Development of an Online Tool for Pasteurella multocida Genotyping and Genotypes of Pasteurella multocida From Different Hosts

Pasteurella multocida is a versatile zoonotic pathogen. Multiple systems have been applied to type P. multocida from different diseases in different hosts. Recently, we found that assigning P. multocida strains by combining their capsular, lipopolysaccharide, and MLST genotypes (marked as capsular: lipopolysaccharide: MLST genotype) could help address the biological characteristics of P. multocida circulation in different hosts. However, there is still lack of a rapid and efficient tool to diagnose P. multocida according to this system. Here, we developed an intelligent genotyping platform PmGT for P. multocida strains according to their whole genome sequences using the web 2.0 technologies. By using PmGT, we determined capsular genotypes, LPS genotypes, and MLST genotypes as well as the main virulence factor genes (VFGs) of P. multocida isolates from different host species based on their whole genome sequences published on NCBI. The results revealed a closer association between the genotypes and pasteurellosis rather than between genotypes and host species. With the advent of high-quality, inexpensive DNA sequencing, PmGT represents a more efficient tool for P. multocida diagnosis in both epidemiological studies and clinical settings.

Establishing Analytical Validity of BeadChip Array Genotype Data by Comparison to Whole-Genome Sequence and Standard Benchmark Datasets

Background Clinical use of genotype data requires high positive predictive value (PPV) and thorough understanding of the genotyping platform characteristics. BeadChip arrays, such as the Global Screening Array (GSA), potentially offer a high-throughput, low-cost clinical screen for known variants. We hypothesize that quality assessment and comparison to whole-genome sequence and benchmark data establish the analytical validity of GSA genotyping.Methods To test this hypothesis, we selected 263 samples from Coriell, generated GSA genotypes in triplicate, generated whole genome sequence (rWGS) genotypes, assessed the quality of each set of genotypes, and compared each set of genotypes to each other and to the 1000 Genomes Phase 3 (1KG) genotypes, a performance benchmark. For 59 genes (MAP59), we also performed theoretical and empirical evaluation of variants deemed medically actionable predispositions.Results Quality analyses detected sample contamination and increased assay failure along the chip margins. Comparison to benchmark data demonstrated that > 82% of the GSA assays had a PPV of 1. GSA assays targeting transitions, genomic regions of high complexity, and common variants performed better than those targeting transversions, regions of low complexity, and rare variants. Comparison of GSA data to rWGS and 1KG data showed >99.3% concordance across all measured parameters. GSA detection of variation within the MAP59 genes was 3/261 consistent with predictions from prior studies.Conclusion We establish the analytical validity of GSA assays using quality analytics and comparison to benchmark and rWGS data. GSA assays meet the standards of a clinical screen although assays interrogating rare variants, transversions, and variants within low-complexity regions require careful evaluation.

Applying targeted genotyping by sequencing with a new set of nuclear and plastid SNP and indel loci for Quercus robur and Quercus petraea

We used Double Digest Restriction site associated DNA sequencing (ddRAD) and Miseq to develop new geographically informative nuclear and plastid SNP and indel loci in Quercus robur and Q. petraea. Genotypes derived from sequence data of 95 individuals and two pools of 20 individuals each of Q. robur and Q. mongolica covering the distribution range of the species, were analysed to select geographically informative and polymorphic loci within Germany and Russia. We successfully screened a selected set of 431 nuclear single nucleotide polymorphism (nSNP), six nuclear Indel, six mitochondrial single nucleotide polymorphism (mtSNP) and ten chloroplast single nucleotide polymorphism (cpSNP) loci with a SeqSNP genotyping platform on 100 individuals Quercus petraea from 10 locations in Germany, 100 individuals Quercus robur from ten locations in Germany and 100 individuals Quercus robur from ten locations in Russia. The newly developed loci are useful for species identification and genetic studies on the genetic diversity and genetic differentiation of Quercus robur and Quercus petraea in Europe.

Genetic Mapping by Sequencing More Precisely Detects Loci Responsible for Anaerobic Germination Tolerance in Rice

Direct seeded rice (DSR) is a mainstay for planting rice in the Americas, and it is rapidly becoming more popular in Asia. It is essential to develop rice varieties that are suitable for this type of production system. ASD1, a landrace from India, possesses several traits desirable for direct-seeded fields, including tolerance to anaerobic germination (AG). To map the genetic basis of its tolerance, we examined a population of 200 F2:3 families derived from a cross between IR64 and ASD1 using the restriction site-associated DNA sequencing (RAD-seq) technology. This genotyping platform enabled the identification of 1921 single nucleotide polymorphism (SNP) markers to construct a high-resolution genetic linkage map with an average interval of 0.9 cM. Two significant quantitative trait loci (QTLs) were detected on chromosomes 7 and 9, qAG7 and qAG9, with LOD scores of 7.1 and 15.0 and R2 values of 15.1 and 29.4, respectively. Here, we obtained more precise locations of the QTLs than traditional simple sequence repeat and low-density SNP genotyping methods and may help further dissect the genetic factors of these QTLs.

Genotyping tools and resources to assess peanut germplasm: smut-resistant landraces as a case study

Peanut smut caused by Thecaphora frezii is a severe fungal disease currently endemic to Argentina and Brazil. The identification of smut resistant germplasm is crucial in view of the potential risk of a global spread. In a recent study, we reported new sources of smut resistance and demonstrated its introgression into elite peanut cultivars. Here, we revisited one of these sources (line I0322) to verify its presence in the U.S. peanut germplasm collection and to identify single nucleotide polymorphisms (SNPs) potentially associated with resistance. Five accessions of Arachis hypogaea subsp. fastigiata from the U.S. peanut collection, along with the resistant source and derived inbred lines were genotyped with a 48K SNP peanut array. A recently developed SNP genotyping platform called RNase H2 enzyme-based amplification (rhAmp) was further applied to validate selected SNPs in a larger number of individuals per accession. More than 14,000 SNPs and nine rhAmp assays confirmed the presence of a germplasm in the U.S. peanut collection that is 98.6% identical (P < 0.01, bootstrap t-test) to the resistant line I0322. We report this germplasm with accompanying genetic information, genotyping data, and diagnostic SNP markers.

Liver Cirrhosis in Chronic Hepatitis B Patients Is Associated with Genetic Variations in DNA Repair Pathway Genes

Liver cirrhosis (LC), contributing to more than 1 million of deaths annually, is a major healthcare concern worldwide. Hepatitis B virus (HBV) is a major LC etiological factor, and 15% of patients with chronic HBV infection (CHB) develop LC within 5 years. Recently, novel host genetic determinants were shown to influence HBV lifecycle and CHB course. DNA repair enzymes can affect dynamics of liver damage and are involved in HBV covalently closed circular DNA (cccDNA) formation, an essential step for viral replication. This study aimed to evaluate the possible role of genes representing key DNA-repair pathways in HBV-induced liver damage. MALDI-TOF MS genotyping platform was applied to evaluate variations within XRCC1, XRCC4, ERCC2, ERCC5, RAD52, Mre11, and NBN genes. Apart from older age (p < 0.001), female sex (p = 0.021), portal hypertension (p < 0.001), thrombocytopenia (p < 0.001), high HBV DNA (p = 0.001), and high aspartate aminotransferase (AST) (p < 0.001), we found that G allele at rs238406 (ERCC2, p = 0.025), T allele at rs25487 (XRCC1, p = 0.012), rs13181 GG genotype (ERCC2, p = 0.034), and C allele at rs2735383 (NBN, p = 0.042) were also LC risk factors. The multivariate logistic regression model showed that rs25487 CC (p = 0.005) and rs238406 TT (p = 0.027) were independently associated with lower risk of LC. This study provides evidence for the impact of functional and potentially functional variations in key DNA-repair genes XRCC1 and ERCC2 in HBV-induced liver damage in a Caucasian population.

Malignant hyperthermia susceptibility: utilization of genetic results in an electronic medical record to increase safety

Aim: This manuscript describes implementation of clinical decision support for providers concerned with perioperative complications of malignant hyperthermia susceptibility. Materials & methods: Clinical decision support for malignant hyperthermia susceptibility was implemented in 2018 based around our pre-emptive genotyping platform. We completed a brief descriptive review of patients who underwent pre-emptive testing, focused particularly on RYR1 and CACNA1S genes. Results: To date, we have completed pre-emptive genetic testing on more than 10,000 patients; 13 patients having been identified as a carrier of a pathogenic or likely pathogenic variant of RYR1 or CACNA1S. Conclusion: An alert system for malignant hyperthermia susceptibility – as an extension of our pre-emptive genomics platform – was implemented successfully. Implementation strategies and lessons learned are discussed herein.

A Data-Driven Approach to Carrier Screening for Common Recessive Diseases

Genetic screening is an advanced tool for reducing recessive disease burden. Nowadays, it is still unclear as to the number of genes or their variants that are necessary for effective screening. This paper describes the development of a carrier screening custom panel for cystic fibrosis, phenylketonuria, alpha-1 antitrypsin deficiency, and sensorineural hearing loss consisting of 116 variants in the CFTR, PAH, SERPINA1, and GJB2 genes. The approach is based on the cheapest and fastest method, on using a small number of genes, and on the estimation of the effectiveness of carriers’ detection. The custom panel was tested on a population-based cohort that included 1244 participants. Genotypes were determined by the TaqMan OpenArray Genotyping platform on the QuantStudio 12K Flex Real-Time PCR System. The frequency of heterozygotes in the Russian population was 16.87% or 1:6 (CI95%: 14.76–19.00% by Clopper-Pearson exact method): in CFTR—2.81% (1:36), PAH—2.33% (1:43), SERPINA1—4.90% (1:20), and GJB2—6.83% (1:15). The data on allele frequencies were obtained for the first time on a Russian population. The panel allows us to identify the vast majority of carriers of recessive diseases in the population. It is an effective approach to carrier screening for common recessive diseases.