Identifying genes within pathways in unannotated genomes with PaGeSearch.

In biological research, the identification and comparison of genes within specific pathways across the genomes of various species are invaluable. However, annotating the entire genome is resource intensive, and sequence similarity searches often yield results that are not actually genes. To address these limitations, we introduce Pathway Gene Search (PaGeSearch), a tool designed to identify genes from predefined lists, especially those in specific pathways, within genomes. The tool uses an initial sequence similarity search to identify relevant genomic regions, followed by targeted gene prediction and neural network-based result filtering. PaGeSearch suggests the regions that are most likely the orthologs of the genes in the query and is designed to be applicable for species within five classes: mammals, fish, birds, eudicotyledons, and Liliopsida. Compared with GeMoMa and miniprot, PaGeSearch generally outperforms in terms of sensitivity and positive predictive value, as well as negative predictive value. Also, the exon coverage of gene models from PaGeSearch is higher compared with those in GeMoMa and miniprot. Although its performance shows increased variability when applied to actual biological pathways, it nonetheless maintains an acceptable level of accuracy. Evaluating PaGeSearch across different assembly levels, chromosome, scaffold, and contig shows minimal variation in outcomes, indicating that PaGeSearch is resilient to variations in assembly quality.

Selection and evaluation of bi-allelic autosomal SNP markers for paternity testing in Koreans.

Due to the advantages of single-nucleotide polymorphisms (SNPs) in forensic science, many forensic SNP panels have been developed. However, the existing SNP panels have a problem that they do not reflect allele frequencies in Koreans or the number of markers is not sufficient to perform paternity testing. Here, we filtered candidate SNPs from the Ansan-Ansung cohort data and selected 200 SNPs with high allele frequencies. To reduce the risk of false inclusion and false exclusion, we calculated likelihood ratios of alleged father-child pairs from simulated families when the alleged father is the true father, the close relative of the true father, and the random man. As a result, we estimated that 160 SNPs were needed to perform paternity testing. Furthermore, we performed validation using Twin-Family cohort data. When 160 selected SNPs were used to calculate the likelihood ratio, paternity and non-paternity were accurately distinguished. Our set of 160 SNPs could be useful for paternity testing in Koreans.

Whole-Transcriptome Sequencing-Based Analysis of DAZL and Its Interacting Genes during Germ Cells Specification and Zygotic Genome Activation in Chickens.

The deleted in azoospermia like (DAZL) is required for germ cells development and maintenance. In chickens, the mRNA and protein of DAZL, a representative maternally inherited germ plasm factor, are detected in the germ plasm of oocyte, zygote, and all stages of the intrauterine embryos. However, it is still insufficient to explain the origin and specification process of chicken germ cells, because the stage at which the zygotic transcription of DAZL occurs and the stage at which the maternal DAZL RNA/protein clears have not yet been fully identified. Moreover, a comprehensive understanding of the expression of DAZL interacting genes during the germ cells specification and development and zygotic genome activation (ZGA) is lacking in chickens. In this study, we identified a set of DAZL interacting genes in chickens using in silico prediction method. Then, we analyzed the whole-transcriptome sequencing (WTS)-based expression of DAZL and its interacting genes in the chicken oocyte, zygote, and Eyal-Giladi and Kochav (EGK) stage embryos (EGK.I to EGK.X). In the results, DAZL transcripts are increased in the zygote (onset of transcription), maintained the increased level until EGK.VI, and decreased from EGK.VIII (possible clearance of maternal RNAs). Among the DAZL interacting genes, most of them are increased either at 1st ZGA or 2nd ZGA, indicating their involvement in germ cells specification and development.

Identification of genes related to intramuscular fat content of pigs using genome-wide association study.

OBJECTIVE: The aim of this study is to identify single nucleotide polymorphisms (SNPs) and genes related to pig IMF and estimate the heritability of intramuscular fat content (IMF). METHODS: Genome-wide association study (GWAS) on 704 inbred Berkshires was performed for IMF. To consider the inbreeding among samples, associations of the SNPs with IMF were tested as random effects in a mixed linear model using the genetic relationship matrix by GEMMA. Significant genes were compared with reported pig IMF quantitative trait loci (QTL) regions and functional classification of the identified genes were also performed. Heritability of IMF was estimated by GCTA tool. RESULTS: Total 365 SNPs were found to be significant from a cutoff of p-value <0.01 and the 365 significant SNPs were annotated across 120 genes. Twenty five genes were on pig IMF QTL regions. Bone morphogenetic protein-binding endothelial cell precursor-derived regulator, forkhead box protein O1, ectodysplasin A receptor, ring finger protein 149, cluster of differentiation, tyrosine-protein phosphatase non-receptor type 1, SRY (sex determining region Y)-box 9 (SOX9), MYC proto-oncogene, and macrophage migration inhibitory factor were related to mitogen-activated protein kinase pathway, which regulates the differentiation to adipocytes. These genes and the genes mapped on QTLs could be the candidate genes affecting IMF. Heritability of IMF was estimated as 0.52, which was relatively high, suggesting that a considerable portion of the total variance of IMF is explained by the SNP information. CONCLUSION: Our results can contribute to breeding pigs with better IMF and therefore, producing pork with better sensory qualities.

The idiosyncratic genome of Korean long-tailed chicken as a valuable genetic resource.

Today, breeds with ornamental traits such as exceptionally long tail feathers are economically valuable. However, the genetic basis of long-tail feathers is yet to be understood. To provide better understanding of long tail feathers, we sequenced Korean long-tailed chicken (KLC) genomes and compared them with genomes of other chicken breeds. We first analyzed the genome structure of KLC and its genomic relationship with other chickens and observed unique characteristics. Subsequently, we searched for genomic regions under selection. Feather keratin 1-like enriched region and several genes were found to have novel putative functions and effects on the long tail trait in KLC. Our findings support the value of KLC as a unique genetic resource and cast light on the genetic basis of long tail traits in avian species. We expect this novel knowledge to provide new genomic evidence and options for designing and implementing genetic improvements of ornamental chicken productivity through precision crossbreeding aids.

Chicken blastoderms and primordial germ cells possess a higher expression of DNA repair genes and lower expression of apoptosis genes to preserve their genome stability.

DNA is susceptible to damage by various sources. When the DNA is damaged, the cell repairs the damage through an appropriate DNA repair pathway. When the cell fails to repair DNA damage, apoptosis is initiated. Although several genes are involved in five major DNA repair pathways and two major apoptosis pathways, a comprehensive understanding of those gene expression is not well-understood in chicken tissues. We performed whole-transcriptome sequencing (WTS) analysis in the chicken embryonic fibroblasts (CEFs), stage X blastoderms, and primordial germ cells (PGCs) to uncover this deficiency. Stage X blastoderms mostly consist of undifferentiated progenitor (pluripotent) cells that have the potency to differentiate into all cell types. PGCs are also undifferentiated progenitor cells that later differentiate into male and female germ cells. CEFs are differentiated and abundant somatic cells. Through WTS analysis, we identified that the DNA repair pathway genes were expressed more highly in blastoderms and high in PGCs than CEFs. Besides, the apoptosis pathway genes were expressed low in blastoderms and PGCs than CEFs. We have also examined the WTS-based expression profiling of candidate pluripotency regulating genes due to the conserved properties of blastoderms and PGCs. In the results, a limited number of pluripotency genes, especially the core transcriptional network, were detected higher in both blastoderms and PGCs than CEFs. Next, we treated the CEFs, blastoderm cells, and PGCs with hydrogen peroxide (H2O2) for 1 h to induce DNA damage. Then, the H2O2 treated cells were incubated in fresh media for 3-12 h to observe DNA repair. Subsequent analyses in treated cells found that blastoderm cells and PGCs were more likely to undergo apoptosis along with the loss of pluripotency and less likely to undergo DNA repair, contrasting with CEFs. These properties of blastoderms and PGCs should be necessary to preserve genome stability during the development of early embryos and germ cells, respectively.

False gene and chromosome losses in genome assemblies caused by GC content variation and repeats.

BACKGROUND: Many short-read genome assemblies have been found to be incomplete and contain mis-assemblies. The Vertebrate Genomes Project has been producing new reference genome assemblies with an emphasis on being as complete and error-free as possible, which requires utilizing long reads, long-range scaffolding data, new assembly algorithms, and manual curation. A more thorough evaluation of the recent references relative to prior assemblies can provide a detailed overview of the types and magnitude of improvements. RESULTS: Here we evaluate new vertebrate genome references relative to the previous assemblies for the same species and, in two cases, the same individuals, including a mammal (platypus), two birds (zebra finch, Anna's hummingbird), and a fish (climbing perch). We find that up to 11% of genomic sequence is entirely missing in the previous assemblies. In the Vertebrate Genomes Project zebra finch assembly, we identify eight new GC- and repeat-rich micro-chromosomes with high gene density. The impact of missing sequences is biased towards GC-rich 5'-proximal promoters and 5' exon regions of protein-coding genes and long non-coding RNAs. Between 26 and 60% of genes include structural or sequence errors that could lead to misunderstanding of their function when using the previous genome assemblies. CONCLUSIONS: Our findings reveal novel regulatory landscapes and protein coding sequences that have been greatly underestimated in previous assemblies and are now present in the Vertebrate Genomes Project reference genomes.

Corrigendum: Genomic Prediction Accuracy Using Haplotypes Defined by Size and Hierarchical Clustering Based on Linkage Disequilibrium.

[This corrects the article DOI: 10.3389/fgene.2020.00134.].

Haplotype-Based Genome-Wide Association Study and Identification of Candidate Genes Associated with Carcass Traits in Hanwoo Cattle.

Hanwoo, is the most popular native beef cattle in South Korea. Due to its extensive popularity, research is ongoing to enhance its carcass quality and marbling traits. In this study we conducted a haplotype-based genome-wide association study (GWAS) by constructing haplotype blocks by three methods: number of single nucleotide polymorphisms (SNPs) in a haplotype block (nsnp), length of genomic region in kb (Len) and linkage disequilibrium (LD). Significant haplotype blocks and genes associated with them were identified for carcass traits such as BFT (back fat thickness), EMA (eye Muscle area), CWT (carcass weight) and MS (marbling score). Gene-set enrichment analysis and functional annotation of genes in the significantly-associated loci revealed candidate genes, including PLCB1 and PLCB4 present on BTA13, coding for phospholipases, which might be important candidates for increasing fat deposition due to their role in lipid metabolism and adipogenesis. CEL (carboxyl ester lipase), a bile-salt activated lipase, responsible for lipid catabolic process was also identified within the significantly-associated haplotype block on BTA11. The results were validated in a different Hanwoo population. The genes and pathways identified in this study may serve as good candidates for improving carcass traits in Hanwoo cattle.

Genomic Prediction Accuracy Using Haplotypes Defined by Size and Hierarchical Clustering Based on Linkage Disequilibrium.

Genomic prediction is an effective way to estimate the genomic breeding values from genetic information based on statistical methods such as best linear unbiased prediction (BLUP). The used of haplotype, clusters of linked single nucleotide polymorphism (SNP) as markers instead of individual SNPs can improve the accuracy of genomic prediction. Since the probability of a quantitative trait loci to be in strong linkage disequilibrium (LD) with a cluster of markers is higher compared to an individual marker. To make haplotypes efficient in genomic prediction, finding optimal ways to define haplotypes is essential. In this study, 770K or 50K SNP chip data was collected from Hanwoo (Korean cattle) population consisted of 3,498 cattle. Using SNP chip data, haplotype was defined in three different ways based on 1) the number of SNPs included, 2) length of haplotypes (bp), and 3) agglomerative hierarchical clustering based on LD. To compare the methods in parallel, haplotypes defined by all methods were set to have comparable sizes; 5, 10, 20 or 50 SNPs on average per haplotype. A linear mixed model using haplotype to calculated the covariance matrix was applied for testing the prediction accuracy of each haplotype size. Also, conventional SNP-based linear mixed model was tested to evaluate the performance of the haplotype sets on genomic prediction. Carcass weight (CWT), eye muscle area (EMA) and backfat thickness (BFT) were used as the phenotypes. This study reveals that using haplotypes generally showed increased accuracy compared to conventional SNP-based model for CWT and EMA, but found to be small or no increase in accuracy for BFT. LD clustering-based haplotypes specifically the five SNPs size showed the highest prediction accuracy for CWT and EMA. Meanwhile, the highest accuracy was obtained when length-based haplotypes with five SNPs were used for BFT. The maximum gain in accuracy was 1.3% from cross-validation and 4.6% from forward validation for EMA, suggesting that genomic prediction accuracy can be increased by using haplotypes. However, the improvement from using haplotypes may depend on the trait of interest. In addition, when the number of alleles generated by each haplotype defining methods was compared, clustering by LD generated the least number of alleles, thereby reducing computational costs. Therefore, finding optimal ways to define haplotypes and using the haplotype alleles as markers can improve the accuracy of genomic prediction.