Sequence-based Genotyping Research Articles

Sequence-based autosomal STR characterization in four US populations using PowerSeq™ Auto/Y system

The forensic science community is poised to utilize modern advances in massively parallel sequencing (MPS) technologies to better characterize biological samples with higher resolution. A critical component towards the advancement of forensic DNA analysis with these technologies is a comprehensive understanding of the diversity and population distribution of sequence-based short tandem repeat (STR) alleles. Here we analyzed 786 samples of individuals from different population groups, including four of the mostly commonly encountered in forensic casework in the USA. DNA samples were amplified with the PowerSeq™ Auto/Y System Prototype Kit (Promega Corp.), and sequencing was performed on an Illumina® MiSeq instrument. Sequence data were analyzed using a bioinformatics processing tool, Altius. For additional data analysis and profile comparison, capillary electrophoresis (CE) size-based STR genotypes were generated for a subset of individuals, and where possible, also with a second commercially available MPS STR assay. Autosomal STR loci were analyzed and frequencies were calculated based on sequence composition. Also, population genetics studies were performed, with Hardy–Weinberg equilibrium, polymorphic information content (PIC), and observed and expected heterozygosity all assessed. Overall, sequence-based allelic variants of the repeat region were observed in 20 out of 22 different STR loci commonly used in forensic DNA genotyping, with the highest number of sequence variation observed at locus D12S391. The highest increase in allelic diversity and in PIC through sequence-based genotyping was observed at loci D3S1358 and D8S1179. Such detailed sequence analysis, as the one performed in the present study, is important to help understand the diversity of sequence-based STR alleles across different populations and to demonstrate how such allelic variation can improve statistics used for forensic casework.

Fast sequence-based microsatellite genotyping development workflow.

Application of high-throughput sequencing technologies to microsatellite genotyping (SSRseq) has been shown to remove many of the limitations of electrophoresis-based methods and to refine inference of population genetic diversity and structure. We present here a streamlined SSRseq development workflow that includes microsatellite development, multiplexed marker amplification and sequencing, and automated bioinformatics data analysis. We illustrate its application to five groups of species across phyla (fungi, plant, insect and fish) with different levels of genomic resource availability. We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped. In contrast, de novo ad hoc primer designs gives highly multiplexed microsatellite assays that can be sequenced to produce high quality genotypes for 20–40 loci. We highlight critical upfront development factors to consider for effective SSRseq setup in a wide range of situations. Sequence analysis accounting for all linked polymorphisms along the sequence quickly generates a powerful multi-allelic haplotype-based genotypic dataset, calling to new theoretical and analytical frameworks to extract more information from multi-nucleotide polymorphism marker systems.

Distribution of rotavirus genotypes in Japan from 2015 to 2018: Diversity in genotypes before and after introduction of rotavirus vaccines

BackgroundDiversity in group A rotavirus (RVA) strains after introduction of RV-vaccines remains an emerging concern worldwide. In this study, we investigated the prevalence and distribution of RVA genotypes in Japanese children with acute gastroenteritis (AGE) from 2015 to 2018. In addition, a comparison of the genotypes in pre-vaccination (2006–2012) and post-vaccination (2012–2018) periods was conducted to understand the impact of these vaccines on genotype distribution. MethodsFecal samples were collected regularly from outpatient clinics in six localities: Hokkaido, Tokyo, Shizuoka, Osaka, Kyoto, and Saga. RVA were screened and genotyped by RT-PCR and sequence-based genotyping. ResultsDuring the period 2015–2018, RVA was detected in 307 (19.7%) samples out of 1557 specimens: 29.9% (95% CI: 25.8% to 34.3%), 17.9% (95% CI: 14.7% to 21.5%), and 13% (95% CI: 10.3% to 16.0%) were detected RVA-positive in 2015–2016, 2016–2017 and 2017–2018, respectively. The average detection of RVA in pre-vaccination (2006–2012) and post-vaccination (2012–2018) era remained almost similar (18%-20%). The G2P[4]I2 (52.1%, 95% CI: 43.5%-60.6%) remained the most common genotype in 2015–2016, whereas G8P[8]I2 (55.9%, 95% CI: 45.2%-66.2%) dominated in 2016–2017. In 2017–2018, G9P[8]I2 (42.0%, 95% CI: 30.5%-53.9%) prevailed, followed by G9P[8]I1 (23.0%, 95% CI: 14.0%-34.2%). The detection rate of some common genotypes of pre-vaccination era like G1P[8] and G3P[8] has been reduced after introduction of RV-vaccine, whereas genotypes that were sporadic before the introduction of vaccines like G2P[4], G2P[8], G9P[8] and G8P[8] were emerged/reemerged in post-vaccination period. ConclusionsOur study presented the diversity in circulating RVA genotypes in Japan before and after introduction of RV-vaccines. Sudden emergence of DS-1-like (I2) unusual strains in post-vaccination era remains alarming. Continuous monitoring of RVA genotypes is therefore indispensable to refine future vaccine strategy.

SUN-296 CYP3A5 AND ABCB1 GENETIC POLYMORPHISMS IN RENAL TRANSPLANT RECIPIENT PATIENTS AND ITS RELATION WITH TACROLIMUS DOSING AND COMPLICATIONS

Tacrolimus is the mainstay of immunosuppression in renal transplantation (RT). However, it has highly variable bioavailability and a narrow therapeutic range. Its metabolism depends upon CYP3A4 and ABCB1gene polymorphism. There is limited data regarding the influence of this polymorphisms on TAC drug level and complications from India. This is a prospective study done over a period of 2 years (2016-2018) from NIMS Hyderabad. A total of 152 patients were recruited. Frequency of CYP3AB(A6986>G) & ABCB1 (C1236>T, G2677>T and C3435>T) polymorphisms were detected by PCR followed by Sanger’s sequence-based genotype & RFLP. Accordingly patients were divided into wild variant(CYP3A5-AA) where there is full expression of the enzyme, heterozygous( CYP3A5-AG), homozygous group who were non expressors (CYP3A5-GG). Day 3, 30,60,90 tacrolimus concentrations and tacrolimus concentration/drug (C/D) ratio were measured. Tacrolimus related toxicity, infections and rejections and the serum creatinine at the time of discharge were compared among the groups Out of 152 renal transplant recipients, 120(78%) were males. 37 patients received deceased donor grafts. Immediate graft function was seen in 117(76%). Tacrolimus C/D ratio was less in wild (AA) variety compared to both AG ( 0.006) and GG ( P<o.ooo1). Tacrolimus toxicity was noted in 28(18%) patients, of which ATN 24(85%), TMA 3(11%), Pancreatitis in 1(3.5%). Most common infectious complication was UTI seen in 22(14%) patients (AA-3(10%), AG-9(14%), GG-10 (16.7%) followed by CMV disease. BPAR were seen in 5 patients. ABCB1 polymorphisms and showed no correlation with tacrolimus C/D ratio. Mean serum creatinine at the time of discharge was 1±0.2 in AA, 1.3±0.8 in AG (AA vs AG P < 0.002), 1.27±0.47 in GG(AA vs GG P <0.004). Our study shows that genetic polymorphism effects the tacrolimus dose requirement. Enzyme expressor (AA) is associated with low C/D ratio and higher risk of acute rejection. Heterozygous (AG) and non expressors (GG) are at higher risk of developing tacrolimus related nephrotoxicity and infections. ABCB1 polymorphisms has no significant impact on tacrolimus C/D ratio

Resolving fine-scale population structure and fishery exploitation using sequenced microsatellites in a northern fish.

The resiliency of populations and species to environmental change is dependent on the maintenance of genetic diversity, and as such, quantifying diversity is central to combating ongoing widespread reductions in biodiversity. With the advent of next‐generation sequencing, several methods now exist for resolving fine‐scale population structure, but the comparative performance of these methods for genetic assignment has rarely been tested. Here, we evaluate the performance of sequenced microsatellites and a single nucleotide polymorphism (SNP) array to resolve fine‐scale population structure in a critically important salmonid in north eastern Canada, Arctic Charr (Salvelinus alpinus). We also assess the utility of sequenced microsatellites for fisheries applications by quantifying the spatial scales of movement and exploitation through genetic assignment of fishery samples to rivers of origin and comparing these results with a 29‐year tagging dataset. Self‐assignment and simulation‐based analyses of 111 genome‐wide microsatellite loci and 500 informative SNPs from 28 populations of Arctic Charr in north‐eastern Canada identified largely river‐specific genetic structure. Despite large differences (~4X) in the number of loci surveyed between panels, mean self‐assignment accuracy was similar with the microsatellite loci and the SNP panel (>90%). Subsequent analysis of 996 fishery‐collected samples using the microsatellite panel revealed that larger rivers contribute greater numbers of individuals to the fishery and that coastal fisheries largely exploit individuals originating from nearby rivers, corroborating results from traditional tagging experiments. Our results demonstrate the efficacy of sequence‐based microsatellite genotyping to advance understanding of fine‐scale population structure and harvest composition in northern and understudied species.

Homotypic and Heterotypic Protection and Risk of Reinfection Following Natural Norovirus Infection in a Highly Endemic Setting.

BackgroundNorovirus is a leading cause of acute gastroenteritis worldwide, yet there is limited information on homotypic or heterotypic protection following natural infection to guide vaccine development.MethodsA total of 6020 stools collected from 299 Peruvian children between 2010 and 2014 were tested by norovirus real-time reverse-transcription polymerase chain reaction followed by sequence-based genotyping. Cox proportional hazards models were used to derive adjusted hazard ratios (HRs) of infection among children with vs without prior exposure.ResultsNorovirus was detected in 1288 (21.3%) samples. GII.4 (26%), GII.6 (19%), and GI.3 (9%) viruses accounted for 54% of infections. Homotypic protection for GI.3 (HR, 0.35; P = .015), GI.7 (HR, 0.19; P = .022), GII.4 (HR, 0.39; P < .001), and GII.6 (HR, 0.52; P = .006) infections was observed. Hazard analysis showed that children with prior GII.4 infection exhibited heterotypic protection with a 48% reduction of subsequent GI.3 infection (HR, 0.52; P = .005). Prior exposure to GI.3, GII.2, and GII.17 infections enhanced susceptibility to subsequent infections with several other norovirus genotypes.ConclusionsChildren up to 2 years of age infected with GII.4 noroviruses demonstrated both homotypic and heterotypic protection to reinfection with other genotypes. These data support the need for ongoing vaccine development efforts with GII.4 as the main component and caution the inclusion of genotypes that may enhance susceptibility to infections.

New Subgenotyping and Consensus Real-Time Reverse Transcription-PCR Assays for Hepatitis A Outbreak Surveillance

Laboratory surveillance plays an important role in the detection and control of hepatitis A outbreaks and requires the application of rapid and accurate molecular diagnostic tools for hepatitis A virus (HAV) RNA detection, subgenotype identification, and sequence-based genotyping. We describe the development and validation of a triplex real-time, reverse transcription-PCR (triplex rRT-PCR) assay for the identification and discrimination of HAV subgenotypes IA, IB, and IIIA and a singleplex rRT-PCR assay designed to detect all HAV genotypes infecting humans. Overall, the accuracy, sensitivity, and specificity of the new assays were >97% for serum and plasma specimens collected during unrelated outbreaks of HAV in California and Michigan compared to a nested RT-PCR genotyping assay and the ISO 15216-1 rRT-PCR method for HAV detection. The new assays will permit the rapid detection of HAV RNA and discrimination among subgenotypes IA, IB, and IIIA in serum and plasma specimens, which will strengthen public health surveillance efforts for HAV outbreak detection and response.

HLA‐II genes are associated with outcomes of specific immunotherapy for allergic rhinitis

Although the precise mechanisms underlying the efficacy of allergen-specific immunotherapy (AIT) are not clear, some evidence suggests that this may be linked to polymorphisms in HLA-II gene. We aimed to investigate the correlation between HLA-II gene polymorphisms and house dust mite (HDM)-specific immunotherapy efficacy, and evaluate specific polymorphisms as potential biomarkers in allergic rhinitis (AR) patients who would benefit most from AIT. Fifty-one Han Chinese patients with AR receiving HDM AIT were recruited. Genomic DNA was extracted from venous blood samples and genotyped for HLA-DRB1 and HAL-DQB1 alleles using the polymerase chain reaction sequence-based genotyping method. Nasal and eye symptoms were investigated based on visual analogue scale and rhinoconjunctivitis quality of life. Allele DRB1*04:06; DRB1*14:05 showed a positive correlation with improvements in nasal blockage, nasal itching, eye itching, and activities. Similarly, DQB1*03:02:01; DQB1*05:03: 01 was positively correlated with improvements in nose blocking, nasal itching, eye itching, behavioral problems, and nasal symptoms scores; and DRB1*03:01; DRB1*04:06 positively correlated with nasal symptoms scores. In contrast, DRB1*07:01:01; DRB1*11:01 was negatively correlated with non-pollen symptoms, behavioral problems, and nasal symptoms. HLA-DRB1 and HLA-DQB1 gene polymorphism are associated with AIT efficacy in HDM-sensitive Chinese patients with AR, of which DRB1*03:01; DRB1*04:06 and DQB1*03:02:01; DQB1*05:03:01 may be useful biomarkers of AR patient candidacy for effective AIT.

Intestinal parasitic infections and associated factors in children of three rural schools in Colombia. A cross-sectional study.

Rural children are one of the populations that are most vulnerable to gastrointestinal parasite infections. Such diseases decrease the quality of life and result in growth and cognitive delays in the long term. This cross-sectional study was conducted to determine the frequency of intestinal parasite infections among rural schoolchildren in the municipality of Apulo, Colombia. A total of 97 stool samples from children aged between 5 and 15 years were collected and examined via direct light microscopy. Microscopic examination was repeated with sediments obtained using a fecal parasite concentrator, and the Kato–Katz test was performed. Frequency of intestinal parasite infection was 100%. Endolimax nana (77.35%), Blastocystis sp. (71.1%), Giardia intestinalis (39.1%), Entamoeba coli (25.7%), and the Entamoeba histolytica/dispar/moshkovskii complex (9.2%) were the most prevalent protozoa. Trichuris trichiura was the most prevalent helminth (12.3%), followed by Enterobius vermicularis (6.15%) and Ascaris lumbricoides (5.1%). Among the analyzed associated factors, consumption of untreated water increased the risk of acquiring pathogenic intestinal parasites. Finally, because G. intestinalis was the most prevalent pathogenic protozoan, molecular analysis was conducted to establish genetic assemblages and subassemblages of Giardia through sequence-based genotyping of the glutamate dehydrogenase, triose phosphate isomerase, and beta-giardin genes. A total of 14 G. intestinalis-positive samples were genotyped, which revealed the presence of subassemblages AI (n = 1), AII (n = 7), BIII (n = 2), BIV (n = 2), and BIII/BIV (n = 1) as well as a mixed subassemblage AII + BIII (n = 1). Our results indicate that gastrointestinal parasite infections in the tested population were mainly caused by suboptimal water quality. Moreover, molecular typing of G. intestinalis suggested contamination of water by animal- and human-derived cysts.

Dissecting Resistance to Phytophthora cinnamomi in Interspecific Hybrid Chestnut Crosses Using Sequence-Based Genotyping and QTL Mapping

The soilborne oomycete Phytophthora cinnamomi-which causes root rot, trunk cankers, and stem lesions on an estimated 5,000 plant species worldwide-is a lethal pathogen of American chestnut (Castanea dentata) as well as many other woody plant species. P. cinnamomi is particularly damaging to chestnut and chinquapin trees (Castanea spp.) in the southern portion of its native range in the United States due to relatively mild climatic conditions that are conductive to disease development. Introduction of resistant genotypes is the most practical solution for disease management in forests because treatment with fungicides and eradication of the pathogen are neither practical nor economically feasible in natural ecosystems. Using backcross families derived from crosses of American chestnuts with two resistant Chinese chestnut cultivars Mahogany and Nanking, we constructed linkage maps and identified quantitative trait loci (QTLs) for resistance to P. cinnamomi that had been introgressed from these Chinese chestnut cultivars. In total, 957 plants representing five cohorts of three hybrid crosses were genotyped by sequencing and phenotyped by standardized inoculation and visual examination over a 6-year period from 2011 to 2016. Eight parental linkage maps comprising 7,715 markers were constructed, and 17 QTLs were identified on four linkage groups (LGs): LG_A, LG_C, LG_E, and LG_K. The most consistent QTLs were detected on LG_E in seedlings from crosses with both 'Mahogany' and 'Nanking' and LG_K in seedlings from 'Mahogany' crosses. Two consistent large and medium effect QTLs located ∼10 cM apart were present in the middle and at the lower end of LG_E; other QTLs were considered to have small effects. These results imply that the genetic architecture of resistance to P. cinnamomi in Chinese chestnut × American chestnut hybrid progeny may resemble the P. sojae-soybean pathosystem, with a few dominant QTLs along with quantitatively inherited partial resistance conferred by multiple small-effect QTLs.

Single primer enrichment technology as a tool for massive genotyping: a benchmark on black poplar and maize.

The advent of molecular breeding is advocated to improve the productivity and sustainability of second-generation bioenergy crops. Advanced molecular breeding in bioenergy crops relies on the ability to massively sample the genetic diversity. Genotyping-by-sequencing has become a widely adopted method for cost-effective genotyping. It basically requires no initial investment for design as compared with array-based platforms which have been shown to offer very robust assays. The latter, however, has the drawback of being limited to analyse only the genetic diversity accounted during selection of a set of polymorphisms and design of the assay. In contrast, genotyping-by-sequencing with random sampling of genomic loci via restriction enzymes or random priming has been shown to be fast and convenient but lacks the ability to target specific regions of the genome and to maintain high reproducibility across laboratories. Here we present a first adoption of single-primer enrichment technology (SPET) which provides a highly efficient and scalable system to obtain targeted sequence-based large genotyping data sets, bridging the gaps between array-based systems and traditional sequencing-based protocols. To fully explore SPET performance, we conducted a benchmark study in ten Zea mays lines and a large-scale study of a natural black poplar population of 540 individuals with the aim of discovering polymorphisms associated with biomass-related traits. Our results showed the ability of this technology to provide dense genotype information on a customized panel of selected polymorphisms, while yielding hundreds of thousands of untargeted variable sites. This provided an ideal resource for association analysis of natural populations harbouring unexplored allelic diversities and structure such as in black poplar. The improvement of sequencing throughput and the development of efficient library preparation methods has made it feasible to carry out targeted genotyping-by-sequencing experiments cost-competitively with either random complexity reduction systems or traditional array-based platforms, while maintaining the key advantages of both technologies.

Characterisation of major histocompatibility complex class IIa haplotypes in an island sheep population

The ovine MHC class IIa is known to consist of six to eight loci located in close proximity on chromosome 20, forming haplotypes that are typically inherited without recombination. Here, we characterise the class IIa haplotypes within the Soay sheep (Ovis aries) on St. Kilda to assess the diversity present within this unmanaged island population. We used a stepwise sequence-based genotyping strategy to identify alleles at seven polymorphic MHC class IIa loci in a sample of 118 Soay sheep from four cohorts spanning 15 years of the long-term study on St. Kilda. DRB1, the most polymorphic MHC class II locus, was characterised first in all 118 sheep and identified six alleles. Using DRB1 homozygous animals, the DQA (DQA1, DQA2 and DQA2-like) and DQB (DQB1, DQB2 and DQB2-like) loci were sequenced, revealing eight haplotypes. Both DQ1/DQ2 and DQ2/DQ2-like haplotype configurations were identified and a single haplotype carrying three DQB alleles. A test sample of 94 further individuals typed at the DRB1 and DQA loci found no exceptions to the eight identified haplotypes and a haplotype homozygosity of 21.3%. We found evidence of historic positive selection at DRB1, DQA and DQB. The limited variation at MHC class IIa loci in Soay sheep enabled haplotype characterisation but showed that no single locus could capture the full extent of the expressed variation in the region.

Calling known variants and identifying new variants while rapidly aligning sequence data

Whole-genome sequencing studies can identify causative mutations for subsequent use in genomic evaluations. Speed and accuracy of sequence alignment can be improved by accounting for known variant locations during alignment instead of calling the variants after alignment as in previous programs. The new programs Findmap and Findvar were compared with alignment using Burrows-Wheeler alignment (BWA) or SNAP and variant identification using Genome Analysis ToolKit (GATK) or SAMtools. Findmap stores the reference map and any known variant locations while aligning reads and counting reference and alternate alleles for each DNA source. Findmap also outputs potential new single nucleotide variant, insertion, and deletion alleles. Findvar separates likely true variants from read errors and outputs genotype probabilities. Strategies were tested using cattle, human, and a completely random reference map and simulated or actual data. Most tests simulated 10 bulls, each with 10× simulated sequence reads containing 39 million variants from the 1000 Bull Genomes Project. With 10 processors, clock times for processing 100× data were 105 h for BWA, 25 h for GATK, and 11 h for SAMtools but only about 4 h for SNAP, 3 h for Findmap, and 1 h for Findvar. Alignment programs required about the same total memory; BWA used 46 GB (4.6 GB/processor), whereas >10 processors can share the same memory in SNAP and Findmap, which used 40 and 46 GB, respectively. Findmap correctly mapped 92.9% of reads (compared with 92.6% from SNAP and 90.5% from BWA) and had high accuracy of calling alleles for known variants. For new variants, Findvar found 99.8% of single nucleotide variants, 79% of insertions, and 67% of deletions; GATK found 99.4, 95, and 90%, respectively; and SAMtools found 99.8, 12, and 16%, respectively. False positives (as percentages of true variants) were 11% of single nucleotide variants, 0.4% of insertions, and 0.3% of deletions from Findvar; 12, 8.4, and 2.9%, respectively, from GATK; and 37, 1.3, and 0.4%, respectively, from SAMtools. Advantages of Findmap and Findvar are fast processing, precise alignment, more useful data summaries, more compact output, and fewer steps. Calling known variants during alignment allows more efficient and accurate sequence-based genotyping.

Platforms and Analytical Tools Used in Nucleic Acid Sequence-Based Microbial Genotyping Procedures.

In the decade and a half since the introduction of next-generation sequencing (NGS), the technical feasibility, cost, and overall utility of sequencing have changed dramatically, including applications for infectious disease epidemiology. Massively parallel sequencing technologies have decreased the cost of sequencing by more than 6 orders or magnitude over this time, with a corresponding increase in data generation and complexity. This review provides an overview of the basic principles, chemistry, and operational mechanics of current sequencing technologies, including both conventional Sanger and NGS approaches. As the generation of large amounts of sequence data becomes increasingly routine, the role of bioinformatics in data analysis and reporting becomes all the more critical, and the successful deployment of NGS in public health settings requires careful consideration of changing information technology, bioinformatics, workforce, and regulatory requirements. While there remain important challenges to the sustainable implementation of NGS in public health, in terms of both laboratory and bioinformatics capacity, the impact of these technologies on infectious disease surveillance and outbreak investigations has been nothing short of revolutionary. Understanding the important role that NGS plays in modern public health laboratory practice is critical, as is the need to ensure appropriate workforce, infrastructure, facilities, and funding consideration for routine NGS applications, future innovation, and rapidly scaling NGS-based infectious disease surveillance and outbreak response activities. *This article is part of a curated collection.

Klossiella equi Infecting Kidneys of Ontario Horses: Life Cycle Features and Multilocus Sequence-Based Genotyping Confirm the Genus Klossiella Belongs in the Adeleorina (Apicomplexa: Coccidia)

Species in the genus Klossiella Smith and Johnson, 1902 are unique among the suborder Adeleorina because they are monoxenous in mammals exclusively, whereas all other reported members of the Adeleorina use invertebrates as definitive hosts. Unlike other coccidia, all members of the Adeleorina undergo syzygy, the association of microgamonts and macrogamonts before maturation to gametes and syngamy. After fertilization, many members of the Adeleorina produce thin-walled polysporocystic oocysts. Despite being biologically similar to other members of the Adeleorina, the phylogenetic placement of the genus Klossiella has been questioned based on its unique host affinity. In the present study, 2 cases of Klossiella equi were reported from the kidneys of horses in Ontario. Details of the life cycle as well as mitochondrial and nuclear 18S ribosomal DNA ( 18S rDNA) sequences were analyzed to provide both morphological and molecular evidence for the phylogenetic placement of K. equi. Initially, various stages of the life cycle were identified in histological slides prepared from the kidney tissue, and DNA was isolated from the infected tissue. Polymerase chain reaction and Sanger sequencing were used to generate a complete mitochondrial genome sequence (6,569 bp) and a partial 18S rDNA sequence (1,443 bp). The K. equi 18S rDNA sequence was aligned with various publicly available apicomplexan 18S rDNA sequences. This alignment was used to generate a phylogenetic tree based on Bayesian inference. Multiple K. equi stages were identified including meronts, microgamonts, and macrogamonts associating in syzygy as well as thin-walled oocysts in various stages of sporogonic development. The 18S rDNA sequence of K. equi positioned within the monophyletic Adeleorina clade. The mitochondrial genome of K. equi contained 3 coding sequences for cytochrome c oxidase I, cytochrome c oxidase III, and cytochrome b as well as various fragmented ribosomal sequences. These components were arranged in a unique order that has not been observed in other apicomplexan mitochondrial genomes sequenced to date. Overall, it was concluded that there were sufficient morphological and molecular data to confirm the placement of K. equi and the genus Klossiella among the Adeleorina. The biological and molecular data obtained from these cases may assist with future studies evaluating the prevalence and life history of this seemingly underreported parasite and better define the impact of K. equi on the health of domestic and wild equids.

Genetic characterization of worldwide Prunus domestica (plum) germplasm using sequence-based genotyping

Prunus domestica commonly known as European plum is a hexaploid fruit tree species cultivated around the world. Locally it is used for fresh consumption, in jams or jellies, and the production of spirits while commercially the fruit is primarily sold dried (prunes). Despite its agricultural importance and long history of cultivation, many questions remain about the origin of this species, the relationships among its many pomological types, and its underlying genetics. Here, we used a sequence-based genotyping approach to characterize worldwide plum germplasm including the potential progenitor Eurasian plum species. Analysis of 405 DNA samples established a set of four clades consistent with the pomological groups Greengages, Mirabelles, European plums, and d’Agen (French) prune plums. A number of cultivars from each clade were identified as likely clonal selections, particularly among the “French” type prune germplasm that is widely cultivated today. Overall, there was relatively low genetic diversity across all cultivated plums suggesting they have been largely inbred and/or derived from a limited number of founders. The results agree with P. domestica having originated as an interspecific hybrid of a diploid P. cerasifera and a tetraploid P. spinosa that itself may have been an interspecific hybrid of P. cerasifera and an unknown Eurasian plum species. The low genetic diversity and lack of true wild-types coupled with the known cultivation history of Eurasian plums imply that P. domestica may have been a product of inter-specific cross breeding and artificial selection by early agrarian Eurasian societies.

SNPSelect: A scalable and flexible targeted sequence-based genotyping solution.

In plant breeding the use of molecular markers has resulted in tremendous improvement of the speed with which new crop varieties are introduced into the market. Single Nucleotide Polymorphism (SNP) genotyping is routinely used for association studies, Linkage Disequilibrium (LD) and Quantitative Trait Locus (QTL) mapping studies, marker-assisted backcrosses and validation of large numbers of novel SNPs. Here we present the KeyGene SNPSelect technology, a scalable and flexible multiplexed, targeted sequence-based, genotyping solution. The multiplex composition of SNPSelect assays can be easily changed between experiments by adding or removing loci, demonstrating their content flexibility. To demonstrate this versatility, we first designed a 1,056-plex maize assay and genotyped a total of 374 samples originating from an F2 and a Recombinant Inbred Line (RIL) population and a maize germplasm collection. Next, subsets of the most informative SNP loci were assembled in 384-plex and 768-plex assays for further genotyping. Indeed, selection of the most informative SNPs allows cost-efficient yet highly informative genotyping in a custom-made fashion, with average call rates between 88.1% (1,056-plex assay) and 99.4% (384-plex assay), and average reproducibility rates between duplicate samples ranging from 98.2% (1056-plex assay) to 99.9% (384-plex assay). The SNPSelect workflow can be completed from a DNA sample to a genotype dataset in less than three days. We propose SNPSelect as an attractive and competitive genotyping solution to meet the targeted genotyping needs in fields such as plant breeding.

Improved pairwise kinship analysis using massively parallel sequencing

In the present study, 67 individuals from two families were analyzed to explore the efficacy of the ForenSeq™ DNA Signature Prep Kit for pairwise kinship analysis. Six types of pairwise relationships including 81 parent-offspring, 60 full siblings, 48 grandparent-grandchildren, 147 uncle/aunt-nephew/nieces, 97 first cousins and 190 non-relatives were generated from these two families and the corresponding likelihood ratio (LR) was calculated using either sequence-based or length-based STR genotype data (i.e., LRsequence and LRlength). In addition, 10,000 pairs of different relationships were simulated to estimate the system powers of the STRs and SNPs in this panel. The results showed that 54, 9 and 5 additional alleles were observed based on sequence for 27 autosomal STRs, 24 Y-STRs and 7 X-STRs, respectively, compared to those based on length information and 11 novel alleles were identified. Five mutations were found for 58 STRs in 81 parent-offspring but no mutations were observed for SNPs. For 27 autosomal STR loci, the LRs were increased from 9.20, 7.87, 2.01, 2.07, 0.42 for log10LRlength to 11.52, 10.12, 2.61, 2.60, 0.52 for log10LRsequence for paternity index (PI), full siblings index (FSI), grandparent-grandchild index (GI), uncle/aunt-nephew/niece index (UNI) and first cousins index (FCI), respectively. PI values for 94 SNPs separated more than those of 27 STRs if two individuals were non parent-offspring relatives. For the simulation study, the effectiveness was 1 for the parent-offspring relationship at the thresholds of t1 = − 4 and t2 = 4 and was 0.9998 for full siblings (t1 = − 2, t2 = 2). With an error rate of 0.42%, 93.02% of second degree relatives could be identified at the thresholds of t1 = − 1 and t2 = 1. However, the effectiveness was only 0.4300 for first cousins with a relatively high error rate of 2.68% (t1 = − 1, t2 = 1). In conclusion, STR typing according to the sequence information is more polymorphic, which increases the discrimination power for kinship testing. Compared to these 27 STR markers, 94 SNP markers in this panel have advantages in paternity testing especially when mutated STRs are involved or when a relative is an alleged parent. This panel is powerful enough to resolve paternity and full sibling testing. Most of the second degree relationships could be identified with low error rate while more markers are still needed for first cousins testing.

Prevalence of intestinal parasites, with emphasis on the molecular epidemiology of Giardia duodenalis and Blastocystis sp., in the Paranagu\xe1 Bay, Brazil: a community survey

BackgroundIntestinal protozoan parasites are major contributors to the global burden of gastrointestinal disease causing significant socioeconomic consequences. Children living in resource-poor settings with restricted access to water and sanitary services are particularly at risk of these infections.MethodsA prospective, community-based, cross-sectional survey was conducted in Paraná (southern Brazil) between May 2015 and May 2016. A total of 766 stool samples were individually collected from volunteers (male/female ratio: 0.99; age range: 0–76 years) and used for investigating the presence of intestinal helminth and protozoan species by routine microscopic procedures including the Kato-Katz and modified Ritchie concentration methods and the Ziehl-Neelsen stain technique. Quantitative real-time PCR confirmed microscopy-positive samples for Giardia duodenalis and the assemblages and sub-assemblages determined by multilocus sequence-based genotyping of the glutamate dehydrogenase (gdh) and β-giardin (bg) genes of the parasite. Identification of Blastocystis subtypes was carried out by amplification and sequencing of a partial fragment of the small-subunit ribosomal RNA gene (SSU rDNA) of this heterokont microorganism.ResultsOverall, 46.1% (353/766) of the participants were infected/colonised by at least one intestinal parasite/commensal species. Protozoan and helminth species were detected in 42.7% and 10.1% of the surveyed population, respectively. Blastocystis sp. (28.2%), Endolimax nana (14.9%), and Giardia duodenalis (11.0%) were the most prevalent species found among protozoans and Ascaris lumbricoides (5.0%), Trichuris trichiura (4.6%) and hookworms (1.0%) among helminths. A total of 38 G. duodenalis-positive samples were genotyped at gdh and bg markers, revealing the presence of the sub-assemblages AII (47.4%), AII/AIII (2.6%), BIII (5.3%), BIV (26.3%) and BIII/BIV (13.1%). Two samples (5.3%) were only identified as assemblage B. AII was predominantly found in females aged 5–9 years and was associated with a higher likelihood of reporting gastrointestinal symptoms. A total of 102 Blastocystis-positive samples were successfully subtyped at the SSU rRNA gene revealing the presence of ST1 (36.3%), ST2 (15.7%), ST3 (41.2%), ST4 (2.9%), ST6 (1.0%) and ST8 (2.9%).ConclusionsData presented here indicate that enteric parasites still represent a pressing health concern in Paraná, Brazil, probably due to sub-optimal water, sanitation and hygiene conditions. A mostly anthroponotic origin is suspected for G. duodenalis and Blastocystis sp. infections.

The genetic architecture of genome-wide recombination rate variation in allopolyploid wheat revealed by nested association mapping.

SummaryRecombination affects the fate of alleles in populations by imposing constraints on the reshuffling of genetic information. Understanding the genetic basis of these constraints is critical for manipulating the recombination process to improve the resolution of genetic mapping, and reducing the negative effects of linkage drag and deleterious genetic load in breeding. Using sequence‐based genotyping of a wheat nested association mapping (NAM) population of 2,100 recombinant inbred lines created by crossing 29 diverse lines, we mapped QTL affecting the distribution and frequency of 102 000 crossovers (CO). Genome‐wide recombination rate variation was mostly defined by rare alleles with small effects together explaining up to 48.6% of variation. Most QTL were additive and showed predominantly trans‐acting effects. The QTL affecting the proximal COs also acted additively without increasing the frequency of distal COs. We showed that the regions with decreased recombination carry more single nucleotide polymorphisms (SNPs) with possible deleterious effects than the regions with a high recombination rate. Therefore, our study offers insights into the genetic basis of recombination rate variation in wheat and its effect on the distribution of deleterious SNPs across the genome. The identified trans‐acting additive QTL can be utilized to manipulate CO frequency and distribution in the large polyploid wheat genome opening the possibility to improve the efficiency of gene pyramiding and reducing the deleterious genetic load in the low‐recombining pericentromeric regions of chromosomes.