PoolHap: Inferring Haplotype Frequencies from Pooled Samples by Next Generation Sequencing

With the advance of next-generation sequencing (NGS) technologies, increasingly ambitious applications are becoming feasible. A particularly powerful one is the sequencing of polymorphic, pooled samples. The pool can be naturally occurring, as in the case of multiple pathogen strains in a blood sample, multiple types of cells in a cancerous tissue sample, or multiple isoforms of mRNA in a cell. In these cases, it's difficult or impossible to partition the subtypes experimentally before sequencing, and those subtype frequencies must hence be inferred. In addition, investigators may occasionally want to artificially pool the sample of a large number of individuals for reasons of cost-efficiency, e. g., when carrying out genetic mapping using bulked segregant analysis. Here we describe PoolHap, a computational tool for inferring haplotype frequencies from pooled samples when haplotypes are known. The key insight into why PoolHap works is that the large number of SNPs that come with genome-wide coverage can compensate for the uneven coverage across the genome. The performance of PoolHap is illustrated and discussed using simulated and real data. We show that PoolHap is able to accurately estimate the proportions of haplotypes with less than 2% error for 34-strain mixtures with 2X total coverage Arabidopsis thaliana whole genome polymorphism data. This method should facilitate greater biological insight into heterogeneous samples that are difficult or impossible to isolate experimentally. Software and users manual are freely available at http://arabidopsis.gmi.oeaw.ac.at/quan/poolhap/

The Complex Genetic Architecture of the Metabolome

Discovering links between the genotype of an organism and its metabolite levels can increase our understanding of metabolism, its controls, and the indirect effects of metabolism on other quantitative traits. Recent technological advances in both DNA sequencing and metabolite profiling allow the use of broad-spectrum, untargeted metabolite profiling to generate phenotypic data for genome-wide association studies that investigate quantitative genetic control of metabolism within species. We conducted a genome-wide association study of natural variation in plant metabolism using the results of untargeted metabolite analyses performed on a collection of wild Arabidopsis thaliana accessions. Testing 327 metabolites against >200,000 single nucleotide polymorphisms identified numerous genotype–metabolite associations distributed non-randomly within the genome. These clusters of genotype–metabolite associations (hotspots) included regions of the A. thaliana genome previously identified as subject to recent strong positive selection (selective sweeps) and regions showing trans-linkage to these putative sweeps, suggesting that these selective forces have impacted genome-wide control of A. thaliana metabolism. Comparing the metabolic variation detected within this collection of wild accessions to a laboratory-derived population of recombinant inbred lines (derived from two of the accessions used in this study) showed that the higher level of genetic variation present within the wild accessions did not correspond to higher variance in metabolic phenotypes, suggesting that evolutionary constraints limit metabolic variation. While a major goal of genome-wide association studies is to develop catalogues of intraspecific variation, the results of multiple independent experiments performed for this study showed that the genotype–metabolite associations identified are sensitive to environmental fluctuations. Thus, studies of intraspecific variation conducted via genome-wide association will require analyses of genotype by environment interaction. Interestingly, the network structure of metabolite linkages was also sensitive to environmental differences, suggesting that key aspects of network architecture are malleable

Global Analysis of Arabidopsis/Downy Mildew Interactions Reveals Prevalence of Incomplete Resistance and Rapid Evolution of Pathogen Recognition

Interactions between Arabidopsis thaliana and its native obligate oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) represent a model system to study evolution of natural variation in a host/pathogen interaction. Both Arabidopsis and Hpa genomes are sequenced and collections of different sub-species are available. We analyzed ∼400 interactions between different Arabidopsis accessions and five strains of Hpa. We examined the pathogen's overall ability to reproduce on a given host, and performed detailed cytological staining to assay for pathogen growth and hypersensitive cell death response in the host. We demonstrate that intermediate levels of resistance are prevalent among Arabidopsis populations and correlate strongly with host developmental stage. In addition to looking at plant responses to challenge by whole pathogen inoculations, we investigated the Arabidopsis resistance attributed to recognition of the individual Hpa effectors, ATR1 and ATR13. Our results suggest that recognition of these effectors is evolutionarily dynamic and does not form a single clade in overall Arabidopsis phylogeny for either effector. Furthermore, we show that the ultimate outcome of the interactions can be modified by the pathogen, despite a defined gene-for-gene resistance in the host. These data indicate that the outcome of disease and disease resistance depends on genome-for-genome interactions between the host and its pathogen, rather than single gene pairs as thought previously

2015 recommendations for the management of polymyalgia rheumatica: a European League Against Rheumatism/American College of Rheumatology collaborative initiative

Therapy for polymyalgia rheumatica (PMR) varies widely in clinical practice as international recommendations for PMR treatment are not currently available. In this paper, we report the 2015 European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) recommendations for the management of PMR. We used the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) methodology as a framework for the project. Accordingly, the direction and strength of the recommendations are based on the quality of evidence, the balance between desirable and undesirable effects, patients'and clinicians'values and preferences, and resource use. Eight overarching principles and nine specific recommendations were developed covering several aspects of PMR, including basic and follow-up investigations of patients under treatment, risk factor assessment, medical access for patients and specialist referral, treatment strategies such as initial glucocorticoid (GC) doses and subsequent tapering regimens, use of intramuscular GCs and disease modifying anti-rheumatic drugs (DMARDs), as well as the roles of non-steroidal anti-rheumatic drugs and non-pharmacological interventions. These recommendations will inform primary, secondary and tertiary care physicians about an international consensus on the management of PMR. These recommendations should serve to inform clinicians about best practices in the care of patients with PMR

Natural Selection Affects Multiple Aspects of Genetic Variation at Putatively Neutral Sites across the Human Genome

A major question in evolutionary biology is how natural selection has shaped patterns of genetic variation across the human genome. Previous work has documented a reduction in genetic diversity in regions of the genome with low recombination rates. However, it is unclear whether other summaries of genetic variation, like allele frequencies, are also correlated with recombination rate and whether these correlations can be explained solely by negative selection against deleterious mutations or whether positive selection acting on favorable alleles is also required. Here we attempt to address these questions by analyzing three different genome-wide resequencing datasets from European individuals. We document several significant correlations between different genomic features. In particular, we find that average minor allele frequency and diversity are reduced in regions of low recombination and that human diversity, human-chimp divergence, and average minor allele frequency are reduced near genes. Population genetic simulations show that either positive natural selection acting on favorable mutations or negative natural selection acting against deleterious mutations can explain these correlations. However, models with strong positive selection on nonsynonymous mutations and little negative selection predict a stronger negative correlation between neutral diversity and nonsynonymous divergence than observed in the actual data, supporting the importance of negative, rather than positive, selection throughout the genome. Further, we show that the widespread presence of weakly deleterious alleles, rather than a small number of strongly positively selected mutations, is responsible for the correlation between neutral genetic diversity and recombination rate. This work suggests that natural selection has affected multiple aspects of linked neutral variation throughout the human genome and that positive selection is not required to explain these observations

Contributions of protein-coding and regulatory change to adaptive molecular evolution in murid rodents

The contribution of regulatory versus protein change to adaptive evolution has long been controversial. In principle, the rate and strength of adaptation within functional genetic elements can be quantified on the basis of an excess of nucleotide substitutions between species compared to the neutral expectation or from effects of recent substitutions on nucleotide diversity at linked sites. Here, we infer the nature of selective forces acting in proteins, their UTRs and conserved noncoding elements (CNEs) using genome-wide patterns of diversity in wild house mice and divergence to related species. By applying an extension of the McDonald-Kreitman test, we infer that adaptive substitutions are widespread in protein-coding genes, UTRs and CNEs, and we estimate that there are at least four times as many adaptive substitutions in CNEs and UTRs as in proteins. We observe pronounced reductions in mean diversity around nonsynonymous sites (whether or not they have experienced a recent substitution). This can be explained by selection on multiple, linked CNEs and exons. We also observe substantial dips in mean diversity (after controlling for divergence) around protein-coding exons and CNEs, which can also be explained by the combined effects of many linked exons and CNEs. A model of background selection (BGS) can adequately explain the reduction in mean diversity observed around CNEs. However, BGS fails to explain the wide reductions in mean diversity surrounding exons (encompassing ~100 Kb, on average), implying that there is a substantial role for adaptation within exons or closely linked sites. The wide dips in diversity around exons, which are hard to explain by BGS, suggest that the fitness effects of adaptive amino acid substitutions could be substantially larger than substitutions in CNEs. We conclude that although there appear to be many more adaptive noncoding changes, substitutions in proteins may dominate phenotypic evolution

Tree Core Sampling to Assess the Degree of Chlorinated Solvent Contamination in Groundwater

Chlorinated aliphatic hydrocarbons (CAH´s) were used widely within dry cleaning facilities and for metal degreasing until their toxicity was discovered. PCE is still used as dry cleaning liquid. Today CAH´s are found in soil environment at places where they have been used in the past. The CAH-concentration in trees growing on contaminated land has quite recently received attention as a cheap and effective way of assessing the extent of a CAH-contamination. The method has however, not been put into use in Sweden. The aim of the study has been to investigate whether the CAH-concentration in tree cores could be used to delineate the spread of CAH in a soil environment under Swedish conditions in different seasons. The aim has also been to gain an understanding of the uptake process, as well as to identify the limitations of the method and important issues to consider when sampling. Trees were sampled in March and June on Helgö 1:25, 1:26 in Växjö, Småland. Metal degreasing earlier conducted at this site has lead to the CAH contamination of soil and groundwater (PCE, TEC c-DACE). Mostly PCE, TCE, and chloroform were detected in tree cores. The CAH concentration was higher in June. Using the sum of PCE+TCE+c-DCE in trees to delineate the spread gave a result that was quite consistent with a delination done based on groundwater sampling. The uptake of CAH by trees is governed by the uptake of water at the root. The water usage, together with the origin of the water used is important for the ability of the tree to take up CAH. The CAH concentration within trees is also dependent on the chemical properties of the compound (Log kow, solubility etc), the concentration of the compound in the soil as well as degradation processeses. The position and height of sampling in the trees, tree species as well as tree size are important factors to consider when sampling. Sampling during summer is preferred when the concentration of CAH in trees is likely to be higher. The analysis of CAH in tree cores has potential to be used as a screening tool in soil investigations under Swedish conditions. It is a cheap and easy to use method, which would be a good complement to other investigative measures. However, an increased understanding of the processes involved, together with more analysis are needed., as this is a new method.Klorerade lösningsmedel (CAH) användes i stor omfattning som bl a kemtvättmedel och avfettningsmedel tills dess att deras toxiska egenskaper blev kända. Perkloretylen (PCE) används än idag som kemtvättmedel. CAH återfinns ofta i markmiljö på de platser där de tidigare använts. Analys av CAH-koncentration i trädkärnor har uppmärksammats som en billig och effektiv metod för att översiktligt bedöma utbredningen av dessa föroreningar. Metoden har inte tidigare använts i Sverige. Syftet med detta examensarbete är att undersöka om trädprovtagning kan användas för att bedöma utbredningen av föroreningar i markmiljö under svenska förhållanden vid olika årstider. Analyserade CAH-halter i träd har jämförts med tidigare registrerade halter av CAH i grundvatten. Syftet har också varit att beskriva CAH-upptaget i träd, undersöka metodens begränsningar samt att sammanfatta viktiga aspekter vid provtagning. Provtagningar av träd har genomförts under mars och juni på fastigheterna Helgö 1:25 och 1:26 i Växjö, där tidigare metallavfettning har medfört att mark och grundvattnet förorenats av CAH; perkloretylen (PCE), trikloretylen (TCE) och nedbrytningsprodukten dikloretylen (c1,2-DCE). Vid analys av trädkärnor detekterades främst PCE, TCE samt TCM (kloroform). Koncentrationen av CAH var högre i juni. Halten PCE+TCE+c-DCE i trädproverna gav en översiktlig bild av föroreningssituationen som överensstämde väl med de grundvattenprover som tidigare tagits på fastigheten. CAH tas upp i vattenlöst fas vid trädens rötter. Trädets vattenbehov och vilket vatten det utnyttjar är därför viktigt för dess möjlighet att ta upp CAH. Ämnets kemiska egenskaper (log kow, flyktighet, mm.), samt förekomst och nedbrytning påverkar den halt som registreras i trädet. Vid provtagning bör provtagningspunkternas höjd över marken och position, trädart samt trädstorlek beaktas. Provtagning under sommaren är att föredra eftersom halterna då är högre. Jämförelsen med grundvattenprovtagning visar att metoden har potential att användas i Sverige för att bedöma utbredningen av en CAH-förorening i markmiljö. Den är enkel att använda och kan vara ett alternativ på platser där konventionella metoder är svåra att genomföra. En ökad förståelse för involverade processer, samt utökade undersökningar av metoden är nödvändiga då metoden är ny

Possibilities for increased reliability and cost-effectivness in district heating networks : A simulation and optimization study at Kalmar Energi

Since district heating is the most common way of space heatingin Sweden, continual improvements of theefficiency of production and distribution of district heating is crucial. The purposeof this report is therefore to, using Kalmar Energi as an example, study how the production and distribution of district heating is affected by introducing a bypass pipeat a critical point in the network, and to investigate howcost efficiency might be achieved. To fulfill this task, the network simulation software NetSim and the cost optimizing software MODEST was used. Most ofthe heat in the district heating network in Kalmar comesfrom the bio-fueled combined heat and power plant Moskogen and the top load production plantDraken covers the top load when the heat from Moskogen is not enoughto fulfill the heating demand. However, the studied scenario does not represent normal operationbut a temporary solution for when Draken and its circulation pumps for some reason cannot be used.In that scenario, all heat in the city mustbe delivered using the bypass pipeor usingthe two smaller reserve production plantsLindsdal and Dvärgen. The simulations performed in NetSim consist of three primary cases; one reference modelwhich represents the currentsituation, one model with only Moskogenand the bypass and one model with Moskogen, bypass, Lindsdal and Dvärgen. To study the possibility for improvements of the network further simulations were performed with measures like new circulation pumps, upgradeto bigger pipes and addition of new pipes. The optimization in MODEST also consist of three cases; normaloperation, operation using the bypassand possible benefits ofusing RME instead of oil in reserve plants. The simulations in NetSimshows that the bypass pipe enables fulfillment of the heating demand of Kalmar without the use ofDraken or otherreserveproduction plantsdown to an outside temperature around 1°C.If the reserve units in the periphery parts of the city are used, the bypass of Draken enables fulfillmentof the heating demand until an outside temperature of approximately -3°C. The limiting factorsare mainlycirculationpump capacity and the differential pressure. If a higher supply temperature than normal is usedor newcirculationpumps and bigger pipes are introduced, a working network can be achieved for loweroutsidetemperatures. However, a highersupply temperature means higherheatlosses and no additional electricity production and a new pumping station just to handle a worst-case scenario is most likely not economically feasible. New pipes connecting areas with high and low differential pressures was also found to improve the system, but might not be profitable in the short term. If the city is expected to expand in the certain areas that the new pipesweretested, the new pipes might become amore feasiblemeasurein the future.The optimizing in MODEST shows that from a cost minimizing perspective, it is optimal to use the biomass-fueled combined heat and power plant Moskogenas much as possibleand to cover the remaining heat demand using wood powderboilersand if necessary, oilboilers.It is also evident thatregardless ofthe bypass beingused or not, it is always more profitable to lower the electricity production of Moskogen in favor of heat production during periods when the electricity price is low or whentheuse of oilboilerswould otherwise be needed.This measure also lowers the amount ofcarbon dioxideemissionsfor the systemwith up to 250 tons/yearsince oil use can be exchanged for biomass use.An alternative way of eliminating carbon dioxide emissionsis to replace the oilfuelwith a renewable fuel like RME. However, this would be associated with higher costs for Kalmar Energi, since RME is currently more expensive per MWh than the oil that is currentlyused.Med tanke på att fjärrvärme är den vanligaste uppvärmningsprincipen i Sverige är arbete med att förbättra effektiviteten och pålitligheten vid produktion och distribution av värme ett ständigt aktuellt ämne. Syftet med detta arbete är därför att med Kalmar Energi som exempel undersöka hur produktion och distribution av fjärrvärme påverkas av att en förbikoppling av en kritisk sektion har byggts samt att identifiera hur kostnadsoptimal drift av fjärrvärmenätet bör ske. För att uppnå detta har nätverkssimuleringsprogrammet NetSim och kostnadsoptimeringsprogrammet MODEST använts. Majoriteten av värmen i Kalmars fjärrvärmenät kommer från det bioeldade kraftvärmeverket Moskogen. Värmeverket Draken täcker under normal drift upp när Moskogens effekt inte räcker till för att uppfylla värmeefterfrågan. Scenariot som framförallt har studerats representerar dock inte normal drift utan utgör en tillfällig lösning för perioder när värmeverket Draken med tillhörande pumpar av någon anledning inte är tillgängliga. All Kalmars fjärrvärme måste i detta läge levereras från Moskogen via den nybyggda förbikopplingen eller med hjälp av de två mindre reservanläggningarna Lindsdal och Dvärgen. De simuleringar som gjorts i NetSim består av tre primära fall; en referensmodell som illustrerar nuläget, en modell med endast Moskogen via förbikopplingen samt en modell med Moskogen via förbikopplingen i kombination med Dvärgen och Lindsdal. För att studera förbättringsmöjligheter i systemet genomfördes även simuleringar på hur förändringar så som nya pumpar och utbyte av gamla rör påverkar systemet. Arbetet i MODEST består också av tre olika delar; normal drift, drift vid användning av förbikopplingen samt nyttan med ett bränslebyte från olja till RME. För normal drift och förbikopplingsdrift utvärderas lönsamheten med att minska elproduktionen och därmed få ut mer värme till fjärrvärmenätet.   Simuleringarna i NetSim visar att Moskogen tack vare förbikopplingen ensam kan uppfylla Kalmars värmebehov ner till utomhustemperaturer kring 1 °C. Vid användning av reservanläggningarna i kombination med förbikopplingen kan värmebehovet i dagsläget uppfyllas fullständigt ner till utomhustemperaturer kring -3 °C. De begränsande faktorerna för nätet är framförallt kapacitet hos cirkulationspumpar och differenstryck. Om högre framledningstemperatur används eller om nya cirkulationspumpar införs och begränsande rör görs större kan ett väl fungerande system fås utan Draken även vid lägre utomhustemperaturer. Värt att notera är dock att höjd framledningstemperatur ökar värmeförlusterna i nätet samt att införskaffandet av nya pumpar endast för att hantera ett reservfall bedöms bli dyrt. Nya rör som kopplar samman områden med höga och låga differenstryck förbättrar också systemet men det bedöms på kort sikt inte vara lönsamt att implementera men kan kanske bli aktuellt i samband med framtida expansion av staden.  Optimeringen av driften i MODEST visar att det ur produktionskostnadsperspektiv är optimalt att använda det bioeldade kraftvärmeverket Moskogen så mycket som möjligt och sedan täcka upp med träpulver och därefter olja när det behövs. Optimeringen visar också att det både vid utnyttjande av förbikopplingen och vid normal drift är ekonomiskt lönsamt att minska på elproduktionen i Moskogen till fördel för ökad värmeproduktion under perioder när elpriset är lågt eller när olja annars måste användas för värmeproduktion. Denna åtgärd minskar även utsläppen av fossil koldioxid med upp till 250 ton koldioxid per år eftersom oljeanvändning kan ersättas med Moskogens biobränsle. Ett alternativt sätt att eliminera utsläpp av fossil koldioxid vore att ersätta oljan med ett förnybart alternativ, exempelvis RME. Detta medför dock i Kalmars fall att den årliga driftskostnaden ökar med 179 miljoner SEK per år eftersom RME i dagsläget är dyrare per MWh än den olja som idag används

Possibilities for increased reliability and cost-effectivness in district heating networks : A simulation and optimization study at Kalmar Energi

Since district heating is the most common way of space heatingin Sweden, continual improvements of theefficiency of production and distribution of district heating is crucial. The purposeof this report is therefore to, using Kalmar Energi as an example, study how the production and distribution of district heating is affected by introducing a bypass pipeat a critical point in the network, and to investigate howcost efficiency might be achieved. To fulfill this task, the network simulation software NetSim and the cost optimizing software MODEST was used. Most ofthe heat in the district heating network in Kalmar comesfrom the bio-fueled combined heat and power plant Moskogen and the top load production plantDraken covers the top load when the heat from Moskogen is not enoughto fulfill the heating demand. However, the studied scenario does not represent normal operationbut a temporary solution for when Draken and its circulation pumps for some reason cannot be used.In that scenario, all heat in the city mustbe delivered using the bypass pipeor usingthe two smaller reserve production plantsLindsdal and Dvärgen. The simulations performed in NetSim consist of three primary cases; one reference modelwhich represents the currentsituation, one model with only Moskogenand the bypass and one model with Moskogen, bypass, Lindsdal and Dvärgen. To study the possibility for improvements of the network further simulations were performed with measures like new circulation pumps, upgradeto bigger pipes and addition of new pipes. The optimization in MODEST also consist of three cases; normaloperation, operation using the bypassand possible benefits ofusing RME instead of oil in reserve plants. The simulations in NetSimshows that the bypass pipe enables fulfillment of the heating demand of Kalmar without the use ofDraken or otherreserveproduction plantsdown to an outside temperature around 1°C.If the reserve units in the periphery parts of the city are used, the bypass of Draken enables fulfillmentof the heating demand until an outside temperature of approximately -3°C. The limiting factorsare mainlycirculationpump capacity and the differential pressure. If a higher supply temperature than normal is usedor newcirculationpumps and bigger pipes are introduced, a working network can be achieved for loweroutsidetemperatures. However, a highersupply temperature means higherheatlosses and no additional electricity production and a new pumping station just to handle a worst-case scenario is most likely not economically feasible. New pipes connecting areas with high and low differential pressures was also found to improve the system, but might not be profitable in the short term. If the city is expected to expand in the certain areas that the new pipesweretested, the new pipes might become amore feasiblemeasurein the future.The optimizing in MODEST shows that from a cost minimizing perspective, it is optimal to use the biomass-fueled combined heat and power plant Moskogenas much as possibleand to cover the remaining heat demand using wood powderboilersand if necessary, oilboilers.It is also evident thatregardless ofthe bypass beingused or not, it is always more profitable to lower the electricity production of Moskogen in favor of heat production during periods when the electricity price is low or whentheuse of oilboilerswould otherwise be needed.This measure also lowers the amount ofcarbon dioxideemissionsfor the systemwith up to 250 tons/yearsince oil use can be exchanged for biomass use.An alternative way of eliminating carbon dioxide emissionsis to replace the oilfuelwith a renewable fuel like RME. However, this would be associated with higher costs for Kalmar Energi, since RME is currently more expensive per MWh than the oil that is currentlyused.Med tanke på att fjärrvärme är den vanligaste uppvärmningsprincipen i Sverige är arbete med att förbättra effektiviteten och pålitligheten vid produktion och distribution av värme ett ständigt aktuellt ämne. Syftet med detta arbete är därför att med Kalmar Energi som exempel undersöka hur produktion och distribution av fjärrvärme påverkas av att en förbikoppling av en kritisk sektion har byggts samt att identifiera hur kostnadsoptimal drift av fjärrvärmenätet bör ske. För att uppnå detta har nätverkssimuleringsprogrammet NetSim och kostnadsoptimeringsprogrammet MODEST använts. Majoriteten av värmen i Kalmars fjärrvärmenät kommer från det bioeldade kraftvärmeverket Moskogen. Värmeverket Draken täcker under normal drift upp när Moskogens effekt inte räcker till för att uppfylla värmeefterfrågan. Scenariot som framförallt har studerats representerar dock inte normal drift utan utgör en tillfällig lösning för perioder när värmeverket Draken med tillhörande pumpar av någon anledning inte är tillgängliga. All Kalmars fjärrvärme måste i detta läge levereras från Moskogen via den nybyggda förbikopplingen eller med hjälp av de två mindre reservanläggningarna Lindsdal och Dvärgen. De simuleringar som gjorts i NetSim består av tre primära fall; en referensmodell som illustrerar nuläget, en modell med endast Moskogen via förbikopplingen samt en modell med Moskogen via förbikopplingen i kombination med Dvärgen och Lindsdal. För att studera förbättringsmöjligheter i systemet genomfördes även simuleringar på hur förändringar så som nya pumpar och utbyte av gamla rör påverkar systemet. Arbetet i MODEST består också av tre olika delar; normal drift, drift vid användning av förbikopplingen samt nyttan med ett bränslebyte från olja till RME. För normal drift och förbikopplingsdrift utvärderas lönsamheten med att minska elproduktionen och därmed få ut mer värme till fjärrvärmenätet.   Simuleringarna i NetSim visar att Moskogen tack vare förbikopplingen ensam kan uppfylla Kalmars värmebehov ner till utomhustemperaturer kring 1 °C. Vid användning av reservanläggningarna i kombination med förbikopplingen kan värmebehovet i dagsläget uppfyllas fullständigt ner till utomhustemperaturer kring -3 °C. De begränsande faktorerna för nätet är framförallt kapacitet hos cirkulationspumpar och differenstryck. Om högre framledningstemperatur används eller om nya cirkulationspumpar införs och begränsande rör görs större kan ett väl fungerande system fås utan Draken även vid lägre utomhustemperaturer. Värt att notera är dock att höjd framledningstemperatur ökar värmeförlusterna i nätet samt att införskaffandet av nya pumpar endast för att hantera ett reservfall bedöms bli dyrt. Nya rör som kopplar samman områden med höga och låga differenstryck förbättrar också systemet men det bedöms på kort sikt inte vara lönsamt att implementera men kan kanske bli aktuellt i samband med framtida expansion av staden.  Optimeringen av driften i MODEST visar att det ur produktionskostnadsperspektiv är optimalt att använda det bioeldade kraftvärmeverket Moskogen så mycket som möjligt och sedan täcka upp med träpulver och därefter olja när det behövs. Optimeringen visar också att det både vid utnyttjande av förbikopplingen och vid normal drift är ekonomiskt lönsamt att minska på elproduktionen i Moskogen till fördel för ökad värmeproduktion under perioder när elpriset är lågt eller när olja annars måste användas för värmeproduktion. Denna åtgärd minskar även utsläppen av fossil koldioxid med upp till 250 ton koldioxid per år eftersom oljeanvändning kan ersättas med Moskogens biobränsle. Ett alternativt sätt att eliminera utsläpp av fossil koldioxid vore att ersätta oljan med ett förnybart alternativ, exempelvis RME. Detta medför dock i Kalmars fall att den årliga driftskostnaden ökar med 179 miljoner SEK per år eftersom RME i dagsläget är dyrare per MWh än den olja som idag används