Heterozygous Positions Research Articles

SARS-CoV-2 spike protein diversity at an intra-host level, among SARS-CoV-2 infected individuals in South Africa, 2020 to 2022.

Intra-host diversity studies are used to characterise the mutational heterogeneity of SARS-CoV-2 infections in order to understand the impact of virus-host adaptations. This study investigated the frequency and diversity of the spike (S) protein mutations within SARS-CoV-2 infected South African individuals. The study included SARS-CoV-2 respiratory samples, from individuals of all ages, received at the National Health Laboratory Service at Charlotte Maxeke Johannesburg Academic hospital, Gauteng, South Africa, from June 2020 to May 2022. Single nucleotide polymorphism (SNP) assays and whole genome sequencing were performed on a random selection of SARS-CoV-2 positive samples. The allele frequency (AF) was determined using TaqMan Genotyper software for SNP PCR analysis and galaxy.eu for analysis of FASTQ reads from sequencing. The SNP assays identified 5.3% (50/948) of Delta cases with heterogeneity at delY144 (4%; 2/50), E484Q (6%; 3/50), N501Y (2%; 1/50) and P681H (88%; 44/50), however only heterogeneity for E484Q and delY144 were confirmed by sequencing. From sequencing we identified 9% (210/2381) of cases with Beta, Delta, Omicron BA.1, BA.2.15, and BA.4 lineages that had heterogeneity in the S protein. Heterogeneity was primarily identified at positions 19 (1.4%) with T19IR (AF 0.2-0.7), 371 (92.3%) with S371FP (AF 0.1-1.0), and 484 (1.9%) with E484AK (0.2-0.7), E484AQ (AF 0.4-0.5) and E484KQ (AF 0.1-0.4). Mutations at heterozygous amino acid positions 19, 371 and 484 are known antibody escape mutations, however the impact of the combination of multiple substitutions identified at the same position is unknown. Therefore, we hypothesise that intra-host SARS-CoV-2 quasispecies with heterogeneity in the S protein facilitate competitive advantage of variants that can completely/partially evade host's natural and vaccine-induced immune responses.

Abstract 3124: Identification of sub-clonal loss of heterozygosity (LOH) in glioblastomas analyzed with spatial transcriptomics

Abstract We propose a method to leverage spatial transcriptomics and bioinformatic analysis to identify sub-clonal loss of heterozygosity that could otherwise be missed by standard bulk sequencing in glioblastoma tumors. Glioblastomas are highly invasive and aggressive brain tumors with a low five-year survival rate of less than 10%. Current treatment strategies are not sufficient for long term disease outcome, and innovative approaches to analyze glioblastoma are imperative for future improved options. Many factors contribute to difficulty of treatment, though one critical consideration is the diffuse nature and known intratumoral heterogeneity of this cancer. It is important to determine LOH heterogeneity, as this genomic alteration is associated with copy number variation and specific classifications of gliomas. Loss of heterozygosity is an irreversible deletion of DNA that is a common alteration in cancer. Application of spatial transcriptomics can increase our understanding of genomic complexity across a tissue, and unsupervised graph clustering can computationally separate spatial regions with similar gene expression patterns. Our method investigates spatial clusters individually at known heterozygous SNP sites to identify evidence for LOH with Bayesian inference and a hidden Markov model. Our analysis centers on the balance of reference (A) and non-reference (B) spatial transcriptomics read counts at SNPs of interest. B allele frequency is the number of reads aligned to the alternative allele divided by the total number of reads counted at the SNP. At a normal two-copy state at a heterozygous site, B allele frequency is ideally 50 percent. LOH can be identified as this value deviates toward 0 or 100, and copy number alterations impact this frequency. Our method utilizes Bayes factor values, or likelihood ratios between two models, using allele counts to provide support for either LOH or a heterozygous event. For our analysis, spatial transcriptomics BAMs are processed with a graph clustering algorithm and split into cluster-specific BAMs. We calculate allele count coverage for each cluster at pre-selected heterozygous SNP positions identified through germline exome sequencing. Bayes factor values are then calculated at each SNP, resulting in evidence for LOH or a heterozygous event. Chromosomal regions are segmented by a hidden Markov approach. Cumulative metrics for each segment are evaluated to determine a “state” label of heterozygous, LOH, or undefined. We tested our method on three WHO grade 4 IDH wild-type EGFRvIII positive fresh frozen glioblastoma tissue samples. Sub-clonal heterogeneity was identified in two samples and confirmed by bulk exome sequencing, bulk RNA sequencing, and analysis of initial clinical reports. Citation Format: Michelle G. Webb, Frances Chow, Carmel McCullough, Bohan Zhang, Kyle Hurth, John Carpten, Gabriel Zada, David W. Craig. Identification of sub-clonal loss of heterozygosity (LOH) in glioblastomas analyzed with spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3124.

TrioPhaser: using Mendelian inheritance logic to improve genomic phasing of trios

BackgroundWhen analyzing DNA sequence data of an individual, knowing which nucleotide was inherited from each parent can be beneficial when trying to identify certain types of DNA variants. Mendelian inheritance logic can be used to accurately phase (haplotype) the majority (67–83%) of an individual's heterozygous nucleotide positions when genotypes are available for both parents (trio). However, when all members of a trio are heterozygous at a position, Mendelian inheritance logic cannot be used to phase. For such positions, a computational phasing algorithm can be used. Existing phasing algorithms use a haplotype reference panel, sequencing reads, and/or parental genotypes to phase an individual; however, they are limited in that they can only phase certain types of variants, require a specific genotype build, require large amounts of storage capacity, and/or require long run times. We created trioPhaser to address these challenges.ResultstrioPhaser uses gVCF files from an individual and their parents as initial input, and then outputs a phased VCF file. Input trio data are first phased using Mendelian inheritance logic. Then, the positions that cannot be phased using inheritance information alone are phased by the SHAPEIT4 phasing algorithm. Using whole-genome sequencing data of 52 trios, we show that trioPhaser, on average, increases the total number of phased positions by 21.0% and 10.5%, respectively, when compared to the number of positions that SHAPEIT4 or Mendelian inheritance logic can phase when either is used alone. In addition, we show that the accuracy of the phased calls output by trioPhaser are similar to linked-read and read-backed phasing.ConclusiontrioPhaser is a containerized software tool that uses both Mendelian inheritance logic and SHAPEIT4 to phase trios when gVCF files are available. By implementing both phasing methods, more variant positions are phased compared to what either method is able to phase alone.

HaplotypeTools: a toolkit for accurately identifying recombination and recombinant genotypes

BackgroundIdentifying haplotypes is central to sequence analysis in diploid or polyploid genomes. Despite this, there remains a lack of research and tools designed for physical phasing and its downstream analysis.ResultsHaplotypeTools is a new toolset to phase variant sites using VCF and BAM files and to analyse phased VCFs. Phasing is achieved via the identification of reads overlapping ≥ 2 heterozygous positions and then extended by additional reads, a process that can be parallelized across a computer cluster. HaplotypeTools includes various utility scripts for downstream analysis including crossover detection and phylogenetic placement of haplotypes to other lineages or species. HaplotypeTools was assessed for accuracy against WhatsHap using simulated short and long reads, demonstrating higher accuracy, albeit with reduced haplotype length. HaplotypeTools was also tested on real Illumina data to determine the ancestry of hybrid fungal isolate Batrachochytrium dendrobatidis (Bd) SA-EC3, finding 80% of haplotypes across the genome phylogenetically cluster with parental lineages BdGPL (39%) and BdCAPE (41%), indicating those are the parental lineages. Finally, ~ 99% of phasing was conserved between overlapping phase groups between SA-EC3 and either parental lineage, indicating mitotic gene conversion/parasexuality as the mechanism of recombination for this hybrid isolate. HaplotypeTools is open source and freely available from https://github.com/rhysf/HaplotypeTools under the MIT License.ConclusionsHaplotypeTools is a powerful resource for analyzing hybrid or recombinant diploid or polyploid genomes and identifying parental ancestry for sub-genomic regions.

MR1 encompasses at least six allele groups with coding region alterations.

Unlike classical HLA class I genes, MR1 is assumed to have limited polymorphic positions. We developed a MR1 specific PCR assay and sequenced 56 DNA samples from cells with a diverse set of HLA genotypes. In this relatively small panel we found six allele groups encoding for different MR1 proteins. The two most frequent allele groups found in this panel had a frequency of 71% (MR1*01) and 25% (MR1*02), respectively. Moreover, the panel contained many intronic SNPs and silent variants, with individual samples containing up to 15 heterozygous positions. The data presented here is consistent with marked variation in MR1.

Phasing and imputation of single nucleotide polymorphism data of missing parents of biparental plant populations.

This paper presents an extension to a heuristic method for phasing and imputation of genotypes of descendants in biparental populations so that it can phase and impute genotypes of parents that are ungenotyped or partially genotyped. The imputed genotypes of the parent are used to impute low‐density (Ld) genotyped descendants to high density (Hd). The extension was implemented as part of the AlphaPlantImpute software and works in three steps. First, it identifies whether a parent has no or Ld genotypes and identifies its relatives that have Hd genotypes. Second, using the Hd genotypes of relatives, it determines whether the parent is homozygous or heterozygous for a given locus. Third, it phases heterozygous positions of the parent by matching haplotypes to its relatives. We measured the accuracy (correlation between true and imputed genotypes) of imputing parent genotypes in simulated biparental populations from different scenarios. We tested the imputation accuracy of the missing parent's descendants using the true genotype of the parent and compared this with using the imputed genotypes of the parent. Across all scenarios, the imputation accuracy of a parent was >0.98 and did not drop below ∼0.96. The imputation accuracy of a parent was always higher when it was inbred than outbred. Including ancestors of the parent at Hd, increasing the number of crosses and the number of Hd descendants increased the imputation accuracy. The high imputation accuracy achieved for the parent translated to little or no impact on the imputation accuracy of its descendants.

Factors that influence bidirectional long-tract homozygosis due to double-strand break repair in Candida albicans.

Genomic rearrangements have been associated with the acquisition of adaptive phenotypes, allowing organisms to efficiently generate new favorable genetic combinations. The diploid genome of Candida albicans is highly plastic, displaying numerous genomic rearrangements that are often the by-product of the repair of DNA breaks. For example, DNA double-strand breaks (DSB) repair using homologous-recombination pathways are a major source of loss-of-heterozygosity (LOH), observed ubiquitously in both clinical and laboratory strains of C. albicans. Mechanisms such as break-induced replication (BIR) or mitotic crossover (MCO) can result in long tracts of LOH, spanning hundreds of kilobases until the telomere. Analysis of I-SceI-induced BIR/MCO tracts in C. albicans revealed that the homozygosis tracts can ascend several kilobases toward the centromere, displaying homozygosis from the break site toward the centromere. We sought to investigate the molecular mechanisms that could contribute to this phenotype by characterizing a series of C. albicans DNA repair mutants, including pol32-/-, msh2-/-, mph1-/-, and mus81-/-. The impact of deleting these genes on genome stability revealed functional differences between Saccharomyces cerevisiae (a model DNA repair organism) and C. albicans. In addition, we demonstrated that ascending LOH tracts toward the centromere are associated with intrinsic features of BIR and potentially involve the mismatch repair pathway which acts upon natural heterozygous positions. Overall, this mechanistic approach to study LOH deepens our limited characterization of DNA repair pathways in C. albicans and brings forth the notion that centromere proximal alleles from DNA break sites are not guarded from undergoing LOH.

Extending long-range phasing and haplotype library imputation algorithms to large and heterogeneous datasets

BackgroundWe describe the latest improvements to the long-range phasing (LRP) and haplotype library imputation (HLI) algorithms for successful phasing of both datasets with one million individuals and datasets genotyped using different sets of single nucleotide polymorphisms (SNPs). Previous publicly available implementations of the LRP algorithm implemented in AlphaPhase could not phase large datasets due to the computational cost of defining surrogate parents by exhaustive all-against-all searches. Furthermore, the AlphaPhase implementations of LRP and HLI were not designed to deal with large amounts of missing data that are inherent when using multiple SNP arrays.MethodsWe developed methods that avoid the need for all-against-all searches by performing LRP on subsets of individuals and then concatenating the results. We also extended LRP and HLI algorithms to enable the use of different sets of markers, including missing values, when determining surrogate parents and identifying haplotypes. We implemented and tested these extensions in an updated version of AlphaPhase, and compared its performance to the software package Eagle2.ResultsA simulated dataset with one million individuals genotyped with the same 6711 SNPs for a single chromosome took less than a day to phase, compared to more than seven days for Eagle2. The percentage of correctly phased alleles at heterozygous loci was 90.2 and 99.9% for AlphaPhase and Eagle2, respectively. A larger dataset with one million individuals genotyped with 49,579 SNPs for a single chromosome took AlphaPhase 23 days to phase, with 89.9% of alleles at heterozygous loci phased correctly. The phasing accuracy was generally lower for datasets with different sets of markers than with one set of markers. For a simulated dataset with three sets of markers, 1.5% of alleles at heterozygous positions were phased incorrectly, compared to 0.4% with one set of markers.ConclusionsThe improved LRP and HLI algorithms enable AlphaPhase to quickly and accurately phase very large and heterogeneous datasets. AlphaPhase is an order of magnitude faster than the other tested packages, although Eagle2 showed a higher level of phasing accuracy. The speed gain will make phasing achievable for very large genomic datasets in livestock, enabling more powerful breeding and genetics research and application.

Lost in translation: Population genomics and long-read sequencing reveals relaxation of concerted evolution of the ribosomal DNA cistron

Concerted evolution of the ribosomal DNA array has been studied in numerous eukaryotic taxa, yet is still poorly understood. rDNA genes are repeated dozens to hundreds of times in the eukaryotic genome (Eickbush and Eickbush, 2007) and it is believed that these arrays are homogenized through concerted evolution (Zimmer et al., 1980; Dover, 1993) preventing the accumulation of intragenomic, and intraspecific, variation. However, numerous studies have reported rampant intragenomic and intraspecific variation in the rDNA array (Ganley and Kobayashi, 2011; Naidoo et al., 2013; Hughes and Petersen, 2001; Lindner and Banik, 2011; Li et al., 2013; Lindner et al., 2013; Hughes et al., 2018), contradicting our current understanding of concerted evolution. The internal transcribed spacers (ITS) of the rDNA cistron are the most commonly used DNA barcoding region in Fungi (Schoch et al., 2012), and rely on concerted evolution to homogenize the rDNA array leading to a “barcode gap” (Puillandre et al., 2012). Here we show that in Boletus edulis Bull., ITS intragenomic variation persists at low allele frequencies throughout the rDNA array, this variation does not correlate with genomic relatedness between populations, and rDNA genes may not evolve in a strictly concerted fashion despite the presence of unequal recombination and gene conversion. Under normal assumptions, heterozygous positions found in ITS sequences represent hybridization between populations, yet through allelic mapping of the rDNA array we found numerous heterozygous alleles to be stochastically introgressed throughout, presenting a dishonest signal of gene flow. Moreover, despite the signal of gene flow in ITS, our organisms were highly inbred, indicating a disconnect between true gene flow and barcoding signals. In addition, we show that while the mechanisms of concerted evolution are ongoing in pseudo-heterozygous individuals, they are not fully homogenizing the ITS array. Concerted evolution of the rDNA array may insufficiently homogenize the ITS gene, allowing for misleading signals of gene flow to persist, vastly complicating the use of the ITS locus for DNA barcoding in Fungi.

Resolving MiSeq-Generated Ambiguities in HLA-DPB1 Typing by Using the Oxford Nanopore Technology

The technical limitations of current next-generation sequencing technologies, combined with an ever-increasing number of human leukocyte antigen (HLA) alleles, form the basis for the additional ambiguities encountered at an increasing rate in clinical practice. HLA-DPB1 characterization, particularly, generates a significant percentage of ambiguities (25.5%), posing a challenge for accurate and unambiguous HLA-DPB1 genotyping. Phasing of exonic heterozygous positions between exon 2 and all other downstream exons has been the major cause of ambiguities. In this study, the Oxford Nanopore MinION, a third-generation sequencing technology, was used to resolve the phasing. The accurate MiSeq sequencing data, combined with the long reads obtained from the MinION platform, allow for the resolution of the tested ambiguities.

Resequencing of four novel alleles with nanopore technology.

Long nanopore reads allow phasing of heterozygous positions but show limitations in sequencing of homopolymers.

P030How do different testing platforms influence novel allele characterization? A case study of a novel DPB1 allele

A renal transplant patient sample was submitted in order to characterize an unresolved HLA-DPB1 allele. Sanger sequence-based typing (SBT) resulted in DPB1*04:02:01 and a number of equally possible candidates for the second allele, each with a single nucleotide mismatch. Removing all exons except exon 2 from analysis resulted in a perfect match with homozygous DPB1*04:02:01G. However, several heterozygous positions leading to non-synonymous mutations were present in the excluded exons, contradicting this apparent homozygosity. One next-generation sequencing (NGS) platform then typed the sample as DPB1*04:02:01 for the first allele and various ambiguous alleles for the second. However, this was due to low coverage in a 19-nucleotide region of exon 2 rather than a mismatch. The low coverage pattern was replicated on a repeat run. DPB1*414:01 with one mismatch in exon 2 was called as the second allele when the minimum read depth was lowered. Interestingly, none of the possible second alleles were part of DPB1*04:02:01G. Upon testing on a second NGS platform, DPB1*04:02:01, DPB1*463:01:01 was obtained with novel mutations in exon 4 and introns. Comparing this result to the Sanger SBT data revealed the same heterozygous mismatch in exon 4. A third NGS platform verified this typing and associated mismatches. Therefore, if potential intronic mutations are ignored, the typing may be reported in two ways: as DPB1*04:02:01, DPB1*463:01 with a mutation in exon 4, or as DPB1*04:02:01, DPB1*414:01 with a mutation in exon 2. The first combination may lead to the assumption of homozygosity for treatment purposes, since DPB1*463:01 is part of DPB1*04:02:01G. This is currently the practice in the HLA field when exons 3 and 4 are not included in analysis. Ignoring heterozygous positions in exons that make up regions outside the antigen binding groove could lead to development of antibodies. This may be an important consideration in the renal transplant setting, depending on patient-donor matching and determination of immunogenicity.

Inferring Variation in Copy Number Using High Throughput Sequencing Data in R.

Inference of copy number variation presents a technical challenge because variant callers typically require the copy number of a genome or genomic region to be known a priori. Here we present a method to infer copy number that uses variant call format (VCF) data as input and is implemented in the R package vcfR. This method is based on the relative frequency of each allele (in both genic and non-genic regions) sequenced at heterozygous positions throughout a genome. These heterozygous positions are summarized by using arbitrarily sized windows of heterozygous positions, binning the allele frequencies, and selecting the bin with the greatest abundance of positions. This provides a non-parametric summary of the frequency that alleles were sequenced at. The method is applicable to organisms that have reference genomes that consist of full chromosomes or sub-chromosomal contigs. In contrast to other software designed to detect copy number variation, our method does not rely on an assumption of base ploidy, but instead infers it. We validated these approaches with the model system of Saccharomyces cerevisiae and applied it to the oomycete Phytophthora infestans, both known to vary in copy number. This functionality has been incorporated into the current release of the R package vcfR to provide modular and flexible methods to investigate copy number variation in genomic projects.

Transcriptome of the bivalve Limecola balthica L. from Western Pacific: A new resource for studies of European populations

The Baltic clam Limecola balthica L. (Tellinidae) is broadly used in ecophysiological, toxicological, evolutionary and environmental monitoring studies. However, it is poorly studied in respect of genome and gene functions. We obtained a transcriptome of Limecola b. balthica from Kamchatka (Western Pacific) generated with the use of Illumina high-throughput sequencing. We annotated 11,374 proteins, including 53 from the oxidative phosphorylation pathway and a number of pollution-stress biomarkers, recovered 254,540 single nucleotide variants within two annotated transcriptomes including 25,330 scorable in the previously published European data. Our results confirmed the available allozyme data indicating that nuclear genomes of the clams from the Baltic Sea were intermediate in their genetic composition between the Pacific (L. b. balthica) and the Atlantic (L. b. rubra) subspecies. At the same time, the mitochondrial genomes of Limecola from Kamchatka were nearly identical to the single published genome from the Baltic. The genomic diversity in Limecola was found to be high and comparable with that of other marine mollusks (0.0138 and 0.0142 heterozygous positions in the two studied transcriptomes). The data obtained in our study are a valuable resource for further development of genomic markers for evolutionary genetic and ecophysiological studies of L. balthica complex.

P100 A novel sequence enrichment protocol allowing HLA-typing on 5-cells

Aim Limited genomic DNA template in a sample can make accurate HLA-typing difficult. We therefore aimed to develop a method that allows HLA-typing using next generation sequencing (NGS) up to p-group resolution with starting DNA template of only 5 cells. Methods Aliquots of 5 cells were sorted from aneuploid and euploid cell lines (Coriell Institute of Medical Research, USA). Whole genome amplification was performed using the DOPlify™ TSE (Targeted Sequence Enrichment) protocol (RHS Ltd, Australia) with the inclusion of SBTessenz® primers (GenDx, the Netherlands) to increase amplification of exon 2 to 3 of the HLA-A locus. Enrichment was determined using semi-quantitative PCR for HLA-A. The HLA-A amplicon was subsequently pooled with the original DOPlify™ TSE WGA DNA in a 1:10 and 1:20 ratio before library preparation. After indexing and multiplexing of 40 samples for sequencing on a MiSeq platform according to standard 2 × 75 bp protocol (Illumina), the NGS data was analyzed and typed by the HLA-typing software NGSengine®. Results Targeted sequence enrichment increased the breadth and depth of coverage of exon 2, intron 2 and exon 3 for the HLA-A locus, with concurrent detection of aneuploidy. When the HLA-A amplicon was pooled with the DOPlify™ TSE WGA DNA, coverage of 100% was achieved and the average read depth following NGS was greater than 200. Amplification of both alleles was confirmed with a ratio of approximately 30:70% which enabled clear detection and analysis of the heterozygous positions. The quality of the sequence data allowed resolution of all ambiguities that are present in exon 2, intron 2 and exon 3; including the sequence up front of exon 2 required to exclude several null alleles. Conclusions We developed a novel sequence enrichment protocol to amplify and type HLA-A from a 5 cell sample. The data quality meets general laboratory standards for HLA-typing and allows typing up to p-group resolution. This protocol has application in preimplantation genetic diagnosis and low template input HLA typing such as buccal swabs.

Regulatory Variants Other Than VKORC1 rs9923231 May Explain The Effect on Warfarin Dose

Warfarin is the most commonly used oral anticoagulant for thrombotic disorders and atrial fibrillation. The dose is influenced by CYP2C9 and VKORC1. CYP2C9 variants change the protein sequence, but VKORC1 variants are non-coding and thought to act through regulation of gene expression. Which VKORC1 variant that mediates the effect has not been resolved due to high linkage disequilibrium (LD). We used ENCODE data on regulatory transcription factor binding sites, and heterozygous positions in a liver cell line to determine variants that regulate VKORC1. We sequenced the liver cell line HepG2 to locate all heterozygous positions in LD r2>0.8 with the GWAS top hits, and used ENCODE data to find variants that bind transcription factors in an allele-specific way. The functional effect of these variants was evaluated using luciferase assays and over-expression of candidate transcription factors. VKORC1 rs9923231 was not located in an ENCODE regulatory element, and luciferase assays did not show any difference in transcriptional activity between the two alleles. One allele-specific SNP, rs56314408, was on the same haplotype as rs9923231, and rs2032915 was located in the same liver enhancer 24 bp away. The C alleles of rs56314408 and rs2032915 showed higher transcriptional activity, which was further increased after over-expression of the transcription factors YY1 and USF1. We are currently evaluating the potential regulatory effect of rs56314408 and rs2032915 using CRISPR/Cas9. The conventionally analyzed VKORC1 -1639 G>A (rs9923231) is not located in a regulatory element in the liver, and has no evidence of being functional. The LD between rs9923231 and both functional candidates rs56314408 and rs2032915 is known to be high in Europeans. We propose that rs9923231 predicts warfarin dose adequately only in populations where it is in high LD with functional variants such as the candidates rs56314408 and rs2032915.

Short communication: Identification of variation in the ovine prolactin gene of Chios sheep with a cost-effective sequence-based typing assay

The present study identified single nucleotide polymorphisms (SNP) in the coding and untranslated regions of the ovine prolactin gene of Chios sheep. By developing a cost-effective direct sequence-based typing assay, around 600 bp of reliable sequencing data and clear identification of heterozygous positions was achieved. Five SNP were found, located in exons 2 (KC764410:g.567G>A, g.625C>T, g.683C>A) and 3 (KC764410:g.2015C>A, g.2101G>A), whereas the remaining exons were monomorphic. The identified SNP were synonymous, with the exception of the g.567G>A SNP, which results in an Arg to His amino acid change. As the sequencing cost of the sequence-based typing assay was 20 orders of magnitude lower compared with a standard Sanger method, the assay was also used as a genotyping tool. The identified polymorphism was genotyped for 247 ewes and was subsequently used in mixed model association analyses of milk yield, milk fat content, and litter size at birth. The association analysis revealed a significant dominance effect of 0.17 ± 0.07 of the g.2015C>A SNP on milk fat percentage, whereas a dominance effect of -21.33 ± 10.51 of the same SNP on total lactation milk yield was also estimated. The g.2015C>A SNP explained 2.47 and 3.68% of the total phenotypic variance of milk yield and milk fat percentage, respectively, whereas the corresponding values for the animal variance were 7.14 and 11.75%. A suggestive association of the nonsynonymous g.567G>A SNP with litter size at birth was also detected.

Six new Phytophthora species from ITS Clade 7a including two sexually functional heterothallic hybrid species detected in natural ecosystems in Taiwan.

During a survey of Phytophthora diversity in natural ecosystems in Taiwan six new species were detected. Multigene phylogeny based on the nuclear ITS, ß-tubulin and HSP90 and the mitochondrial cox1 and NADH1 gene sequences demonstrated that they belong to ITS Clade 7a with P. europaea, P. uniformis, P. rubi and P. cambivora being their closest relatives. All six new species differed from each other and from related species by a unique combination of morphological characters, the breeding system, cardinal temperatures and growth rates. Four homothallic species, P. attenuata, P. flexuosa, P. formosa and P. intricata, were isolated from rhizosphere soil of healthy forests of Fagus hayatae, Quercus glandulifera, Q. tarokoensis, Castanopsis carlesii, Chamaecyparis formosensis and Araucaria cunninghamii. Two heterothallic species, P. xheterohybrida and P. xincrassata, were exclusively detected in three forest streams. All P. xincrassata isolates belonged to the A2 mating type while isolates of P. xheterohybrida represented both mating types with oospore abortion rates according to Mendelian ratios (4-33 %). Multiple heterozygous positions in their ITS, ß-tubulin and HSP90 gene sequences indicate that P. xheterohybrida, P. xincrassata and P. cambivora are interspecific hybrids. Consequently, P. cambivora is re-described as P. xcambivora without nomenclatural act. Pathogenicity trials on seedlings of Castanea sativa, Fagus sylvatica and Q. suber indicate that all six new species might pose a potential threat to European forests.

CloneCNA: detecting subclonal somatic copy number alterations in heterogeneous tumor samples from whole-exome sequencing data.

BackgroundCopy number alteration is a main genetic structural variation that plays an important role in tumor initialization and progression. Accurate detection of copy number alterations is necessary for discovering cancer-causing genes. Whole-exome sequencing has become a widely used technology in the last decade for detecting various types of genomic aberrations in cancer genomes. However, there are several major issues encountered in these detection problems, including normal cell contamination, tumor aneuploidy, and intra-tumor heterogeneity. Especially, deciphering the intra-tumor heterogeneity is imperative for identifying clonal and subclonal copy number alterations.ResultsWe introduce CloneCNA, a novel bioinformatics tool for efficiently addressing these issues and automatically detecting clonal and subclonal somatic copy number alterations from heterogeneous tumor samples. CloneCNA fully explores the log ratio of read counts between paired tumor-normal samples and tumor B allele frequency of germline heterozygous SNP positions, further employs efficient statistical models to quantitatively represent copy number status of tumor sample containing multiple clones. We examine CloneCNA on simulated heterogeneous and real tumor samples, and the results demonstrate that CloneCNA has higher power to detect copy number alterations than existing methods.ConclusionsCloneCNA, a novel algorithm is developed to efficiently and accurately identify somatic copy number alterations from heterogeneous tumor samples. We demonstrate the statistical framework of CloneCNA represents a remarkable advance for tumor whole-exome sequencing data. We expect that CloneCNA will promote cancer-focused studies for investigating the role of clonal evolution and elucidating critical events benefiting tumor tumourigenesis and progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1174-7) contains supplementary material, which is available to authorized users.

Genomic diversity and geographical structure of the Pyrenean desman

The Pyrenean desman (Galemys pyrenaicus) is a small semi-aquatic mammal endemic to the Iberian Peninsula. The species has recently experienced a strong decline and some of its populations are severely threatened with extinction. To help in the preservation of this species, it is critical to understand its genetic structure and main evolutionary units, as these may have specific local adaptations and could be of great conservation value. Sequencing reduced representation libraries (ddRAD) from 26 specimens selected from across its entire range, we obtained around 45,000 loci per specimen and 1185 single nucleotide polymorphisms. Heterozygosity varied substantially among individuals from different areas. Interestingly, specimens from the southeastern Pyrenees had some of the lowest proportions of heterozygous positions inferred from genome-wide data in mammals so far. In addition, we estimated a tree reflecting genomic divergence, performed a principal component analysis, and carried out a Bayesian analysis of the population structure. Combined evidence supported the existence of five distinct genomic clusters largely coincident with the main mountain ranges where the species occurs, with few specimens presenting relevant admixture levels. There was good correspondence between these populations and the mitochondrial lineages detected in a previous study, yet substantial differences in some areas demonstrate the importance of performing genomic analysis to reveal the whole population history. Although the analysis of further specimens is necessary to better characterize the distribution of the different evolutionary units, the distinctive geographical structure of this species revealed by the genomic data should be considered in future conservation plans.