QuantPrime – a flexible tool for reliable high-throughput primer design for quantitative PCR

<p>Abstract</p> <p>Background</p> <p>Medium- to large-scale expression profiling using quantitative polymerase chain reaction (qPCR) assays are becoming increasingly important in genomics research. A major bottleneck in experiment preparation is the design of specific primer pairs, where researchers have to make several informed choices, often outside their area of expertise. Using currently available primer design tools, several interactive decisions have to be made, resulting in lengthy design processes with varying qualities of the assays.</p> <p>Results</p> <p>Here we present QuantPrime, an intuitive and user-friendly, fully automated tool for primer pair design in small- to large-scale qPCR analyses. QuantPrime can be used online through the internet <url>http://www.quantprime.de/</url> or on a local computer after download; it offers design and specificity checking with highly customizable parameters and is ready to use with many publicly available transcriptomes of important higher eukaryotic model organisms and plant crops (currently 295 species in total), while benefiting from exon-intron border and alternative splice variant information in available genome annotations. Experimental results with the model plant <it>Arabidopsis thaliana</it>, the crop <it>Hordeum vulgare </it>and the model green alga <it>Chlamydomonas reinhardtii </it>show success rates of designed primer pairs exceeding 96%.</p> <p>Conclusion</p> <p>QuantPrime constitutes a flexible, fully automated web application for reliable primer design for use in larger qPCR experiments, as proven by experimental data. The flexible framework is also open for simple use in other quantification applications, such as hydrolyzation probe design for qPCR and oligonucleotide probe design for quantitative <it>in situ </it>hybridization. Future suggestions made by users can be easily implemented, thus allowing QuantPrime to be developed into a broad-range platform for the design of RNA expression assays.</p

Species Identification in Malaise Trap Samples by DNA Barcoding Based on NGS Technologies and a Scoring Matrix

The German Barcoding initiatives BFB and GBOL have generated a reference library of more than 16,000 metazoan species, which is now ready for applications concerning next generation molecular biodiversity assessments. To streamline the barcoding process, we have developed a meta-barcoding pipeline: We pre-sorted a single malaise trap sample (obtained during one week in August 2014, southern Germany) into 12 arthropod orders and extracted DNA from pooled individuals of each order separately, in order to facilitate DNA extraction and avoid time consuming single specimen selection. Aliquots of each ordinal-level DNA extract were combined to roughly simulate a DNA extract from a non-sorted malaise sample. Each DNA extract was amplified using four primer sets targeting the CO1-5' fragment. The resulting PCR products (150-400bp) were sequenced separately on an Illumina Mi-SEQ platform, resulting in 1.5 million sequences and 5,500 clusters (coverage >10;CD-HIT-EST, 98%). Using a total of 120,000 DNA barcodes of identified, Central European Hymenoptera, Coleoptera, Diptera, and Lepidoptera downloaded from BOLD we established a reference sequence database for a local CUSTOM BLAST. This allowed us to identify 529 Barcode Index Numbers (BINs) from our sequence clusters derived from pooled Malaise trap samples. We introduce a scoring matrix based on the sequence match percentages of each amplicon in order to gain plausibility for each detected BIN, leading to 390 high score BINs in the sorted samples;whereas 268 of these high score BINs (69%) could be identified in the combined sample. The results indicate that a time consuming pre-sorting process will yield approximately 30% more high score BINs compared to the nonsorted sample in our case. These promising results indicate that a fast, efficient and reliable analysis of next generation data from malaise trap samples can be achieved using this pipeline

Construction and evaluation of a whole genome microarray of Chlamydomonas reinhardtii

Toepel J, Albaum S, Arvidsson S, et al. Construction and evaluation of a whole genome microarray of Chlamydomonas reinhardtii. BMC Genomics. 2011;12(1): 579.ABSTRACT: BACKGROUND: Chlamydomonas reinhardtii is widely accepted as a model organism regarding photosynthesis, circadian rhythm, cell mobility, phototaxis, and biotechnology. The complete annotation of the genome allows transcriptomic studies, however a new microarray platform was needed. Based on the completed annotation of Chlamydomonas reinhardtii a new microarray on an Agilent platform was designed using an extended JGI 3.1 genome data set which included 15000 transcript models. RESULTS: In total 44000 probes were determined (3 independent probes per transcript model) covering 93% of the transcriptome. Alignment studies with the recently published AUGUSTUS 10.2 annotation confirmed 11000 transcript models resulting in a very good coverage of 70% of the transcriptome (17000). Following the estimation of 10000 predicted genes in Chlamydomonas reinhardtii our new microarray, nevertheless, covers the expected genome by 90-95%. CONCLUSIONS: To demonstrate the capabilities of the new microarray, we analyzed transcript levels for cultures grown under nitrogen as well as sulfate limitation, and compared the results with recently published microarray and RNA-seq data. We could thereby confirm previous results derived from data on nutrient-starvation induced gene expression of a group of genes related to protein transport and adaptation of the metabolism as well as genes related to efficient light harvesting, light energy distribution and photosynthetic electron transport

Efficacy of dust binding agents : a laboratory study

I arbetet har kalciumklorid respektive magnesiumklorid utvärderats i kombination med fyra olika finmaterial (granit, amfibolit, metagråvacka samt naturlig silt) vilka används vid underhåll av grusvägar. Vattenlösningar av respektive salt har tillförts finmaterialet varefter regn och upptorkning har simulerats i laboratoriemiljö. Utvärdering har gjorts med avseende på mängden kvarvarande salt efter regnsimulering samt svepelektronmikroskopi (SEM) och optisk mikroskopi på finmaterialet efter upptorkning. Det har varit svårt att dra några slutsatser. Den framtagna försöksmetodiken har inte kunnat påvisa agglomerering i finfraktion av grusslitlager eller ge ledning till doseringsråd. Man kan se att kalciumklorid är effektivare per giva än magnesiumklorid. Det krävs mindre mängd salt (flingor) för att binda en viss mängd vatten med kalciumklorid än med magnesiumklorid vilket dock är känt sedan tidigare. Agglomerering kunde inte bevisas med upprepade försök. Kemiskt var det ingen större skillnad, salterna betedde sig snarlikt och enligt tidigare känt beteende. Skillnader av beteende beroende på typ av bergmaterial var försumbart i förhållande till salternas inverkan. Den allra största delen (som minst 80 %) av tillfört salt har lakats ur efter regnsimulering. Ingen skillnad mellan kalcium- och magnesiumklorid eller de olika finmaterialen kunde konstateras avseende urlakning. En liten skillnad mellan urlakning av katjoner och anjoner kunde konstateras där katjonerna hölls kvar i finmaterialet. Detta kan röra sig om en jonbytareffekt. Analys med SEM och optisk mikroskopi kunde inte påvisa några egentliga skillnader mellan de olika kombinationerna av salt respektive finmaterial. Vid upptorkning av finmaterialet kunde konstateras att kalciumklorid, räknat som handelsvara, kan absorbera mer vatten än magnesiumklorid. Denna effekt kvarstod även efter att regnsimulering har utförts. Den metod som utvecklades i samband med arbetet gav inga påvisbara skillnader, som inte var kända sedan tidigare, mellan de olika salterna och finmaterialen. Troligen har systemet förenklats så långt att relevanta parametrar tagits bort eller inte reflekterar verkliga fältförhållanden.  In this study, calcium chloride and magnesium chloride have been evaluated in combination with four different fine materials (granite, amphibolite, meta greywacke and naturally occurring silt), which are used for gravel road maintenance. Aqueous solutions of the two salts have been added to the fine material where after rain and drying has been simulated under laboratory conditions. Evaluation has been performed in terms of residual amount of salt after rain simulation, and SEM and optical microscopy on the fine material after drying, respectively It has been difficult to draw any conclusions that would yield new information from the tests. The methodology developed was not able to show agglomeration in the fine fraction of the gravel road material and it was not possible to provide recommendations regarding the dosage of dust binding agents. The tests show that calcium chloride is more efficient in retaining moisture than magnesium chloride when the comparison is based on the amount of flakes used. Agglomeration could not be proven despite several repeated tests. The chemical properties did not differ significantly, the salts behaved similarly and according to earlier known data. Differences in the origin of rock material was negligible compared with the effects of the salts. The added salt was for the most part (with a minimum of 80%) leached out during the rain simulation. No differences between calcium chloride and magnesium chloride or between the different fine materials were observed in terms of leaching. A small difference between leaching of cations and anions was recorded where the cations were retained in the fine material. This might be an ion-exchange effect. Analysis with SEM and optical microscopy did not yield any significant differences between the different combinations of salt and fine materials, respectively. During drying of the fine material it was observed that calcium chloride, calculated as commercial product, could absorb more water than magnesium chloride. This effect persisted also after the rain simulation. The experimental method that was developed during the study did not give any, previously not known, differences between the different salts and fine materials. Probably the system has been simplified to such an extent that relevant parameters were removed or do not reflect real field conditions

Efficacy of dust binding agents : a laboratory study

I arbetet har kalciumklorid respektive magnesiumklorid utvärderats i kombination med fyra olika finmaterial (granit, amfibolit, metagråvacka samt naturlig silt) vilka används vid underhåll av grusvägar. Vattenlösningar av respektive salt har tillförts finmaterialet varefter regn och upptorkning har simulerats i laboratoriemiljö. Utvärdering har gjorts med avseende på mängden kvarvarande salt efter regnsimulering samt svepelektronmikroskopi (SEM) och optisk mikroskopi på finmaterialet efter upptorkning. Det har varit svårt att dra några slutsatser. Den framtagna försöksmetodiken har inte kunnat påvisa agglomerering i finfraktion av grusslitlager eller ge ledning till doseringsråd. Man kan se att kalciumklorid är effektivare per giva än magnesiumklorid. Det krävs mindre mängd salt (flingor) för att binda en viss mängd vatten med kalciumklorid än med magnesiumklorid vilket dock är känt sedan tidigare. Agglomerering kunde inte bevisas med upprepade försök. Kemiskt var det ingen större skillnad, salterna betedde sig snarlikt och enligt tidigare känt beteende. Skillnader av beteende beroende på typ av bergmaterial var försumbart i förhållande till salternas inverkan. Den allra största delen (som minst 80 %) av tillfört salt har lakats ur efter regnsimulering. Ingen skillnad mellan kalcium- och magnesiumklorid eller de olika finmaterialen kunde konstateras avseende urlakning. En liten skillnad mellan urlakning av katjoner och anjoner kunde konstateras där katjonerna hölls kvar i finmaterialet. Detta kan röra sig om en jonbytareffekt. Analys med SEM och optisk mikroskopi kunde inte påvisa några egentliga skillnader mellan de olika kombinationerna av salt respektive finmaterial. Vid upptorkning av finmaterialet kunde konstateras att kalciumklorid, räknat som handelsvara, kan absorbera mer vatten än magnesiumklorid. Denna effekt kvarstod även efter att regnsimulering har utförts. Den metod som utvecklades i samband med arbetet gav inga påvisbara skillnader, som inte var kända sedan tidigare, mellan de olika salterna och finmaterialen. Troligen har systemet förenklats så långt att relevanta parametrar tagits bort eller inte reflekterar verkliga fältförhållanden.  In this study, calcium chloride and magnesium chloride have been evaluated in combination with four different fine materials (granite, amphibolite, meta greywacke and naturally occurring silt), which are used for gravel road maintenance. Aqueous solutions of the two salts have been added to the fine material where after rain and drying has been simulated under laboratory conditions. Evaluation has been performed in terms of residual amount of salt after rain simulation, and SEM and optical microscopy on the fine material after drying, respectively It has been difficult to draw any conclusions that would yield new information from the tests. The methodology developed was not able to show agglomeration in the fine fraction of the gravel road material and it was not possible to provide recommendations regarding the dosage of dust binding agents. The tests show that calcium chloride is more efficient in retaining moisture than magnesium chloride when the comparison is based on the amount of flakes used. Agglomeration could not be proven despite several repeated tests. The chemical properties did not differ significantly, the salts behaved similarly and according to earlier known data. Differences in the origin of rock material was negligible compared with the effects of the salts. The added salt was for the most part (with a minimum of 80%) leached out during the rain simulation. No differences between calcium chloride and magnesium chloride or between the different fine materials were observed in terms of leaching. A small difference between leaching of cations and anions was recorded where the cations were retained in the fine material. This might be an ion-exchange effect. Analysis with SEM and optical microscopy did not yield any significant differences between the different combinations of salt and fine materials, respectively. During drying of the fine material it was observed that calcium chloride, calculated as commercial product, could absorb more water than magnesium chloride. This effect persisted also after the rain simulation. The experimental method that was developed during the study did not give any, previously not known, differences between the different salts and fine materials. Probably the system has been simplified to such an extent that relevant parameters were removed or do not reflect real field conditions.On the Roa

Molekylär klassificering av tjocktarmscancer : PAM-klusteranalys för identifiering av undergrupper

The main objective of this study is to divide a number of colorectal cancer cases into subgroups based on their molecular features using cluster analysis. The data used is supplied by a research group at Pathology, the Department of Medical Biosciences, Umeå University, and consists, after some preparation, of 455 observations which is a larger data set than many similar studies. The molecular variables that the clustering is based on are CIMP (CpG Island Methylator Phenotype), MSI (Micro Satellite Instability), BRAF- and KRAS-mutations. These are categorical variables and consequently the clustering method used is PAM (Partitioning Around Medoids) which is particularly useful with data on diverse variable level. The final analysis results in four subgroups that are represented by different combinations of attributes on the aforementioned variables. The disparity between the clusters are then evaluated by, for instance, comparing the survival time for their pertaining patients and it appears that two of the clusters are significantly different in this aspect. Other patient related and tumor specific characteristics are also linked with the separate cancer types and tested if they occur in varying extent. The locations of the tumors in the colon are for instance significantly different between the groups. Cluster analyses are exploratory tools so the choice of useful variables and subsequent interpretation of the results can be complicated and require relevant subject knowledge.Huvudsyftet med denna studie är att med hjälp av klusteranalys dela in en mängd tjocktarmscancerfall i undergrupper baserat på deras molekylära egenskaper. Materialet som används tillhandahålls av en forskningsgrupp vid Patologi, Institutionen för medicinsk biovetenskap, Umeå universitet, och består efter viss bearbetning av 455 observationer vilket är en större datamängd än flera liknande studier. De molekylära variabler som ligger till grund för klusterindelningen är nivån på CIMP (CpG Island Methylator Phenotype), MSI (Mikrosatellitinstabillitet), BRAF- och KRAS-mutationer. Dessa är kategoriska variabler och därför används PAM (Partitioning Around Medoids) som är en särskild klusterteknik lämpad vid data på varierade variabelnivåer. I det slutliga resultatet fås fyra undergrupper som representeras av olika kombinationer av utfallen på ovannämnda variabler. Klustren utvärderas bland annat genom att jämföra överlevnadstiden för varje kluster, och det visar sig att två av klustren skiljer sig signifikant åt i detta avseende. Även andra patientrelaterade och tumörspecifika egenskaper kopplas samman till de olika cancertyperna och testas om de förekommer i varierande utsträckning. Var någonstans tumören är placerad är till exempel signifikant skilt mellan grupperna. Klusteranalyser är explorativa redskap så valet av variabler och sedermera tolkningar av resultat kan vara komplicerade och kräva stor sakkunskap