Strategy and Performance Evaluation of Low-Frequency Variant Calling for SARS-CoV-2 Using Targeted Deep Illumina Sequencing

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, constitutes a tremendous global health issue. Continuous monitoring of the virus has become a cornerstone to make rational decisions on implementing societal and sanitary measures to curtail the virus spread. Additionally, emerging SARS-CoV-2 variants have increased the need for genomic surveillance to detect particular strains because of their potentially increased transmissibility, pathogenicity and immune escape. Targeted SARS-CoV-2 sequencing of diagnostic and wastewater samples has been explored as an epidemiological surveillance method for the competent authorities. Currently, only the consensus genome sequence of the most abundant strain is taken into consideration for analysis, but multiple variant strains are now circulating in the population. Consequently, in diagnostic samples, potential co-infection(s) by several different variants can occur or quasispecies can develop during an infection in an individual. In wastewater samples, multiple variant strains will often be simultaneously present. Currently, quality criteria are mainly available for constructing the consensus genome sequence, and some guidelines exist for the detection of co-infections and quasispecies in diagnostic samples. The performance of detection and quantification of low-frequency variants using whole genome sequencing (WGS) of SARS-CoV-2 remains largely unknown. Here, we evaluated the detection and quantification of mutations present at low abundances using the mutations defining the SARS-CoV-2 lineage B.1.1.7 (alpha variant) as a case study. Real sequencing data were in silico modified by introducing mutations of interest into raw wild-type sequencing data, or by mixing wild-type and mutant raw sequencing data, to construct mixed samples subjected to WGS using a tiling amplicon-based targeted metagenomics approach and Illumina sequencing. As anticipated, higher variation and lower sensitivity were observed at lower coverages and allelic frequencies. We found that detection of all low-frequency variants at an abundance of 10, 5, 3, and 1%, requires at least a sequencing coverage of 250, 500, 1500, and 10,000×, respectively. Although increasing variability of estimated allelic frequencies at decreasing coverages and lower allelic frequencies was observed, its impact on reliable quantification was limited. This study provides a highly sensitive low-frequency variant detection approach, which is publicly available at https://galaxy.sciensano.be, and specific recommendations for minimum sequencing coverages to detect clade-defining mutations at certain allelic frequencies. This approach will be useful to detect and quantify low-frequency variants in both diagnostic (e.g., co-infections and quasispecies) and wastewater [e.g., multiple variants of concern (VOCs)] samples.

GENETIC CONNECTIVITY OF MULLETS (Mugil liza) BETWEEN RODRIGO DE FREITAS LAGOON AND A CONSERVATION UNIT IN STATE OF RIO DE JANEIRO, SOUTHEASTERN BRAZIL

We analyzed the genetic connectivity between mullets (Mugil liza) captured around the protected Natural Monument of Cagarras Islands (MoNa Cagarras) and inside Rodrigo de Freitas Lagoon, using microsatellite markers polymorphisms. Our data revealed the occurrence of 31 shared alleles (from 41 sampled), a high similarity in both allelic frequencies and genetic diversity and lack of differentiation between collection points (FST = 0.000, p > 0.05, STRUCTURE best estimative K = 1), results which, analyzed together, are strongly indicative of panmixia. We conclude that individuals collected inside the Rodrigo de Freitas Lagoon are genetically similar to those individuals collected around MoNa Cagarras. Given the importance of estuaries for the reproduction and development of individuals of M. liza, it is recommended that the Rodrigo de Freitas Lagoon to be managed in order to maintain genetic connectivity and diversity between the two ecosystems.

Strategy and performance evaluation of low-frequency variant calling for SARS-CoV-2 in wastewater using targeted deep Illumina sequencing

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, constitutes a tremendous global health issue. Continuous monitoring of the virus has become a cornerstone to make rational decisions on implementing societal and sanitary measures to curtail the virus spread. Additionally, emerging SARS-CoV-2 variants have increased the need for genomic surveillance to detect particular strains because of their potentially increased transmissibility, pathogenicity and immune escape. Targeted SARS-CoV-2 sequencing of wastewater has been explored as an epidemiological surveillance method for the competent authorities. Few quality criteria are however available when sequencing wastewater samples, and those available typically only pertain to constructing the consensus genome sequence. Multiple variants circulating in the population can however be simultaneously present in wastewater samples. The performance, including detection and quantification of low-abundant variants, of whole genome sequencing (WGS) of SARS-CoV-2 in wastewater samples remains largely unknown. Here, we evaluated the detection and quantification of mutations present at low abundances using the SARS-CoV-2 lineage B.1.1.7 (alpha variant) defining mutations as a case study. Real sequencing data were in silico modified by introducing mutations of interest into raw wild-type sequencing data, or by mixing wild-type and mutant raw sequencing data, to mimic wastewater samples subjected to WGS using a tiling amplicon-based targeted metagenomics approach and Illumina sequencing. As anticipated, higher variation, lower sensitivity and more false negatives, were observed at lower coverages and allelic frequencies. We found that detection of all low-frequency variants at an abundance of 10%, 5%, 3% and 1%, requires at least a sequencing coverage of 250X, 500X, 1500X and 10,000X, respectively. Although increasing variability of estimated allelic frequencies at decreasing coverages and lower allelic frequencies was observed, its impact on reliable quantification was limited. This study provides a highly sensitive low-frequency variant detection approach, which is publicly available at https://galaxy.sciensano.be, and specific recommendations for minimum sequencing coverages to detect clade-defining mutations at specific allelic frequencies.

Functional Polymorphisms in the p53 Pathway Genes on the Genetic Susceptibility to Zika Virus Teratogenesis

Congenital Zika Syndrome (CZS) occurs in up to 42% of individuals exposed to ZIKV prenatally. Deregulation in gene expression and protein levels of components of the p53 signaling pathway, such as p53 and MDM2, due to ZIKV infection has been reported. Here, we evaluate functional polymorphisms in genes of the p53 signaling pathway as risk factors to CZS. Forty children born with CZS and forty-eight children exposed to ZIKV, but born without congenital anomalies were included in this study. Gestational and sociodemographic information as well as the genotypic and allelic frequencies of functional polymorphisms in TP53, MDM2, MIR605 and LIF genes were compared between the two groups. We found children with CZS exposed predominantly in the first trimester and controls in the third trimester (p<0.001). Moreover, children with CZS were predominantly from families with a lower socioeconomic level (p=0.008). We did not find a statistically significant association between the investigated polymorphisms and development of CZS; however, by comparing individuals with CZS and lissencephaly or without lissencephaly, we found a significative difference in the allelic frequencies of the TP53 rs1042522, which is associated with a more potent p53-induced apoptosis (p=0.007). Our findings suggest that the TP53 rs1042522 polymorphism should be better investigate as a genetic risk factor for the development of lissencephaly in children with CZS.

Predictive Ability of a Clinical-Genetic Risk Score for Venous Thromboembolism in Northern and Southern European Populations

Venous thromboembolism (VTE) is a complex, multifactorial problem, the development of which depends on a combination of genetic and acqfiguired risk factors. In a Spanish population, the Thrombo inCode score (or TiC score), which combines clinical and genetic risk components, was recently proven better at determining the risk of VTE than the commonly used model involving the analysis of two genetic variants associated with thrombophilia: the Factor V Leiden (F5 rs6025) and the G20210A prothrombin (F2 rs1799963).The aim of the present case–control study was to validate the VTE risk predictive capacity of the TiC score in a Northern European population (from Sweden).The study included 173 subjects with VTE and 196 controls. All were analyzed for the genetic risk variants included in the TiC gene panel. Standard measures —receiver operating characteristic (ROC) area under the curve (AUC), sensitivity, specificity, and odds ratio (OR)—were calculated.The TiC score returned an AUC value of 0.673, a sensitivity of 72.25%, a specificity of 60.62%, and an OR of 4.11. These AUC, sensitivity, and OR values are all greater than those associated with the currently used combination of genetic variants. A TiC version adjusted for the allelic frequencies of the Swedish population significantly improved its AUC value (0.783).In summary, the TiC score returned more reliable risk estimates for the studied Northern European population than did the analysis of the Factor V Leiden and the G20210A genetic variations in combination. Thus, the TiC score can be reliably used with European populations, despite differences in allelic frequencies.

Genomic miscellany and allelic frequencies of Plasmodium falciparum msp-1, msp-2 and glurp in parasite isolates

Plasmodium falciparum, the main causative agent of malaria is an important public health vector in Khyber Pakhtunkhwa, Pakistan. Identification of the genetic diversity of malaria parasites can inform the intensity of transmission and identify potential deficiencies in malaria control programs. The aim of this study was to investigate the genetic diversity, allele frequencies and multiplicity of infection (MOI) of P. falciparum in Khyber Pakhtunkhwa, Pakistan. Methods: A total of 85 isolates from patients presenting to the local health centers with P. falciparum species were collected from 2017 to 2019. Parasite DNA was extracted from a total of 200 micro litter whole blood per patient using the Qiagen DNA extraction kit according to manufactures instructions. The polymorphic region of msp-1, msp-2 and glurp loci were genotyped by using nested polymerase chain reactions followed by gel electrophoresis for fragment analysis. Results: Genetic diversity and allelic frequencies of msp-1, msp-2 and glurp were identified in 85 blood samples. A total of 62 msp alleles were detected in which 30 for msp-1 and 32 for msp-2. For msp-1 the successful amplification occurred in (75/85) 88.23% isolates for msp-1, 78.9% (67/85) for msp-2 and 70% (60/85) for glurp. For msp-1, the K1 allelic family was predominant at 66.66% (50/75), followed by RO33 and MAD20. The frequency of samples having only K1, MAD20 and RO33 were 21.34% (16/75), 8% (6/75) and 10.67% (8/75) respectively. In msp-2, the FC27 allelic family was the most abundant with 70.14% (47/67) compared to 3D7 with 67.16% (45/67). Nine glurp RII region genotypes were identified. The overall mean multiplicity of infection was 2.6 with1.8 and 1.4 for msp-1 and msp-2 respectively while for glurp RII genes (MOI=1.03). There was no significant association between multiplicity of infection and age group (Spearman rank coefficient = 0.050; P = 0.6). There was significant correlation between MOI and parasite density for msp-2 allelic family. Conclusion: Our study showed high genetic diversity and allelic frequency with multiple clones of msp-1, msp-2 and glurp in P. falciparum isolates from malaria patients in Khyber Pakhtunkhwa Pakistan. In the present study the genotype data provided the valuable information which is essential for monitoring the impact of malaria eradication efforts in this region.

Identification of genomic regions affecting production traits in pigs divergently selected for feed efficiency

Background Feed efficiency is a major driver of the sustainability of pig production systems. Understanding the biological mechanisms that underlie these agronomic traits is an important issue for environment questions and farms' economy. This study aimed at identifying genomic regions that affect residual feed intake (RFI) and other production traits in two pig lines divergently selected for RFI during nine generations (LRFI, low RFI; HRFI, high RFI). Results We built a whole dataset of 570,447 single nucleotide polymorphisms (SNPs) in 2426 pigs with records for 24 production traits after both imputation and prediction of genotypes using pedigree information. Genome-wide association studies (GWAS) were performed including both lines (global-GWAS) or each line independently (LRFI-GWAS and HRFI-GWAS). Forty-five chromosomal regions were detected in the global-GWAS, whereas 28 and 42 regions were detected in the HRFI-GWAS and LRFI-GWAS, respectively. Among these 45 regions, only 13 were shared between at least two analyses, and only one was common between the three GWAS but it affects different traits. Among the five quantitative trait loci (QTL) detected for RFI, two were close to QTL for meat quality traits and two pinpointed novel genomic regions that harbor candidate genes involved in cell proliferation and differentiation processes of gastrointestinal tissues or in lipid metabolism-related signaling pathways. In most cases, different QTL regions were detected between the three designs, which suggests a strong impact of the dataset structure on the detection power and could be due to the changes in allelic frequencies during the establishment of lines. Conclusions In addition to efficiently detecting known and new QTL regions for feed efficiency, the combination of GWAS carried out per line or simultaneously using all individuals highlighted chromosomal regions that affect production traits and presented significant changes in allelic frequencies across generations. Further analyses are needed to estimate whether these regions correspond to traces of selection or result from genetic drift.

Interleukin 6 (rs1800795) and Pentraxin 3 (rs2305619) Polymorphisms - Association with Inflammation and All-Cause Mortality in End-stage-renal Disease Patients on Dialysis

Chronic inflammation plays an important role in the progression and outcome of chronic kidney disease (CKD). The inflammatory biomarkers interleukin-6 (IL6) and pentraxin 3 (PTX3) are enhanced in CKD patients and associated with progression of the disease and higher risk for cardiovascular events, the major cause of death in these patients. Our aim was to study how the polymorphisms of their encoding genes affect the inflammatory response and outcome of end-stage renal disease (ESRD) patients on dialysis. We analyzed two single nucleotide polymorphisms (SNP), the IL6 (rs1800795) polymorphism in the promoter region (-174G/C), and the PTX3 polymorphism in the intron 1 (+ 281A/G), in ESRD patients on dialysis and in heathy individuals. The allelic frequencies, genotype distribution and their association with the circulating levels of the inflammatory markers high sensitivity C-reactive protein (hsCRP), interleukin (IL6), growth differentiation factor 15 (GDF15) and PTX3, were determined in ESRD patients; events of death were recorded along one year to evaluate all-cause mortality and the association between inflammation and the studied polymorphisms. The allelic frequencies and genotyping distribution for IL6 and PTX3 in controls and ESRD patients were similar and in agreement with European reports. For the IL6 polymorphism, we found an association of the GG and CC genotype with higher IL6 levels; the CC genotype showed also high PTX3, hsCRP and GDF15 levels. For the PTX3 polymorphism, the AA genotype was linked to the highest values of hsCRP and IL6. The mortality rate after 1-year follow-up was 10.4%. The CC genotype (IL6 polymorphism), in deceased patients, was associated to increased levels of hsCRP, IL6 and PTX3, with low levels of GDF15 and with a highest mortality risk. The AA genotype for PTX3 polymorphism, in spite of the enhancement in inflammation, showed no significant impact on mortality. Our results show that the CC genotype of the IL6 polymorphism was associated with an enhanced inflammatory state and a poorer survival rate. Both IL6 and PTX3 polymorphisms seem to modulate the inflammatory response and, therefore, disease progression and outcome. Our data also highlights the importance of research on genetic variants that, although less frequent, may have significant biological value.

The Polymorphism Analyses of Short Tandem Repeats as a Basis for Understanding the Genetic Characteristics of the Guanzhong Han Population

The short tandem repeat (STR) loci are polymorphic markers in the combined DNA index system (CODIS) and non-CODIS STR loci. Due to the highly polymorphic characteristic of STR loci, they are popular and widely used in forensic DNA typing laboratories. In this study, 22 STR loci (1 CODIS, 21 non-CODIS STR loci) and an Amelogenin locus were genotyped and analyzed in 590 unrelated individuals of the Guanzhong Han population. None of the 22 STR loci deviated from the Hardy–Weinberg equilibrium, and all the loci were in the linkage equilibrium state. We observed 247 alleles, and the corresponding allelic frequencies ranged from 0.0008 to 0.3695 in the Guanzhong Han population. The combined power of discrimination and the cumulative exclusion probability was 0.999 999 999 999 999 999 999 999 999 346 36 and 0.999 999 999 709 74, respectively. The results including Nei’s D A genetic distance, multidimensional scaling analysis, and principal component analysis showed that the Guanzhong Han population has closer genetic affinities with Northern Han, Chengdu Han, and Xinjiang Hui groups from China based on allelic frequencies of 15 overlapped STR loci from Guanzhong Han and 13 reference groups. The present results indicated that Microreader™ 23sp ID kit included highly polymorphic loci, and it could be well used for individual identification, paternity testing, and population genetics in the Guanzhong Han population.