Rapid Detection of Circulating Tumour DNA using Allele Specific Mini-Loop Mediated Isothermal Amplification

Isothermal amplification is an emerging approach for non-invasive, rapid and cost-effective real-time monitoring of cancer specific mutations through circulating tumour DNA (ctDNA). This study demonstrates a compact allele specific (AS) loop mediated isothermal amplification (LAMP) strategy, termed ‘AS-Mini-LAMP’, modelled using wild type (WT) and mutation specific reactions targeting the estrogen receptor ESR1 c.1138G>C (p.E380Q) missense mutation. Allele selectivity, encoded at the 5’-end of the forward and backward inner primers (FIP and BIP) promotes enhanced selectivity upon self-hybridisation, loop formation and self-primed exponential amplification. Inclusion of unmodified self-stabilising (USS) primers aimed to reduce the likelihood of non-specific allele amplification through competitive inhibition and to enhance reaction velocity through an assisted strand displacement ‘swarm’ priming effect. The two assays were optimised using short synthetic WT and E380Q mutant DNA templates, and subsequently validated to a limit of detection of 500 mutant copies in under 25 minutes in ddPCR-confirmed positive (20.7% variant allele frequency) and negative patient plasma cfDNA samples. These results demonstrate the ability of AS-Mini-LAMP to achieve sensitive and selective amplification of actionable mutations present within plasma ctDNA.

Identification of Lens Cultivars in Market by Molecular Tools: DNA Barcoding and SSRs

Substitution of plant cultivars of high commercial value with a cheaper, lower-quality one is a common fraud committed against consumers and producers. Since it is one of the most widely grown legumes, lentil (Lens culinaris Medik.) are a suitable for such frauds. This study aimed to identify lentil cultivars which are registered and authorized in the market in Turkey by using current molecular methods. For this purpose, 26 lentil cultivars were analyzed for 15 SSR markers and 2 DNA barcode loci (trnH-psbA and matK). A high allele diversity was observed by 12 scorable SSR markers, and the average number of alleles was determined to be 16. One of the important findings was the presence of “cultivar-specific alleles” that can be used to identify each cultivar in the lentil market in Turkey. At least one “cultivar-specific allele” was obtained for each cultivar. The lentil cultivars were analyzed in terms of 2 DNA barcode regions as trnH-psbA and matK. Sequences that could identify 14 of the 26 cultivars were obtained. While the rate of the intra-species variation for the trnH-psbA region was observed to be low, a higher rate was found for matK. Nevertheless, it was observed that intra-species discrimination can be made more effective when both loci are used together. We expect that the results of this study, especially the cultivar-specific SSR alleles and DNA barcoding sequence data may be used routinely to identify on production and packaged products that are commercially available in markets.

Bait-ER: a Bayesian method to detect targets of selection in Evolve-and-Resequence experiments

For over a decade, experimental evolution has been combined with high-throughput sequencing techniques in so-called Evolve-and-Resequence (E&R) experiments. This allows testing for selection in populations kept in the laboratory under given experimental conditions. However, identifying signatures of adaptation in E&R datasets is far from trivial, and it is still necessary to develop more efficient and statistically sound methods for detecting selection in genome-wide data. Here, we present Bait-ER - a fully Bayesian approach based on the Moran model of allele evolution to estimate selection coefficients from E&R experiments. The model has overlapping generations, a feature that describes several experimental designs found in the literature. We tested our method under several different demographic and experimental conditions to assess its accuracy and precision, and it performs well in most scenarios. However, some care must be taken when analysing specific allele trajectories, particularly those where drift largely dominates and starting frequencies are low. We compare our method with other available software and report that ours has generally high accuracy even for very difficult trajectories. Furthermore, our approach avoids the computational burden of simulating an empirical null distribution, outperforming available software in terms of computational time and facilitating its use on genome-wide data. We implemented and released our method in a new open-source software package that can be accessed at https://github.com/mrborges23/Bait-ER.

Assessment of tea plant (Camellia sinensis L.) accessions for pollen sources in natural crossing by using microsatellites

Tea (Camellia sinensis L. [O.] Kuntze) is a highly cross-pollinated and self-incompatible plant. Seeds can be harvested from specific individual mother plants in polyclonal tea gardens. Whether the pollen donor plays an important role in seed formation remains unclear. This study aimed to identify the male parents of 72 natural hybridized progenies (F1) from one female parent on the basis of a putative specific allele by using simple-sequence repeat (SSR) markers and the exclusion-likelihood method with Cervus 3.0 software. The genetic material, which comprised seven accessions of C. sinensis L., was acquired from Assamica planted in the Kayulandak polyclonal seed garden of the Pagilaran tea plantation in Batang District, Central Java, Indonesia, and was studied during 2019 and 2020. The genotype PGL-15 was used as the female parent, whereas the six candidate genotypes PGL-10, GMB-9, GMB-7, TPS-93, GMB-11, and TRI-2025 were used as the male parents. In this study, 13 SSR loci were used to identify the male parents of the F1 progenies obtained through natural hybridization between one female and six male tea accessions. Results indicated that the exclusion-likelihood method, which correctly predicted 100% of the male parents, was more effective than the putative specific allele approach, which correctly predicted only 34.72% of the male parents in the 72 hybridized F1 progenies of tea plants.

Expanding the Russian allele frequency reference via cross-laboratory data integration: insights from 6,096 exome samples

The frequency of a genetic variant in a population is crucially important for accurate interpretation of known and novel variant effects in medical genetics. Recently, several large allele frequency databases, such as Genome Aggregation Database (gnomAD), have been created to serve as a global reference for such studies. However, frequencies of many rare alleles vary dramatically between populations, and population-specific allele frequency can be more informative than the global one. Many countries and regions (including Russia) remain poorly studied from the genetic perspective. Here, we report the first successful attempt to integrate genetic information between major medical genetic laboratories in Russia. We construct an expanded reference set of genetic variants by analyzing 6,096 exome samples collected in two major Russian cities of Moscow and St. Petersburg. An approximately tenfold increase in sample size compared to previous studies allowed us to identify genetically distinct clusters of individuals within an admixed population of Russia. We show that up to 18 known pathogenic variants are overrepresented in Russia compared to other European countries. We also identify several dozen high-impact variants that are present in healthy donors despite either being annotated as pathogenic in ClinVar or falling within genes associated with autosomal dominant disorders. The constructed database of genetic variant frequencies in Russia has been made available to the medical genetics community through a variant browser available at http://ruseq.ru.

A Founder Pathogenic Variant of PPIB Unique to Chinese Population Causes Osteogenesis Imperfecta IX

Background: Osteogenesis imperfecta (OI) is a heterogeneous genetic disorder characterized by bone fragility. PPIB pathogenic variants cause a perinatal lethal form of OI type IX. A limited number of pathogenic variants have been reported so far worldwide.Methods: We identified a rare pedigree whose phenotype was highly consistent with OI-IX. Exome sequencing was performed to uncover the causal variants. The variant pathogenicity was classified following the ACMG/AMP guidelines. The founder effect and the age of the variant were assessed.Results: We identified a homozygous missense variant c.509G > A/p.G170D in PPIB in an affected fetus. This variant is a Chinese-specific allele and can now be classified as pathogenic. We estimated the allele frequency (AF) of this variant to be 0.0000427 in a Chinese cohort involving 128,781 individuals. All patients and carriers shared a common haplotype, indicative of a founder effect. The estimated age of variant was 65,160 years. We further identified pathogenic variants of PPIB in gnomAD and ClinVar databases, the conserved estimation of OI type IX incidence to be 1/1,000,000 in Chinese population.Conclusion: We reported a founder pathogenic variant in PPIB specific to the Chinese population. We further provided our initial estimation of OI-IX disease incidence in China.

Demographic, Clinical and Immunogenetic Profiles of a Greek Cohort of COVID-19 Patients

The present cross-sectional study consists of a comprehensive analysis of epidemiological, laboratory, and clinical characteristics of COVID-19 patients in relation to their immunogenetic profiles. We studied 125 COVID-19 patients comprising different stages of disease severity; non-hospitalized (mild n = 69) and hospitalized (n = 56). Analysis of disease characteristics revealed no major differences between males and females of each group of patients while hospitalized patients were older and presented with comorbidities. A positive allele association was observed for HLA-DRB1*01 in total COVID-19 patients versus healthy controls. Subgrouping of COVID-19 patients in mild and hospitalized further identified a statistically significant increase in HLA-DRB1*01 in mild COVID-19 patients versus controls. The frequency of A*11, A*23, and DRB1*09 alleles was higher, while the frequency of C*12 was lower, in hospitalized patients versus healthy controls albeit with uncorrected statistical significance. The identification of specific allele associations may provide useful future markers for disease susceptibility in order to allow successful clinical management of COVID-19 patients.

Identification and Molecular Analysis of Putative Self-Incompatibility Ribonuclease Alleles in an Extreme Polyploid Species, Prunus laurocerasus L.

Cherry laurel (Prunus laurocerasus L.) is an extreme polyploid (2n = 22x) species of the Rosaceae family where gametophytic self-incompatibility (GSI) prevents inbreeding. This study was carried out to identify the S-ribonuclease alleles (S-RNases) of P. laurocerasus using PCR amplification of the first and second intron region of the S-RNase gene, cloning and sequencing. A total of 23 putative S-RNase alleles (S1–S20, S5m, S13m, and S18m) were sequenced from the second (C2) to the fifth conserved region (C5), and they shared significant homology to other Prunus S-RNases. The length of the sequenced amplicons ranged from 505 to 1,544 bp, and similar sizes prevented the proper discrimination of some alleles based on PCR analysis. We have found three putatively non-functional alleles (S5m, S18m, and S9) coding for truncated proteins. Although firm conclusions cannot be drawn, our data seem to support that heteroallelic pollen cannot induce self-compatibility in this polyploid Prunus species. The identities in the deduced amino acid sequences between the P. laurocerasus and other Prunus S-RNases ranged between 44 and 100%, without a discontinuity gap separating the identity percentages of trans-specific and more distantly related alleles. The phylogenetic position, the identities in nucleotide sequences of the second intron and in deduced amino acid sequences found one or more trans-specific alleles for all but S10, S14, S18, and S20 cherry laurel RNases. The analysis of mutational frequencies in trans-specific allele pairs indicated the region RC4–C5 accepts the most amino acid replacements and hence it may contribute to allele-specificity. Our results form the basis of future studies to confirm the existence and function of the GSI system in this extreme polyploid species and the alleles identified will be also useful for phylogenetic studies of Prunus S-RNases as the number of S-RNase sequences was limited in the Racemose group of Prunus (where P. laurocerasus belongs to).

A novel method for an unbiased estimate of cross-ancestry genetic correlation using individual-level data

Cross-ancestry genetic correlation is an important parameter to understand the genetic relationship between two ancestry groups for a complex trait. However, existing methods cannot properly account for ancestry-specific genetic architecture, which is diverse across ancestries, producing biased estimates of cross-ancestry genetic correlation. Here, we present a method to construct a genomic relationship matrix (GRM) that can correctly account for the relationship between ancestry-specific allele frequencies and ancestry-specific causal effects. Through comprehensive simulations, we show that the proposed method outperforms existing methods in the estimations of SNP-based heritability and cross-ancestry genetic correlation. The proposed method is further applied to six anthropometric traits from the UK Biobank data across 5 ancestry groups. One of our findings is that for obesity, the estimated genetic correlation between African and European ancestry cohorts is significantly different from unity, suggesting that obesity is genetically heterogenous between these two ancestry groups.

Microsatellites to reveal genetic diversity and to distinguish four mangoes of Tegal District, Central Java, Indonesia

Hidayat A, Rahayu ES, Abdullah M, Retnoningsih A. 2021. Microsatellites to reveal genetic diversity and to distinguish four mangoes of Tegal District, Central Java, Indonesia. Biodiversitas 22: 3467-3473. The Wirasangka mango is Tegal District’s flora of identity, which requires recognition, and its existence deserves to be preserved. However, based on the morphology of the fruit, most of the people of Tegal still have difficulty distinguishing the wirasangka mango from other mangoes. Molecular markers are needed to ascertain the differences between these mango varieties. Microsatellite DNA is a repeating DNA of stable DNA markers with high repeatability and polymorphism. This study analyzes the level of genetic diversity and microsatellite alleles that can differentiate four mangoes from Tegal District, Central Java, Indonesia, i.e. wirasangka, tengkueh, golek, and okyong. Eleven accessions of six locations in Tegal District were analyzed using ten microsatellite loci. The microsatellite amplification result was separated using electrophoresis in 6% polyacrylamide gel and then visualized with silver dye. A total of 35 microsatellite alleles were found measuring 100-1000 bp, ranging from 1-7 alleles for each locus. The average polymorphism information content (PIC) of 0.54 indicates that genetic diversity is relatively high and informative. Therefore, the microsatellite alleles can be used to differentiate mango varieties. The specific allele characteristic of wirasangka mango accession is locus AJ63516, with an allele size of 600-700bp.