SNP discovery using Paired‐End RAD‐tag sequencing on pooled genomic DNA of Sisymbrium austriacum (Brassicaceae)

Single nucleotide polymorphisms SNPs are rapidly replacing anonymous markers in population genomic studies, but their use in non model organisms is hampered by the scarcity of cost‐effective approaches to uncover genome‐wide variation in a comprehensive subset of individuals. The screening of one or only a few individuals induces ascertainment bias. To discover SNPs for a population genomic study of the Pyrenean rocket (Sisymbrium austriacum subsp. chrysanthum), we undertook a pooled RAD‐PE (Restriction site Associated DNA Paired‐End sequencing) approach. RAD tags were generated from the PstI‐digested pooled genomic DNA of 12 individuals sampled across the species distribution range and paired‐end sequenced using Illumina technology to produce ~24.5 Mb of sequences, covering ~7% of the specie's genome. Sequences were assembled into ~76 000 contigs with a mean length of 323 bp (N₅₀ = 357 bp, sequencing depth = 24x). In all, >15 000 SNPs were called, of which 47% were annotated in putative genic regions based on homology with the Arabidopsis thaliana genome. Gene ontology (GO) slim categorization demonstrated that the identified SNPs covered extant genic variation well. The validation of 300 SNPs on a larger set of individuals using a KASPar assay underpinned the utility of pooled RAD‐PE as an inexpensive genome‐wide SNP discovery technique (success rate: 87%). In addition to SNPs, we discovered >600 putative SSR markers.     

Quantitative trait loci for IQ and other complex traits: single-nucleotide polymorphism genotyping using pooled DNA and microarrays

Similar to other complex traits, it is likely that many DNA polymorphisms of small effect size [quantitative trait loci (QTLs)] are responsible for the high heritability of intelligence, in addition to many rare monogenic disorders known to contribute to lowered intelligence. We review the current status of approaches to identify QTLs associations for intelligence employing genome-wide strategies using pooled DNA from many individuals and evaluate the innovative approach of microarray analysis to genotype DNA pools for large numbers of single nucleotide polymorphisms.     

Genome-wide quantitative trait locus association scan of general cognitive ability using pooled DNA and 500K single nucleotide polymorphism microarrays

General cognitive ability ( g ), which refers to what cognitive abilities have in common, is an important target for molecular genetic research because multivariate quantitative genetic analyses have shown that the same set of genes affects diverse cognitive abilities as well as learning disabilities. In this first autosomal genome-wide association scan of g , we used a two-stage quantitative trait locus (QTL) design with pooled DNA to screen more than 500 000 single nucleotide polymorphisms (SNPs) on microarrays, selecting from a sample of 7000 7-year-old children. In stage 1, we screened for allele frequency differences between groups pooled for low and high g . In stage 2, 47 SNPs nominated in stage 1 were tested by individually genotyping an independent sample of 3195 individuals, representative of the entire distribution of g scores in the full 7000 7-year-old children. Six SNPs yielded significant associations across the normal distribution of g , although only one SNP remained significant after a false discovery rate of 0.05 was imposed. However, none of these SNPs accounted for more than 0.4% of the variance of g , despite 95% power to detect associations of that size. It is likely that QTL effect sizes, even for highly heritable traits such as cognitive abilities and disabilities, are much smaller than previously assumed. Nonetheless, an aggregated 'SNP set' of the six SNPs correlated 0.11 ( P  < 0.00000003) with g . This shows that future SNP sets that will incorporate many more SNPs could be useful for predicting genetic risk and for investigating functional systems of effects from genes to brain to behavior.     

Preparation of Pooled Arabidopsis YAC DNAs for PCR-Based Mapping

Physical mapping of an arabidopsis DNA sequence of interest can easily be performed by PCR. This is done by using specific primers and pooled DNA templates isolated from publicly available YAC or BAC libraries. We present simple protocols for preparing pooled YAC DNAs and PCR-based detection of sequences in them by which several sequences can be mapped in a short time.     

Conflicts of interest and the evolution of decision sharing

Social animals regularly face consensus decisions whereby they choose, collectively, between mutually exclusive actions. Such decisions often involve conflicts of interest between group members with respect to preferred action. Conflicts could, in principle, be resolved, either by sharing decisions between members (‘shared decisions’) or by one ‘dominant’ member making decisions on behalf of the whole group (‘unshared decisions’). Both, shared and unshared decisions, have been observed. However, it is unclear as to what favours the evolution of either decision type. Here, after a brief literature review, we present a novel method, involving a combination of self-organizing system and game theory modelling, of investigating the evolution of shared and unshared decisions. We apply the method to decisions on movement direction. We find that both, shared and unshared, decisions can evolve without individuals having a global overview of the group''s behaviour or any knowledge about other members'' preferences or intentions. Selection favours unshared over shared decisions when conflicts are high relative to grouping benefits, and vice versa. These results differ from those of group decision models relating to activity timings. We attribute this to fundamental differences between collective decisions about modalities that are disjunct (here, space) or continuous (here, time) with respect to costs/benefits.     

Case study and application of process analytical technology (PAT) towards bioprocessing: Use of tryptophan fluorescence as at‐line tool for making pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining momentum in the biopharmaceutical community due to the potential for continuous real time quality assurance resulting in improved operational control and compliance. Two imperatives for implementing any PAT tool are that “variability is managed by the process” and “product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions.” Recently, we have been examining the feasibility of applying different analytical tools to bioprocessing unit operations. We have previously demonstarted that commercially available online‐high performance liquid chromatography and ultra performance liquid chromatography systems can be used for analysis that can facilitate real‐time decisions for column pooling based on product quality attributes (Rathore et al., 2008 a,b). In this article, we review an at‐line tool that can be used for pooling of process chromatography columns. We have demonstrated that our tryptophan fluorescence method offers a feasible approach and meets the requirements of a PAT application. It is significantly faster than the alternative of fractionation, offline analysis followed by pooling. Although the method as presented here is not an online method, this technique may offer better resolution for certain applications and may be a more optimal approach as it is very conducive to implementation in a manufacturing environment. This technique is also amenable to be used as an online tool via front face fluorescence measurements done concurrently with product concentration determination by UV. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Discovery of rare mutations in extensively pooled DNA samples using multiple target enrichment

Chemical mutagenesis is routinely used to create large numbers of rare mutations in plant and animal populations, which can be subsequently subjected to selection for beneficial traits and phenotypes that enable the characterization of gene functions. Several next‐generation sequencing (NGS)‐based target enrichment methods have been developed for the detection of mutations in target DNA regions. However, most of these methods aim to sequence a large number of target regions from a small number of individuals. Here, we demonstrate an effective and affordable strategy for the discovery of rare mutations in a large sodium azide‐induced mutant rice population (F₂). The integration of multiplex, semi‐nested PCR combined with NGS library construction allowed for the amplification of multiple target DNA fragments for sequencing. The 8 × 8 × 8 tridimensional DNA sample pooling strategy enabled us to obtain DNA sequences of 512 individuals while only sequencing 24 samples. A stepwise filtering procedure was then elaborated to eliminate most of the false positives expected to arise through sequencing error, and the application of a simple Student's t‐test against position‐prone error allowed for the discovery of 16 mutations from 36 enriched targeted DNA fragments of 1024 mutagenized rice plants, all without any false calls.     

Assessment of arbovirus vector infection rates using variable size pooling

Pool testing of vector samples for arboviruses is widely used in surveillance programmes. The proportion of infected mosquitoes (Diptera: Culicidae) is often estimated from the minimum infection rate (MIR), based on the assumption of only one infected mosquito per positive pool. This assumption becomes problematic when pool size is large and/or infection rate is high. By relaxing this constraint, maximum likelihood estimation (MLE) is more useful for a wide range of infection levels that may be encountered in the field. We demonstrate the difference between these two estimation approaches using West Nile virus (WNV) surveillance data from vectors collected by gravid traps in Chicago during 2002. MLE of infection rates of Culex mosquitoes was as high as 60 per 1000 at the peak of transmission in August, whereas MIR was less than 30 per 1000. More importantly, we demonstrate roles of various pooling strategies for better estimation of infection rates based on simulation studies with hypothetical mosquito samples of 18 pools. Variable size pooling (with a serial pool sizes of 5, 10, 20, 30, 40 and 50 individuals) performed consistently better than a constant size pooling of 50 individuals. We conclude that variable pool size coupled with MLE is critical for accurate estimates of mosquito infection rates in WNV epidemic seasons.     

Analysis of genetic relationships of Central American and Mexican pines using RAPD markers that distinguish species

Phylogenetic relationships were inferred for six Central American and Mexican pine species by analysing RAPD marker differences among pooled DNA samples. This population level pooling strategy discounts low-frequency allelic variation within taxa, thus obtaining a 'cumulative genotype' to compare among taxa. We used the morphologically based taxonomy of pines as the basis for inference concerning molecular marker divergence. Only RAPD polymorphisms that were repeatable and intensely amplified were used. The resulting data set was found to have strong hierarchical structure. Phylogenetic analyses were carried out using both neighbour-joining and maximum parsimony. The phylogenetic relationships indicated by analysis of the pine RAPD data provide new insights on pine taxonomy. The phylogenetic analyses of the RAPD marker data placed Pinus tecunumanii and P. patula clearly into two different subgroups, strongly supporting the classification of P. tecunumanii as a distinct species, and raise a new set of issues concerning the distinctions between Pinus tecunumanii and P. caribaea . Phylogenetically informative RAPD markers will be useful tools to address ex-situ conservation issues that involve taxonomic identification and species admixture.     

Massive parallel sequencing in animal genetics: wherefroms and wheretos

Next generation sequencing (NGS) has revolutionized genomics research, making it difficult to overstate its impact on studies of Biology. NGS will immediately allow researchers working in non‐mainstream species to obtain complete genomes together with a comprehensive catalogue of variants. In addition, RNA‐seq will be a decisive way to annotate genes that cannot be predicted purely by computational or comparative approaches. Future applications include whole genome sequence association studies, as opposed to classical SNP‐based association, and implementing this new source of information into breeding programmes. For these purposes, one of the main advantages of sequencing vs. genotyping is the possibility of identifying copy number variants. Currently, experimental design is a topic of utmost interest, and here we discuss some of the options available, including pools and reduced representation libraries. Although bioinformatics is still an important bottleneck, this limitation is only transient and should not deter animal geneticists from embracing these technologies.