From group to individual - Genotyping by pool sequencing eusocial colonies

Background: Eusocial insects play a central role in many ecosystems, and particularly the important pollinator honeybee (Apis mellifera). One approach to facilitate their study in molecular genetics, is to consider whole colonies as single individuals by combining DNA of multiple individuals in a single pool sequencing experiment. Such a technique comes with the drawback of producing data requiring dedicated analytical methods to be fully exploited. Despite this limitation, pool sequencing data has been shown to be informative and cost-effective when working on random mating populations. Here, we present new statistical methods for exploiting pool sequencing data of eusocial colonies in order to reconstruct the genotype of the colony founder, the queen. This leverages the possibility to monitor genetic diversity, perform genomic-based studies or implement selective breeding. Results: Using simulations and honeybee real data, we show that the methods allow for a fast and accurate estimation of the genetic ancestry, with correlations of 0.9 with that obtained from individual genotyping, and for an accurate reconstruction of the queen genotype, with 2% genotyping error. We further validate the inference using experimental data on colonies with both pool sequencing and individual genotyping of drones. Conclusion: In this study we present statistical models to accurately estimate the genetic ancestry and reconstruct the genotype of the queen from pool sequencing data from workers of an eusocial colony. Such information allows to exploit pool sequencing for traditional population genetics, association studies and selective breeding. While validated in Apis mellifera, these methods are applicable to other eusocial hymenoptera species.

Prediction of species composition ratios in pooled specimens of the Anopheles Hyrcanus group using quantitative sequencing

Background Plasmodium vivax is transmitted by members of the Anopheles Hyrcanus Group that includes six species in the Republic of Korea: Anopheles sinensis sensu stricto (s.s.), Anopheles pullus, Anopheles kleini, Anopheles belenrae, Anopheles lesteri, and Anopheles sineroides. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional species identification conducted using individual genotyping (or barcoding) based on species-specific molecular markers requires extensive time commitment and financial resources. Results A population-based quantitative sequencing (QS) protocol developed in this study provided a rapid estimate of species composition ratios among pooled mosquitoes as a cost-effective alternative to individual genotyping. This can be accomplished by using species- or group-specific nucleotide sequences of the mitochondrial cytochrome C oxidase subunit I (COI) and the ribosomal RNA internal transcribed spacer 2 (ITS2) region as species identification alleles in a two-step prediction protocol. Standard genomic DNA fragments of COI and ITS2 genes were amplified from each Anopheles species using group-specific universal primer sets. Following sequencing of the COI or ITS2 amplicons generated from sets of standard DNA mixtures, equations were generated via linear regression to predict species-specific nucleotide sequence frequencies at different positions. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C alleles at the first step, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T alleles. The COI copy number was not significantly different between species, suggesting the reliability of COI-based prediction. In contrast, ITS2 showed a slightly but significantly higher copy number in An. belenrae, requiring an adjustment of its predicted composition ratio. A blind test proved that predicted species composition ratios either from pooled DNA specimens or pooled mosquito specimens were not statistically different from the actual values, demonstrating that the QS-based prediction is accurate and reliable. Conclusions This two-step prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria.

Prediction of species composition ratios in pooled specimens of the Anopheles Hyrcanus Group using quantitative sequencing

Background Vivax malaria is transmitted by members of the Anopheles Hyrcanus Group that includes six species (Anopheles sinensis s.s., An. pullus, An. kleini, An. belenrae, An. lesteri, and An. sineroides) in Republic of Korea. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional species identification conducted using individual genotyping (or barcoding) based on species specific molecular markers requires extensive time commitment and financial resources. Results A population-based quantitative sequencing (QS) protocol developed in this study provided a rapid estimate of species composition ratios among pooled mosquitoes as a cost-effective alternative to individual genotyping. This can be accomplished by using species- or group-specific nucleotide sequences of the mitochondrial cytochrome C oxidase subunit I (COI) and the ribosomal RNA internal transcribed spacer 2 (ITS2) region as species identification markers in a two-step prediction protocol. Standard genomic DNA fragments of COI and ITS2 genes were amplified from each Anopheles species using group-specific universal primer sets. Following sequencing of the COI or ITS2 amplicons generated from sets of standard DNA mixtures, equations were generated via linear regression to predict species-specific nucleotide sequence frequencies at different loci. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C markers at the first step, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T markers. A blind test proved that predicted species composition ratios were not statistically different from the actual values, demonstrating that the QS-based prediction is accurate and reliable. Conclusions This two-step prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria.

ВИЗНАЧЕННЯ ГЕНЕТИЧНОГО ПОЛІМОРФІЗМУ CYP2C9 НА ТЛІ ПРИЗНАЧЕННЯ ВАРФАРИНУ У ПАЦІЄНТІВ НА ІШЕМІЧНУ ХВОРОБУ СЕРЦЯ, УСКЛАДНЕНУ ПОСТІЙНОЮ ФОРМОЮ ФІБРИЛЯЦІЇ ПЕРЕДСЕРДЬ

This article describes the results of a genetic study of the spread of CYP2C9 polymorphic variants in patients in the Podilskyi region of Ukraine. In different ethnic groups, the frequency of cytochrome polymorphic isoenzymes can vary significantly. The presence of genetic mutations of this cytochrome (Arg144Cys, Ile359Leu) is associated with an increased concentration of warfarin in the blood, which can lead to excessive hypocoagulation and risk of bleeding. Particular attention should be paid to patients with a detected mutation in homozygous form, as both alleles of the gene are mutant (their carriers are "slow metabolizers" and therefore require more careful dose selection). Patients are advised to undergo individual genotyping, which will help to predict the risk of each individual patient.

Identification of Susceptibility Genes of Adult Asthma in French Canadian Women

Susceptibility genes of asthma may be more successfully identified by studying subgroups of phenotypically similar asthma patients. This study aims to identify single nucleotide polymorphisms (SNPs) associated with asthma in French Canadian adult women. A pooling-based genome-wide association study was performed in 240 allergic asthmatic and 120 allergic nonasthmatic women. The top associated SNPs were selected for individual genotyping in an extended cohort of 349 asthmatic and 261 nonasthmatic women. The functional impact of asthma-associated SNPs was investigated in a lung expression quantitative trait loci (eQTL) mapping study (n=1035). Twenty-one of the 38 SNPs tested by individual genotyping showedPvalues lower than 0.05 for association with asthma.Cis-eQTL analyses supported the functional contribution of rs17801353 associated withC3AR1(P=7.90E-10). The asthma risk allele for rs17801353 is associated with higher mRNA expression levels ofC3AR1in lung tissue.In silicofunctional characterization of the asthma-associated SNPs also supported the contribution ofC3AR1and additional genes includingSYNE1,LINGO2, andIFNG-AS1. This pooling-based GWAS in French Canadian adult women followed by lung eQTL mapping suggestedC3AR1as a functional locus associated with asthma. Additional susceptibility genes were suggested in this homogenous subgroup of asthma patients.

Genome-wide Association Study Using Pooled DNA to Identify Candidate Markers Mediating Susceptibility to Postoperative Nausea and Vomiting

Background A family history has been established as a risk factor for postoperative nausea and vomiting (PONV), but the identities of susceptibility genes remain unknown. The goal of this study was to identify the genetic loci that may contribute to PONV susceptibility in an adult population. Methods The authors performed a genome-wide association study involving pooling of DNA obtained from 122 patients with severe PONV and 129 matched controls. Each pool was hybridized to a single nucleotide polymorphism (SNP) microarray, and probe intensity was used to predict allele frequency. Differences in allele frequency between SNP in the PONV and control groups were ranked after accounting for the pooling error. The highest ranking SNPs were selected for individual genotyping in the subjects from whom the DNA pool was comprised and in the new verification cohort consisting of 208 subjects (104 PONV patients and 104 controls). Results The authors identified 41 SNP targets showing substantial difference in allelic frequency between pools. These markers were first genotyped in the individual DNA samples from which the pools were comprised. The authors observed evidence for an association between PONV and 19 different loci in the genome. In the separate verification cohort, the association with PONV was observed for four SNPs. This association remained significant after correcting for multiple testing (P < 0.0023) for one SNP (rs2165870), which is located upstream of the promoter for the muscarinic acetylcholine receptor 3 subtype (CHRM3) gene. Conclusions The authors performed the genome-wide association study for PONV using pooled DNA samples. Through individual genotyping, they confirmed association of at least one SNP that is predictive of PONV susceptibility.