to induce sequencing errors. Previous studies showed that some motifs, such as GGT [1,2], induce sequencing errors in the Illumina technologies. However, these studies were dedicated to exact motifs, and did not take into account approximate motifs, limiting the statistical power of such approaches. On the other hand, some tools, such as FIRE [3], DREME [4] and Discrover [5], were developed to search for degenerate motifs over the 15-letter IUPAC alphabet in the context of chip-seq studies. However, these tools use greedy algorithms, implying a lack of sensitivity. So we developed an exact algorithm to search for degenerate motifs by enumerating all possible IUPAC motifs. This algorithm is based on mutual information and uses hashtables with graphs data structure to store the motifs. It is independent from the sequencing technology. Experimental results on real data show that there are many overrepresented motifs upstream of sequencing artifacts. These latter are identified through the strand bias between forward and reverse reads. The homopoly-mer of length 3 CCC seems to be sufficient to induce errors on IonTorrent. On Illumina, motifs are mainly composed of GGC followed by GGT (like: TGGCNGGT) or homopolymers. We have also noticed a base quality fall after the detected motifs. Our exact algorithm requires less than one minute (Intel R Core TM i5-4570 CPU, 3.20GHz), and less than 2GB of RAM to search for full degenerate motifs of length 6 on a dataset of approximately 24000 sequences, extracted from 11 exomes sequenced on IonTorrent Proton

Viability analysis and apoptosis induction of breast cancer cells in a microfluidic device: effect of cytostatic drugs

Breast cancer is the leading cause of cancer deaths among non-smoking women worldwide. At the moment the treatment regime is such that patients receive different chemotherapeutic and/or hormonal treatments dependent on the hormone receptor status, the menopausal status and age. However, in vitro sensitivity testing of tumor biopsies could rationalize and improve the choice of chemo- and hormone therapy. Lab-on-a-Chip devices, using microfluidic techniques, make detailed cellular analysis possible using fewer cells, enabling working with a patients’ own cells and performing chemo- and hormone sensitivity testing in an ex vivo setting. This article describes the development of two microfluidic devices made in poly(dimethylsiloxane) (PDMS) to validate the cell culture properties and analyze the chemosensitivity of MCF-7 cells (estrogen receptor positive human breast cancer cells) in response to the drug staurosporine (SSP). In both cases, cell viability was assessed using the life-stain Calcein-AM (CAAM) and the death dye propidium iodide (PI). MCF-7 cells could be statically cultured for up to 7 days in the microfluidic chip. A 30 min flow with SSP and a subsequent 24 h static incubation in the incubator induced apoptosis in MCF-7 cells, as shown by a disappearance of the aggregate-like morphology, a decrease in CAAM staining and an increase in PI staining. This work provides valuable leads to develop a microfluidic chip to test the chemosensitivity of tumor cells in response to therapeutics and in this way improve cancer treatment towards personalized medicine

2016 Research & Innovation Day Program

A one day showcase of applied research, social innovation, scholarship projects and activities.https://first.fanshawec.ca/cri_cripublications/1003/thumbnail.jp

MONTAGE AUDIOVISUEL DE TROIS CAS D'ASPERGILLOSE PULMONAIRE INVASIVE

LYON1-BU Santé (693882101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Genetic parameters for stillbirth in French beef cattle breeds

International audienc

Mortalité des veaux : analyse phénotypique et étude de la composante génétique

The decrease in calf mortality is a major issue for the cattle sector. The MORPHE project, funded by France Genetique Elevage was aimed at quantifying the genetic impact on calves and heifer mortality from birth to reproduction period and to define relevant phenotypes for new genetic evaluations. The phenotypic analysis of mortality was carried out on nine dairy breeds and 10 beef breeds from birth and death events reported in the national information system between 2005 and 2011. Between zero and six months, the average mortality rate depended on the breeds (from 10.5 to 19.2% in dairy breeds and from 5.1 to 13.7% in beef breeds). It also depended on the sex of the calf (+ 3% mortality for males), calving rank of dam (lower mortality in multiparous), farm size (lower mortality in small herds for dairy breeds) and birth period (higher calf mortality for birth between December and March for dairy breeds). The calf mortality rate between three and 180 days had a high phenotypic variability between sires, ranging from less than 5% for the best bulls to 10 or even 15% for the worse ones. In beef breeds, the genetic study of stillbirth (0-2 days) was a priority. This trait was split into a direct and a maternal effects. Four models were tested on three breeds (Charolaise, Limousine, and Parthenaise) to measure the impact of disconnection between herds and the discrete nature of the phenotype. Heritabilities were low (from 1 to 2%) but consistent between models. The differences of stillbirth for bulls evaluated in extreme classes were important. The comparison of breeding values estimated with the four models showed the importance of connectedness between herds. The model could probably be improved by integrating within herd heterogeneous variances. In dairy breeds, heritability of mortality was estimated with a threshold model at different life stages for Holstein, Montbeliarde, Normandy, Simmental and Brown Swiss breeds. It ranged between 0.1 and 0.9% for the first month of life and between 0.3 and 2.1% between 1 and 6 months of age. Despite these low heritability estimates, the implementation of a geneticLa baisse de la mortalité des veaux est un enjeu majeur pour les filières bovines. Le projet FGE MORPHE, visait à quantifier l’impact génétique sur la mortalité des veaux et des génisses entre leur naissance et leur mise en reproduction et à définir des phénotypes pertinents pour de nouvelles évaluations génétiques. L’analyse phénotypique de la mortalité a été réalisée sur 9 races laitières et 10 races allaitantes à partir des déclarations de naissance et de mortalité des veaux entre 2005 et 2011. Entre 0 et 6 mois, le taux moyen de mortalité est variable selon les races (entre 10,5 et 19,2% en races laitières et entre 5,1 et 13,7% en races allaitantes). Il dépend également du sexe du veau (+3% de mortalité chez les mâles), du rang de vêlage de la mère (moindre mortalité chez les multipares), de la taille des élevages (moindre mortalité dans les petits troupeaux en races laitières), et de la période de naissance (mortalité plus importante des veaux laitiers naissant entre décembre et mars). Sur la période 3-180 jours, le taux de mortalité des veaux présente une variabilité phénotypique importante entre pères, allant de moins de 5% pour les meilleurs à 10 voire 15% pour les moins bons. En races allaitantes, l’étude génétique de la mortinatalité (0-2 jours) a été prioritaire. Ce caractère est décomposé en un effet direct et un effet maternel. 4 modèles ont été testés sur 3 races (Charolaise, Limousine et Parthenaise), afin de mesurer l’impact de la déconnexion entre troupeaux et du caractère discontinu du phénotype. Les héritabilités sont faibles et cohérentes entre modèles, de l’ordre de 1 à 2%. Les écarts de moyennes phénotypiques des taureaux évalués dans les classes extrêmes sont importants. La comparaison des valeurs génétiques estimées avec les 4 modèles montre l’importance de la prise en compte de la déconnexion entre troupeaux. Le modèle pourrait probablement être amélioré en intégrant des variances hétérogènes intra troupeau. Dans les races laitières, l’héritabilité de la mortalité entre 3 et 180 jours a été estimée avec un modèle à seuil à différents stades de vie pour les races Holstein, Montbéliarde, Normande, Simmental et Brune. Elle est généralement comprise entre 0,1 et 0,9% pour les stades de vie définis au cours du 1 er mois et entre 0,3 et 2,1% entre 1 et 6 mois. Ce niveau faible reste cependant compatible avec la mise en œuvre d’une évaluation génétique, dans la perspective d’une prise en compte de ces caractères en sélection génomique

DiNAMO: Exact method for degenerate IUPAC motifs discovery, characterization of sequence-specific errors

National audienceNext generation sequencing technologies are still associated with relatively high error rates, about 1%, which correspond to thousands of errors in the scale of a complete genome. Each region needs therefore to be sequenced several times and variants are usually filtered based on depth criteria. The significant number of artifacts, in spite of those filters, shows the limit of conventional approaches and indicates that some sequencing artifacts are recurrent. This recurrence underlines that sequencing errors can depend on the upstream nucleotide sequence context. Our goal is to search for overrepresented motifs that tend to induce sequencing errors. Previous studies showed that some motifs, such as GGT [1,2], induce sequencing errors in the Illumina technologies. However, these studies were dedicated to exact motifs, and did not take into account approximate motifs, limiting the statistical power of such approaches. On the other hand, some tools, such as FIRE [3], DREME [4] and Discrover [5], were developed to search for degenerate motifs over the 15-letter IUPAC alphabet in the context of chip-seq studies. However, these tools use greedy algorithms, implying a lack of sensitivity. So we developed an exact algorithm to search for degenerate motifs by enumerating all possible IUPAC motifs. This algorithm is based on mutual information and uses hashtables with graphs data structure to store the motifs. It is independent from the sequencing technology. Experimental results on real data show that there are many overrepresented motifs upstream of sequencing artifacts. These latter are identified through the strand bias between forward and reverse reads. The homopoly-mer of length 3 CCC seems to be sufficient to induce errors on IonTorrent. On Illumina, motifs are mainly composed of GGC followed by GGT (like: TGGCNGGT) or homopolymers. We have also noticed a base quality fall after the detected motifs. Our exact algorithm requires less than one minute (Intel R Core TM i5-4570 CPU, 3.20GHz), and less than 2GB of RAM to search for full degenerate motifs of length 6 on a dataset of approximately 24000 sequences, extracted from 11 exomes sequenced on IonTorrent Proton

Individual responses of dairy cows to a 24-hour milking interval

Some dairy farmers opt to omit one milking, either incidentally or weekly, without changing other milking times. This practice entails an extended milking interval of 24 h (24h-MI), which is associated with a decrease in milk yield. This decrease varies among cows and could be partly due to factors such as stage of lactation and milk yield level. The aim of this study was to describe the average and individual responses in terms of loss and carryover effects of a 24h-MI on milk yield. The influence of factors such as parity, stage of lactation, and milk yield potential were investigated, together with response repeatability. Our trial used 292 Holstein-Friesian cows, and consisted of 3 successive periods: 1 wk of twice-daily milking (TDM) as a control, one 24h-MI, and then 13 d of TDM. The number of observations per cow ranged from 1 to 9, with no more than three 24h-MI per lactation. The 24h-MI reduced milk yield by 23% (7.8 kg on average) and milk lactose content by 2.6 g/kg on the 24h-MI day. Milk fat and protein content, and somatic cell score increased by 3.0 g/kg, 0.5 g/kg, and 0.4 units, respectively. No significant carryover effect was found of a 24h-MI on milk yield or milk composition 2 wk after resumption of TDM. Milk yield loss and recovery varied widely (coefficient of variation 62%), and the relationship between milk loss and milk recovery showed substantial variation (residual standard deviation 2.1 kg/d). Cows with a greater milk potential level lost more milk yield but recovered more milk, with no influence on recovery:loss ratio. Cows in early lactation recovered the lost milk yield faster. Repeatability of the responses to a 24h-MI was 44% for milk yield loss (kg/d), 57% for relative milk yield loss (%), 33% for milk yield recovery (kg/d), and 0% for milk recovery:loss ratio (%), suggesting a genetically determined ability to limit loss when one milking is omitted. To conclude, a 24h-MI caused higher milk yield losses than reported in previous studies. Stage of lactation, estimated potential milk yield level, and parity explained the cows' response to the 24h-MI, but did not account for all the individual variability