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The Asian honey bee species i.e., Apis cerana (the eastern honey bee), A. dorsata (the giant honey bee), and the western or European honey bee (A. mellifera) collected from Pakistan were studied using partial sequences from two mitochondrial genes (i) the Cytochrome c oxidase I (COI) and (ii) the mitochondrially encoded NADH dehydrogenase 5 (ND5) and then compared with other honey bees sequences (already submitted from different countries around the globe) obtained after the national center for biotechnology information (NCBI). DNA sequences were analyzed employing molecular evolutionary genetics analysis and Kimura 2-parameter model, neighbor-joining method was applied to investigate phylogenetic relationships, and DNA sequence polymorphism was applied to measure the genetic diversity within the genus Apis. The phylogenetic analyses yielded consistent results. Based on COI gene fragment in two Asian and European honey bee species from Pakistan and from other countries showed considerable genetic diversity levels and deviation among the species. While in contrast the phylogenetic analyses based on ND5 gene fragment in Asian and European honey bee species from Pakistan and other countries showed comparatively higher genetic diversity indices and variations than the COI gene. So, in the genus Apis, the mitochondrial ND5 region has shown the possibility to answer the interactions among species. A further detailed work (by linking the analysis of other genomic and mitochondrial genes) is required for good quality solution to establish the concise genetic diversity and interaction among the Apis species. The objective of this study was to explore the extent of genetic differences and phylogenetic links among the three kinds of honey bee species from Pakistan and comparing them with other bee species around the globe.

Objectives: We determined the whole DNA sequences of plasmids carrying a rare extended-spectrum beta-lactamase gene (bla(TEM-61)) to precisely understand the spread of resistance among nosocomial Serratia marcescens populations. Methods: Twenty non-duplicate ceftazidime-resistant S. marcescens nosocomial isolates (ceftazidime MICs, 32 to >128 mg/L) collected over 1 year were pulsotyped and nucleotide sequences of the bla(TEM-61) gene and its promoter region were determined. Twelve representative isolates were analysed by whole-genome sequencing. Results: The 20 isolates comprised two distinct pulsotypes: I (14 isolates) and II (6 isolates). They all contained the bla(TEM-61) gene. A polymorphism in the repeat number of a 15-nucleotide sequence (5'-ATGTCATGATAATAA-3') was found in the promoter region of bla(TEM-61); two, three and four repeat units were found in 6,12 and 2 isolates, respectively. Single nucleotide polymorphism (SNP)-based phylogenetic analysis of 12 isolates revealed that 7 isolates of pulsotype I (12-44 SNP differences) and 5 isolates of pulsotype II (15-55 SNP differences) formed two distinct clusters of genotypes 1 and 2, respectively. All 12 isolates harboured a plasmid carrying the Tn1-bla(TEM-61) element, although they were slightly different in size (78 883 bp, 78 898 bp and 78 913 bp) owing to differences in the number of 15-bp repetitive sequences. A 42 542-bp broad-host-range plasmid carrying the Tn1-bla(TEM-61) element was also found in one of the isolates. Conclusions: We characterised a plasmid-encoded novel Tn1-bla(TEM-61) element and transposon-dependent mechanisms underlying the propagation of antibiotic resistance, together with repeated new polymorphic 15-bp units in the promoter of bla(TEM-61). (C) 2021 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy., Article, Journal of Global Antimicrobial Resistance 25 : 124-131(2021)

Aconitum carmichaelii is a high-value medicinal herb widely used across China, Japan, and other Asian countries. Aconitine-type diterpene alkaloids (DAs) are the characteristic compounds in Aconitum. Although six transcriptomes, based on short-read next generation sequencing technology, have been reported from the Aconitum species, the terpene synthase (TPS) corresponding to DAs biosynthesis remains unidentified. We apply a combination of Pacbio isoform sequencing and RNA sequencing to provide a comprehensive view of the A. carmichaelii transcriptome. Nineteen TPSs and five alternative splicing isoforms belonging to TPS-b, TPS-c, and TPS-e/f subfamilies were identified. In vitro enzyme reaction analysis functional identified two sesqui-TPSs and twelve diTPSs. Seven of the TPS-c subfamily genes reacted with GGPP to produce the intermediate ent-copalyl diphosphate. Five AcKSLs separately reacted with ent-CPP to produce ent-kaurene, ent-atiserene, and ent-13-epi-sandaracopimaradie: a new diterpene found in Aconitum. AcTPSs gene expression in conjunction DAs content analysis in different tissues validated that ent-CPP is the sole precursor to all DAs biosynthesis, with AcKSL1, AcKSL2s and AcKSL3-1 responsible for C20 atisine and napelline type DAs biosynthesis, respectively. These data clarified the molecular basis for the C20-DAs biosynthetic pathway in A. carmichaelii and pave the way for further exploration of C19-DAs biosynthesis in the Aconitum species., Graphical abstract A Pacbio full-length transcriptome of Aconitum carmichaelii were publicly available. Twelve diterpene synthases were functional identified to responsible for atisine, napelline type diterpene alkaloids (DAs) and ent-13-epi-sandaracopimaradie biosynthesis, respectively.Image 1

The development of next generation sequencing techniques has facilitated the detection of mutations at an unprecedented rate. These efficient tools have been particularly beneficial for extremely heterogeneous disorders such as autosomal recessive non-syndromic hearing loss, the most common form of genetic deafness. GJB2 mutations are the most common cause of hereditary hearing loss. Amongst them the NM_004004.5: c.506G > A (p.Cys169Tyr) mutation has been associated with varying severity of hearing loss with unclear segregation patterns. In this study, we report a large consanguineous Emirati family with severe to profound hearing loss fully segregating the GJB2 missense mutation p.Cys169Tyr. Whole exome sequencing (WES), in silico, splicing and expression analyses ruled out the implication of any other variants and confirmed the implication of the p.Cys169Tyr mutation in this deafness family. We also show preliminary murine expression analysis that suggests a link between the TMEM59 gene and the hearing process. The present study improves our understanding of the molecular pathogenesis of hearing loss. It also emphasizes the significance of combining next generation sequencing approaches and segregation analyses especially in the diagnosis of disorders characterized by complex genetic heterogeneity.

Graphical abstract, Purpose Polydactyly is a highly heterogeneous group of skeletal deformities in clinical and genetic background. The variation spectrum in Chinese sporadic polydactyly has not been comprehensively analyzed. To elucidate genetic variation spectrum and genotype-phenotype correlations in Chinese patients with polydactyly, we conducted comprehensive genetic analysis of patients nationwide using targeted sequencing. Methods A total of 181 patients diagnosed with polydactylies were recruited. We designed a targeted capture panel for sequencing 721 genes that are associated with the pathogenesis of skeletal dysplasia. We performed rigorous variant- and gene-level filtrations to identify potentially damaging variants, followed by enrichment analysis and gene prioritization. Results A total of 568 deleterious variants of 293 genes were identified in 173 of 181 patients with a positive rate of 95.6% by targeted sequencing. For each sample, an average of 3.17 deleterious variants were identified. Especially, 14 pathogenic or likely pathogenic variants were identified in 10 genes in 14 patients out of the 181 patients, providing a positive molecular diagnostic rate of 7.7%. Conclusion Targeted sequencing analysis provides a high efficiency approach for the genetic diagnosis of polydactyly. This is the largest next generation sequencing study performed to date in patients with polydactyly and represents the genetic basis of polydactyly typically encountered in genetics clinics.

Infectious bursal disease (IBD), an acute, highly contagious, and immunosuppressive avian disease, is caused by infectious bursal disease virus (IBDV) and constitutes one of the main threats to the poultry industry, worldwide. This study was performed to isolate and characterize IBDV isolates circulating in Tunisia. Eleven collected bird samples were identified using an SYBR Green–based one-step real-time reverse transcriptase polymerase chain reaction. The full-length genome sequencing of 7 of the 11 IBDV isolates has been realized. VP2 gene data showed limited sequence variations for all the 7 tested samples. The few nucleotide changes were silent and the deduced amino acid sequences were identical with the exception of a unique and characteristic nonsilent mutation (C1203) detected for the TN37/19 isolate, with a change of amino acid (L) to (F) at position 401. In addition, the serine-rich heptapeptide SWSASGS, characteristic of virulent IBDV, as well the amino acid residues, conserved in most very virulent IBDV (vvIBDV) strains, were detected in all the Tunisian tested isolates. Nucleotide sequences of VP5 gene revealed the presence of 5 substitutions leading to changes in the amino acid sequences of the virus. Two of these mutations were unique and characteristic of the Tunisian isolates. Besides, the alternative AUG start codon, characteristic of vvIBDV, was observed in all obtained VP5 gene sequences. The Tunisian protein sequences of VP1 showed E242 and the TDN triplet at positions 145, 146, and 147, a motif specific of vvIBDV. Phylogenetic analyses of the 5 genes confirmed the sequence alignment results and showed that the Tunisian strains are closely related to the very virulent Algerian IBDV strains.

Transcription factors (TFs) as key regulators play crucial roles in biological processes. The identification of TF–target regulatory relationships is a key step for revealing functions of TFs and their regulations on gene expression. The accumulated data of chromatin immunoprecipitation sequencing (ChIP-seq) provide great opportunities to discover the TF–target regulations across different conditions. In this study, we constructed a database named hTFtarget, which integrated huge human TF target resources (7190 ChIP-seq samples of 659 TFs and high-confidence binding sites of 699 TFs) and epigenetic modification information to predict accurate TF–target regulations. hTFtarget offers the following functions for users to explore TF–target regulations: (1) browse or search general targets of a query TF across datasets; (2) browse TF–target regulations for a query TF in a specific dataset or tissue; (3) search potential TFs for a given target gene or non-coding RNA; (4) investigate co-association between TFs in cell lines; (5) explore potential co-regulations for given target genes or TFs; (6) predict candidate TF binding sites on given DNA sequences; (7) visualize ChIP-seq peaks for different TFs and conditions in a genome browser. hTFtarget provides a comprehensive, reliable and user-friendly resource for exploring human TF–target regulations, which will be very useful for a wide range of users in the TF and gene expression regulation community. hTFtarget is available at http://bioinfo.life.hust.edu.cn/hTFtarget.

Background: Whole genome sequencing has revolutionized epidemiological investigations of multidrug-resistant pathogenic bacteria worldwide. Aim of this study was to perform comprehensive characterization of ESBL-positive isolates of Escherichia coli obtained from clinical samples at the King Abdulaziz University Hospital utilizing whole genome sequencing. Methods: Isolates were identified by MALDI-TOF mass spectrometry. Genome sequencing was performed using a paired-end strategy on the MiSeq platform. Results: Nineteen isolates were clustered into different clades in a phylogenetic tree based on single nucleotide polymorphisms in core genomes. Seventeen sequence types were identified in the extended-spectrum β-lactamase (ESBL)-positive isolates, and 11 subtypes were identified based on distinct types of fimH alleles. Forty-one acquired resistance genes were found in the 19 genomes. The blaCTX-M-15 gene, which encodes ESBL, was found in 15 isolates and was the most predominant resistance gene. Other antimicrobial resistance genes (ARGs) found in the isolates were associated with resistance to tetracycline (tetA), aminoglycoside [aph(3″)-Ib, and aph(6)-Id], and sulfonamide (sul1, and sul2). Nonsynonymous chromosomal mutations in the housekeeping genes parC and gyrA were commonly found in several genomes. Conclusion: Several other ARGs were found in CTX-M-positive E. coli isolates confer resistance to clinically important antibiotics used to treat infections caused by Gram-negative bacteria. Keywords: Genome, Antimicrobial resistance gene, Virulence gene, Insertion sequences, Escherichia coli, Saudi Arabia

Graphical abstract, Microsatellite instability (MSI) is a genomic property of the cancers with defective DNA mismatch repair and is a useful marker for cancer diagnosis and treatment in diverse cancer types. In particular, MSI has been associated with the active immune checkpoint blockade therapy response in cancer. Most of computational methods for predicting MSI are based on DNA sequencing data and a few are based on mRNA expression data. Using the RNA-Seq pan-cancer datasets for three cancer cohorts (colon, gastric, and endometrial cancers) from The Cancer Genome Atlas (TCGA) program, we developed an algorithm (PreMSIm) for predicting MSI from the expression profiling of a 15-gene panel in cancer. We demonstrated that PreMSIm had high prediction performance in predicting MSI in most cases using both RNA-Seq and microarray gene expression datasets. Moreover, PreMSIm displayed superior or comparable performance versus other DNA or mRNA-based methods. We conclude that PreMSIm has the potential to provide an alternative approach for identifying MSI in cancer.

Graphical abstract, Highlights • A computational method for identifying non-coding promoters was proposed for the first time. • A high-quality dataset was built to train and test the models for identifying non-coding promoters. • A user-friendly web server was developed to recognize non-coding promoters., The promoter is located near the transcription start sites and regulates transcription initiation of the gene. Accurate identification of promoters is essential for understanding the mechanism of gene regulation. Since experimental methods are costly and ineffective, developing efficient and accurate computational tools to identify promoters are necessary. Although a series of methods have been proposed for identifying promoters, none of them is able to identify the promoters of non-coding RNA (ncRNA). In the present work, a new method called ncPro-ML was proposed to identify the promoter of ncRNA in Homo sapiens and Mus musculus, in which different kinds of sequence encoding schemes were used to convert DNA sequences into feature vectors. To test the length effect, for each species, datasets including sequences with different lengths were built. The results demonstrated that ncPro-ML achieved the best performance based on the dataset with the sequence length of 221 nucleotides for human and mouse. The performances of ncPro-ML were also satisfying from both independent dataset test and cross-species test. The results indicate that the proposed predictor can server as a powerful tool for the discovery of ncRNA promoters. In addition, a web-server for ncPro-ML was developed, which can be freely accessed at http://www.bio-bigdata.cn/ncPro-ML/.

Severe hypertriglyceridemia (HTG) is a relatively common form of dyslipidemia with a complex pathophysiology and serious health complications. HTG can develop in the presence of rare genetic factors disrupting genes involved in the triglyceride (TG) metabolic pathway, including large-scale copy-number variants (CNVs). Improvements in next-generation sequencing technologies and bioinformatic analyses have better allowed assessment of CNVs as possible causes of or contributors to severe HTG. We screened targeted sequencing data of 632 patients with severe HTG and identified partial deletions of the LPL gene, encoding the central enzyme involved in the metabolism of TG-rich lipoproteins, in four individuals (0.63%). We confirmed the genomic breakpoints in each patient with Sanger sequencing. Three patients carried an identical heterozygous deletion spanning the 5′ untranslated region (UTR) to LPL exon 2, and one patient carried a heterozygous deletion spanning the 5′UTR to LPL exon 1. All four heterozygous CNV carriers were determined to have multifactorial severe HTG. The predicted null nature of our identified LPL deletions may contribute to relatively higher TG levels and a more severe clinical phenotype than other forms of genetic variation associated with the disease, particularly in the polygenic state. The identification of novel CNVs in patients with severe HTG suggests that methods for CNV detection should be included in the diagnostic workup and molecular genetic evaluation of patients with high TG levels.

Highlights • 48 SNPs were identified from the genome-wide analysis of 410 South Asian SARS-CoV-2 sequences. • About 85% SNPs are packaged in ORF1ab, spike protein, and nucleocapsid. • South Asian strains are highly related to the South American and European strains according to the phylogenetic analysis. • Unlike other countries, frequency of 1163A>T missense mutation is very high (78.80%) in Bangladeshi samples., Counts for SARS-CoV-2 associated infections and fatalities are on the rise globally even in regions which contained the spread momentarily. The pattern of infections has been found to be controlled by the distinctive selection pressures exerted by fluctuating environmental nature and hosts. A total of 410 whole-genome sequences submitted by the South Asian countries were retrieved from the GISAID database and analyzed to assess the impact and pattern of mutations in this region. Most common and frequent mutations in the South Asian countries are 241C > T, 3037C > T, 14408C > T, and 23403A > G and about 85% SNPs are localized in ORF1ab, spike protein, and nucleocapsid. Among the identified mutations, the proportion of missense type (54.17%) was highest, followed by the synonymous (41.66%) and the non-coding types (4.17%). While analyzing transmission source in terms of geolocation, the largest clustered group from the South Asian countries was based on the G-clade (D614G) (81.7%; 335/410 samples), tracing the inception and transmission of SARS-CoV-2 infections in the South Asian countries from European regions. Phylogenetic analysis also revealed that the South Asian strains are highly related to the South American and European strains. We found that G-clade mutations are more prevalent (96.19%) in the samples of Bangladesh which were also prevalent in the European isolates. Surprisingly, one missense mutation (1163A > T) in ORF1ab gene became dominant only in Bangladesh (78.8%), which led to debates regarding effects on the pathogenicity and transmissibility of the virus. Overall, the findings of this study highlight the frequently mutated SARS-CoV-2 variants among the COVID-19 patients in the South Asian countries which might ease containment of the disease in this region through investigating the virulence reducing factors as the identified mutations are strongly correlated with low infection and mortality rate.

Chromobacterium violaceum is a gram-negative, facultative anaerobic bacillus which is commonly found in soil. It can cause mild diarrhoea upon infection but can progress, although rarely, to fatal multi-organ failure and death. Here we report the whole genome sequence data of Chromobacterium violaceum WCH4 strain, a pathogenic strain that was obtained from a 78 year old male patient suffering from an eye infection. This is a rare case of human infection of the bacteria. Blood culture report as well as 16S rRNA confirmed the presence of C. violaceum WCH4. DNA sequencing using the Illumina HiSeq 4000 system revealed a genome size of 4,637,406 bp with a GC-content of 64.89%. We identified 4,572 protein coding sequences (CDS), 78 transfer RNA (tRNA) genes, and 3 ribosomal RNA (rRNA) genes. The CDS included 1,261 hypothetical proteins and 3,311 proteins with functional assignments. We also identified seven putative genes involved in efflux pump and conferring multidrug antibiotic resistance. The genome data has been deposited at NCBI under the accession number JAFBBB000000000 and consist of full annotated genome and raw sequence data. Our data resource will assist in further downstream analysis and understanding of the mechanism of rare human infection caused by Chromobacterium violaceum WCH4 strain.

We present new complete mitogenome sequences of Silurus glanis (S. glanis) from 4 samples such as male and female individuals from two countries (Hungary, Czech Republic). The complete mitochondria were determined from genome sequencing by using Illumina MiSeq platform resulting in long, 300 bp. paired-end reads. De novo assembly was performed resulting in one nod (scaffold) covering the total mitochondria in each sample. The mitochondrial genomes were circular, double-stranded molecules of 16,524 bp in length and consisted of 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 control region. These sequences were deposited in the NCBI GeneBank under the accession numbers (MW796040, MW796041, MW796042, MW796043) and compared with the only available S. glanis mitochondrial genome (NC_014261.1) sequenced by unidentified technology and showed 99% similarity. We found in seq1 82, in seq2 82, seq3 83, seq4 82 nucleotide alterations involving 10 protein-coding genes and meaning 29 amino acid substitutions as well.

Summary Identifying the molecular mechanisms related to genomic breakage is an important goal of cancer mechanism studies. Among diverse locations of structural variants, fusion genes, which have the breakpoints in the gene bodies and are typically identified from the split reads of RNA-seq data, can provide a highlighted structural variant resource for studying the genomic breakages with expression and potential pathogenic impacts. In this study, we developed FusionAI, which utilizes deep learning to predict gene fusion breakpoints based on DNA sequence and let us identify fusion breakage code and genomic context. FusionAI leverages the known fusion breakpoints to provide a prediction model of the fusion genes from the primary genomic sequences via deep learning, thereby helping researchers a more accurate selection of fusion genes and better understand genomic breakage., Graphical abstract, Highlights • FusionAI predicts fusion gene breakpoints from a DNA sequence • FusonAI reduce the effort for validating fusion genes with other tools • High feature importance regions were apart 100nt from the exon junction BPs • High feature importance regions were overlapped with 44 human genomic features, Genetics; Genomics; Computational bioinformatics; Artificial intelligence applications

Congenital Disorders of Glycosylation (CDG) are an expanding and complex group of rare genetic disorders caused by defects in the glycosylation of proteins and lipids. The genetic spectrum of CDG is extremely broad with mutations in over 140 genes leading to a wide variety of symptoms ranging from mild to severe and life-threatening. There has been an expansion in the genetic complexity of CDG in recent years. More specifically several examples of alternate phenotypes in recessive forms of CDG and new types of CDG following an autosomal dominant inheritance pattern have been identified. In addition, novel genetic mechanisms such as expansion repeats have been reported and several already known disorders have been classified as CDG as their pathophysiology was better elucidated. Furthermore, we consider the future and outlook of CDG genetics, with a focus on exploration of the non-coding genome using whole genome sequencing, RNA-seq and multi-omics technology. ispartof: BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS vol:1865 issue:11 ispartof: location:Netherlands status: published

Trio-next generation sequencing is useful to identify undiagnosed inherited diseases. We have attended a patient with trigenic ADH5/ALDH2/ADGRV1 pathogenic variants, which caused two distinct diseases, myelodysplastic syndrome and Usher syndrome. Whole genome sequencing of peripheral blood from the patient and his parents were applied to identify disease-causing genes. Sanger sequencing was performed to validate the identified ADH5/ALDH2/ADGRV1 variants. Our results identified disease-associated variants in ADGRV1 (disease inheritance autosomal recessive) and in ADH5 (disease inheritance also autosomal recessive) and a variant in ALDH2 (disease inheritance autosomal dominant). Although the variants identified in ADH5 and ALDH2 have been reported, their co-existence in association with disease-causing variation in a third gene has not. They broaden the spectrum of ADGRV1 in Usher syndrome. Findings on next generation sequencing guided rapid and accurate diagnosis, resulting in patient-tailored therapeutic intervention., Highlights • Trigenic ADH5 / ALDH2 / ADGRV1 ​variants in myelodysplastic syndrome with Usher syndrome were identified. • Two novel pathogenic frameshift variants in ​ADGRV1 ​in compound heterozygous state with Usher syndrome type II were described. • Findings on next generation sequencing guided rapid and accurate diagnosis, resulting in patient-tailored therapy., ADH5/ALDH/ADGRV1 variants, Myelodysplastic syndrome, Trigenic mutations, Trio-next generation sequencing, Usher syndrome.

Loss-of-function mutations in Na+/H + exchanger 6 (NHE6) (also termed SLC9A6) cause the X-linked neurogenetic disorder Christianson syndrome (CS). Using peripheral blood mononuclear cells, we developed induced pluripotent stem cell (iPSC) lines from a patient with the NHE6 nonsense mutation c.1569G > A (p.(W523X)) and diagnosed with CS and from a biologically-related control. Using CRISPR/Cas9 gene editing, we generated two isogenic control lines in which the c.1569G > A mutation was corrected. All lines were verified by DNA sequencing and for NHE6 protein expression, pluripotency, and differentiation potential. These lines will serve as a valuable resource for both basic and translational studies in CS.

Summary CRISPR interference is an increasingly popular method for perturbing gene expression. Guided by single-guide RNAs (sgRNAs), nuclease-deficient Cas9 proteins bind to specific DNA sequences and hinder transcription. Specificity is achieved through complementarity of the sgRNAs to the DNA. Changing complementarity by introducing single-nucleotide mismatches can be exploited to tune knockdown. Here, we present a computational pipeline to identify sgRNAs targeting specific genes in a bacterial genome, filter them, and titrate their activity by introducing mismatches. For complete details on the use and execution of this protocol, please refer to Hawkins et al. (2020)., Graphical abstract, Highlights • Identify all CRISPR interference targets for a bacterial gene or genome of interest • Filter associated sgRNAs based on few simple metrics such as potential off-target hits • Predict knockdown efficacies of mismatch sgRNAs for introducing graded knockdowns, CRISPR interference is an increasingly popular method for perturbing gene expression. Guided by single-guide RNAs (sgRNAs), nuclease-deficient Cas9 proteins bind to specific DNA sequences and hinder transcription. Specificity is achieved through complementarity of the sgRNAs to the DNA. Changing complementarity by introducing single-nucleotide mismatches can be exploited to tune knockdown. Here, we present a computational pipeline to identify sgRNAs targeting specific genes in a bacterial genome, filter them, and titrate their activity by introducing mismatches.

Objectives A multidrug-resistant strain of the opportunistic pathogen Streptococcus sanguis (S28) was isolated from a throat swab of a child with scarlet fever as a rare case. Genome sequencing and analysis of strain S28 were performed to gain a better understanding of the multidrug resistance mechanisms of S. sanguis and its relationship with scarlet fever. Methods The genome of S. sanguis S28 was sequenced on a Illumina HiSeq platform. Genome assembly was conducted using SOAPdenovo v.2.04 and the assembled genome sequence was submitted to NCBI for annotation. Results The 1 268 358 696 bp genome of S. sanguis S28 contains 2287 coding sequences (CDS) with a GC content of 43.2%. Strain S28 possesses four antimicrobial resistance genes (ARGs), which is consistent with phenotypic analysis. A novel transposon with three genes conferring resistance to macrolide–lincosamide–streptogramin B (MLSB) antibiotics, tetracyclines and aminoglycosides was discovered. Conclusion To our knowledge, this is the first report of a S. sanguis genome isolated from a throat swab of a child with scarlet fever and the first report of a transposon with three activated ARGs conferring resistance to MLSB, tetracyclines and aminoglycosides together.

Summary Pig has been proved to be a valuable large animal model used for research on diabetic disease. However, their translational value is limited given their distinct anatomy and physiology. For the last 30 years, we have been developing a laboratory Asian miniature pig inbred line (Bama miniature pig [BM]) from the primitive Bama xiang pig via long-term selective inbreeding. Here, we assembled a BM reference genome at full chromosome-scale resolution with a total length of 2.49 Gb. Comparative and evolutionary genomic analyses identified numerous variations between the BM and commercial pig (Duroc), particularly those in the genetic loci associated with the features advantageous to diabetes studies. Resequencing analyses revealed many differentiated gene loci associated with inbreeding and other selective forces. These together with transcriptome analyses of diabetic pig models provide a comprehensive genetic basis for resistance to diabetogenic environment, especially related to energy metabolism., Graphical Abstract, Highlights • Bama miniature pig (BM) is one of the pig lines with the highest inbreeding coefficient • This atlas is a report on the chromosome-level genome assembly of miniature pig • Genomic analyses revealed genetic basis underlying BM's advantages to study diabetes • Some lncRNAs and mRNAs may be linked to resistance to diabetogenic environment, Biological Sciences; Gene Ontology; Genomic Analysis; Genomics; Omics; Sequence Analysis; Transcriptomics

Graphical abstract, Genetically modified model organisms are valuable tools for probing gene function, dissecting complex signaling networks, studying human disease, and more. CRISPR/Cas9 technology has significantly democratized and reduced the time and cost of generating genetically modified models to the point that small gene edits are now routinely and efficiently generated in as little as two months. However, generation of larger and more sophisticated gene-modifications continues to be inefficient. Alternative ways to provide the replacement DNA sequence, method of Cas9 delivery, and tethering the template sequence to Cas9 or the guide RNA (gRNA) have all been tested in an effort to maximize homology-directed repair for precise modification of the genome. We present two CRISPR/Cas9 methods that have been used to successfully generate large and complex gene-edits in mouse. In the first method, the Cas9 enzyme is used in conjunction with two sgRNAs and a long single-stranded DNA (lssDNA) template prepared by an alternative protocol. The second method utilizes a tethering approach to couple a biotinylated, double-stranded DNA (dsDNA) template to a Cas9-streptavidin fusion protein. • Alternative method for generating long, single-stranded DNA templates for CRISPR/Cas9 editing. • Demonstration that using two sgRNAs with Cas9-streptavidin/biotinylated-dsDNA is feasible for large DNA modifications.

Taihu pig breeds are the most prolific breeds of swine in the world, and they also have superior economic traits, including high resistance to disease, superior meat quality, high resistance to crude feed and a docile temperament. The formation of these phenotypic characteristics is largely a result of long-term artificial or natural selection. Therefore, exploring selection signatures in the genomes of the Taihu pigs will help us to identify porcine genes related to productivity traits, disease and behaviour. In this study, we used both intra-population (Relative Extend Haplotype Homozygosity Test (REHH)) and inter-population (the Cross-Population Extend Haplotype Homozygosity Test (XPEHH); F-STATISTICS, F ST ) methods to detect genomic regions that might be under selection process in Taihu pig breeds. As a result, we found 282 (REHH) and 112 (XPEHH) selection signature candidate regions corresponding to 159.78 Mb (6.15%) and 62.29 Mb (2.40%) genomic regions, respectively. Further investigations of the selection candidate regions revealed that many genes under these genomic regions were related to reproductive traits (such as the TLR9 gene), coat colour (such as the KIT gene) and fat metabolism (such as the CPT1A and MAML3 genes). Furthermore, gene enrichment analyses showed that genes under the selection candidate regions were significantly over-represented in pathways related to diseases, such as autoimmune thyroid and asthma diseases. In conclusion, several candidate genes potentially under positive selection were involved in characteristics of Taihu pig. These results will further allow us to better understand the mechanisms of selection in pig breeding.

From the beginning of the genome sequencing era, it has become increasingly evident that genetics plays a role in all diseases, of which only a minority are single-gene disorders, the most common target of current gene therapies. However, the majority of people have some kind of health problems resulting from congenital genetic mutations (over 6000 diseases have been associated to genes, https://www.omim.org/statistics/geneMap) and most genetic disorders are rare and only incompletely understood. The vision and techniques applied to the synthesis of genomes may help to address unmet medical needs from a chromosome and genome-scale perspective. In this chapter, we address the potential therapy of genetic diseases from a different outlook, in which we no longer focus on small gene corrections but on higher-order tools for genome manipulation. These will play a crucial role in the next years, as they prelude to a much deeper understanding of the architecture of the human genome and a more accurate modeling of human diseases, offering new therapeutic opportunities.

The structure and function of every living organism in the biosphere are shaped and directed by its genome, respectively. The genome of an organism is an abode of all the genes and other DNA segments, known and yet to be known, responsible for its metabolism, survival, growth, development, interaction with the environment, defense, reproduction, senescence, etc. To exploit any species as a biological resource at molecular level to serve human kind in form of good(s) and/or service(s), primarily requires a thorough understanding of its genes and their locations in the genome. Genome mapping is a technique that aids in determining the location of genes, and/or its(or their) markers(s), of interest in relation to other gene(s), and/or its (or their) marker(s), within the genome. Since all the genes of commercial interest are nor present in the same individual/taxa, it is essential to move gene(s) of interest from available source(s) to the required target(s), which is made easy with the knowledge of genome map. Genome mapping has wider applications in modern molecular biology: from the applied areas, such as genetic improvement of organisms, to the fundamental research such as genome sequencing. It needs proper tools and techniques such as various kinds of markers and mapping populations. To choose an appropriate tool and method requires thorough knowledge of myriad basket of available genome mapping tools. This chapter brings a complete, comprehensive and updated information that is necessary to understand genome of interest and use appropriate mapping tools, such as markers and mapping populations, for mapping of genes of interest in the genome of an organism of interest, ultimately to help deriving useful product(s) and/or service(s) to meet various needs of humankind in the wake of everchanging environment and never-receding human populations. This chapter also offers future prospects of available genome mapping tools and opportunities to develop new tools in the light of advanced techniques available for DNA sequencing and rapid generation advancement of mapping populations.

Biotechnology has greatly contributed to the improvement of productivity and quality of agricultural and food products. A particularly powerful approach in molecular tools has been genome sequencing and sequencing of individual genes and markers. Genomic tools have been used to explain the genetic basis of variation underlying important traits such as quality of food, yield, and tolerance to biotic and abiotic stress. Nanopore technologies offer a unique way to sequence deoxyribonucleic acid (DNA) and allow for very long strands of DNA to be processed into long-sequence reads. These long reads have several advantages that can be leveraged to improve crop and stock species as well as provide some unique DNA testing methods to identify the species origin of food products or detect locations of genetic modification. Nanopore sequencing involves passing the DNA through the nanopore and detecting each base as it passes through. Types of nanopore that are being investigated are solid-state nanopores and protein-based nanopores, with protein-based nanopores being the only type that is commercially available. In this chapter, we discuss the history of sequencing technologies, the background of nanopore sequencing, and its applications in agriculture and food testing that are currently used for de novo genome assembly, genotyping, transcriptomics, metagenomics, and targeted sequencing.

The genome sequencing revealed that there are limited numbers of protein-coding genes in a genome and that the total size of protein-coding sequences is a small portion of the genome. Coding transcripts, i.e. mRNAs, have the capability to codify proteins but are a small portion. Besides mRNAs, other transcripts do not encode for proteins and hence called non-coding RNAs (ncRNAs) (Cheng et al., 2005; Eddy, 2001; Shahrouki and Larsson, 2012; Todd and Karbstein, 2007). The existing evidence demonstrated that ncRNAs present in all organisms and have a wide range of functions, and hence, are important in biological and biomedical research. Currently, most of these ncRNAs have yet to be biochemically characterized, but the ncRNA field has received great attention in recent years from the general research community, and it shows that ncRNAs have a much richer functional spectrum than anticipated (Amaral et al., 2008; Brosius, 2005; Chadwick and Scott, 2013; Palazzo and Lee, 2015). The progress has been reviewed (Batista and Chang, 2013; Esteller, 2011; Rinn and Chang, 2012; Yang et al., 2014). The rapid advance in genomics technologies and bioinformatics approaches accelerated the ncRNA researches. Especially, in food science, it deserves to discover and characterize ncRNAs in livestock and various studies using modern genomics and bioinformatics methods. In this chapter, methods, software tools, and related databases for discovery and characterization of non-coding RNA, including small RNAs, long non-coding RNAs, and circular RNAs, were reviewed.

It is generally agreed that disease features of systemic lupus erythematosus (SLE) manifest in genetically predisposed individuals after encountering environmental triggers. Genome wide association studies provide an unbiased approach to screening hundreds of thousands of single nucleotide polymorphisms and have enhanced the discovery of genetic risk loci associated with SLE. Further advancements in the field (large-scale replication studies, meta-analysis, next generation sequencing, fine mapping of disease characteristics, and functional studies) have led a rapid increase in the discovery of SLE risk variants, approaching 150 loci identified. Although functions of a number of identified loci remain unknown, most risk gene products participate in various aspects of the immune system, including the clearance of immune complexes, type I interferon and NFkB pathways, B and T cell signaling, and transcriptional regulation, which are highlighted herein. Updated progress in lupus genetics, the identification of new causal variants, the discovery of novel pathways and regulatory mechanisms, and advances in utilizing SLE-risk loci to predict disease susceptibility and therapeutic strategies are reviewed. Here we emphasize the progress made in the last few years that has greatly enhanced our understanding of SLE pathogenesis, which will serve to guide the development of novel therapies to treat the disease.

DNA, RNA, protein, and varieties of chemicals together fulfill the biological functions of all organisms. Accumulative knowledge on these molecules and cells for the past centuries continuously enables scientists to invent tools to manipulate these biomolecules and modify viruses, bacteria, and cells for scientific research, pharmaceutical manufacture, and food production. Taking advantages of these revolutionary technologies, biopharmaceuticals have been created and produced to mimic desired physiological roles or perform novel biological functions. Critical biotechnology, especially gene sequencing and editing, which contribute to the development of biopharmaceuticals, is described in this chapter. Furthermore, general approaches and challenges to discover and develop protein, gene, and cell therapeutics are introduced.

Alternative polyadenylation (APA) is an essential regulatory mechanism for gene expression. The next generation sequencing provides ample opportunity to precisely delineate APA sites genome-wide. Various methods for profiling transcriptome-wide poly(A) sites were developed. By comparing available methods, the ways for adding sequencing adaptors to fit with the Illumina sequencing platform are different. These methods have identified more than 50% genes that undergo APA in eukaryotes. However, due to the unbalanced PCR during library preparation, accurate quantification of poly(A) sites is still a challenge. Here, we describe an updated poly(A) tag sequencing method that incorporates unique molecular identifier (UMI) into the adaptor for removing quantification bias induced by PCR duplicates. Hence, quantification of poly(A) site usages can be achieved by counting UMIs. This protocol, quantifying poly(A) tag sequencing (QPAT-seq), can be finished in 1 day with reduced cost, and is particularly useful for application with a large number of samples.

From the functional standpoint, broadly, three kinds of functional DNA sequences exist: one that encodes proteins, second that encodes only RNA (noncoding DNA), and third that does not transcribe at all (Noncoding DNA) historically people have paid attention to the protein-coding genes. For the last two-and-a-half decades, the noncoding RNA has taken center stage. However, the role of noncoding DNA (the dark matter of genome) is still largely unknown. We asked a simple question: How did nature decide to allocate protein-coding and RNA-coding jobs to a specific set of sequences? Did she sample all possibilities in the neighborhood of protein-coding genes, retaining good results (protein- and RNA-encoding genes), retiring not-so-relevant results (pseudogenes), and leaving some genome sequences untouched (nonexpressive genome) To answer these, we expressed some noncoding intergenic sequences from Escherichia coli into functional proteins. The success of this pilot experiment led us toward building a knowledge base that predicts the outcome of making user-defined genes from nonexpressive DNA sequences. In this chapter, the idea of lab-made genes shall be presented from the original concept to its present state where novel therapeutic molecules against cancer, infectious diseases, and neurodegenerative diseases have been found. We call this new platform “FLAGSHIP”—a novel drug discovery platform originating from the dark matter of the genome.

Genetic improvement with the combined use of conventional breeding and genetic engineering has been reported in most important crop varieties to express human-desired traits, such as resistance to diseases. The appearance of molecular biology in breeding programs in the 1980s enabled knowledge of genetic determinants of phenotypes and marker-assisted selection. Various advanced technologies have been developed that allow accumulation and assessment of large-scale datasets of biological molecules, including DNA sequence (genome), transcripts (transcriptome), proteins (proteome), and metabolites (metabolome). The field of metabolomics is the branch of -omics technology related to the high-throughput identification and quantification of low-molecular-weight molecules (

Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. The resistance of hepatitis C virus (HCV) to direct-acting antiviral agents, used in chronic hepatitis C treatment, consists of a natural process resulting from resistance-associated substitutions (RASs) at specific amino acid regions. To identify and establish the natural prevalence of RASs in the NS3 gene in patients with chronic hepatitis C in the state of Par?, northern Brazil. Molecular analysis was performed on a total of 35 patients infected with HCV genotype 1, who were treatment-naive to protease inhibitors. HCV RNA was extracted from plasma and the NS3 region was amplified and submitted to DNA sequencing (Sanger). The general natural prevalence of RASs in the NS3 gene was 37.5 % (Y56F and S122T). The substitutions Y56F (34.3 %), S122T (3.1 %), V132I (15.6 %) and V170I (9.3 %) were identified. Y56F and S122T provide resistance to the protease inhibitors grazoprevir and simeprevir, respectively. All amino acid substitutions in the NS3 gene, including RASs, identified in patients from the state of Par? were present in other Brazilian studies. The natural presence of RASs in this study reflects the elevated genetic variability of HCV.

HIV-1 is characterized by its ability to mutate and recombine even at polymerase (pol) gene. However, pol-gene diversity is limited due to functional constraints. The aim of this study was to characterize longitudinally, by next-generation sequencing (NGS), HIV-1 variants based on pol-gene sequences, at intra- and inter-host level, from acute/early to chronic stages of infection, in the absence of antiretroviral therapy. Ten men who have sex with men (MSM) were recruited during primary infection and yearly followed for five years. Even after a maximum of a five-year follow-up period, the phylogenetic analysis of HIV-1 pol-gene sequences showed a host-defined structured pattern, with a predominance of purifying selection forces during the follow-up. MSM had been acutely infected by different HIV-1 variants mainly ascribed to pure subtype B, or BF recombinant variants and showed different genetic mosaicism patterns that last until the chronic stage, representing a major challenge for prevention strategies., Genetics; Microbiology; Virology; Viruses; Viral disease; Viral genetics; HIV-1; Phylogeny; Pol-gene; Next-generation-sequencing; Viral diversity; MSM.

Sex-specific markers are extremely important for unisexual breeding and understanding of sex determination mechanisms. Spotted mandarin fish (Siniperca scherzeri) and big eye mandarin fish (Siniperca kneri) are economically important freshwater fish in China. Here, for the first time, we successfully identified three male-specific markers in the two species. Through genome sequencing and bioinformatic analysis of three males, three females, one male mixed pool and one female mixed pool of spotted mandarin fish, we screened 27 potential male-specific sequences. After PCR validation, three male-specific markers were further identified. Moreover, these markers could also be used to identify genetic sex of spotted mandarin fish from different populations with 100 % accuracy. In addition, these markers were also successfully used in the identification of genetic sex of big eye mandarin fish. Notably, the sequences of male-specific markers were highly similar in the two species and the corresponding sequence of one marker was highly similar to partial genome sequence of amhy gene in mandarin fish (Siniperca chuatsi). In all, the verified male-specific markers suggested XY sex determination system existed in spotted mandarin fish and big eye mandarin fish and would play an important role in future work of sex control breeding.

Since the discovery of the ETV6-NTRK3 gene fusion in infantile fibrosarcoma two decades ago, it has become an important diagnostic marker because it is found in the majority of cases. However, the development of new molecular tests, including next generation sequencing, has uncovered additional gene fusions and other oncogenic mutations in tumors with the clinical and morphologic features of infantile fibrosarcoma. We present a case of infantile fibrosarcoma harboring a novel BMPR1A-RAF1 fusion and having a favorable outcome 6 years after surgery. This new structural alteration adds to the list of genetic aberrations in infantile fibrosarcoma and provides another example of the challenge of reconciling classic morphology with novel molecular genetics.

Acholeplasma laidlawii is widespread hypermutable bacteria (class Mollicutes) capable of infecting humans, animals, plants, which is the main contaminant of cell cultures and vaccine preparations. The mechanisms of the development of antimicrobial resistance of this bacterium are associated with the secretion of extracellular vesicles, which can mediate the lateral transfer of antibiotic resistance determinants. We compared the genome profiles of ciprofloxacin-resistant A.laidlawii strains PG8r1 (MIC 10 µg/ml) and PG8r3 (MIC 10 µg/ml) selected under different in vitro conditions - when ciprofloxacin-sensitive (MIC 0.5 µg/ml) A.laidlawii PG8B strain was cultured at increasing concentrations of ciprofloxacin in a broth medium alone, and with vesicles derived from the ciprofloxacin-resistant (MIC 20 µg/ml) A.laidlawii PG8R10c-2 strain, respectively. Genome profiles of PG8c-3 (obtained from a single colony of the strain PG8B) and PG8R10c-2 were analyzed too. Patterns of the quinolone target genes (gyrA, gyrB, parE, parC) containing in extracellular vesicles of PG8c-3, PG8R10c-2, PG8r1 and PG8r3 were determined. Genome sequencing was performed on the NextSeq Illumina platform. Search and annotation of single nucleotide polymorphisms were performed using Samtools and SnpEff, respectively. We also compared cellular proteomes of PG8c-3, PG8r1 and PG8r3. The cellular proteome profiles of the A. laidlawii strains were determined by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. This work presents data on single nucleotide polymorphisms (SNPs) found in the genomes of the ciprofloxacin-resistant strains selected under different in vitro conditions and proteins that were differentially expressed in the cells of ciprofloxacin-resistant strains selected under different conditions in vitro.

Introduction: Pathogenic variants in SURF1, a nuclear-encoded gene encoding a mitochondrial chaperone involved in COX assembly, are one of the most common causes of Leigh syndrome (LS). Material-methods: Sixteen patients diagnosed to have SURF1-related LS between 2012 and 2020 were included in the study. Their clinical, biochemical and molecular findings were recorded. 10/16 patients were diagnosed using whole-exome sequencing (WES), 4/16 by Sanger sequencing of SURF1, 1/16 via targeted exome sequencing and 1/16 patient with whole-genome sequencing (WGS). The pathogenicity of SURF1 variants was evaluated by phylogenetic studies and modelling on the 3D structure of the SURF1 protein. Results: We identified 16 patients from 14 unrelated families who were either homozygous or compound heterozygous for SURF1 pathogenic variants. Nine different SURF1 variants were detected The c.769G > A was the most common variant with an allelic frequency of 42.8% (12/28), c.870dupT [(p.Lys291*); (8/28 28.5%)], c.169delG [(p.Glu57Lysfs*15), (2/24; 7.1%)], c.532 T > A [(p.Tyr178Asn); (2/28, 7.1%)], c.653_654delCT [(p.Pro218Argfs*29); (4/28, 14.2%)] c.595_597delGGA [(p.Gly199del); (1/28, 3.5%)], c.751 + 1G > A (2/28, 4.1%), c.356C > T [(p.Pro119Leu); (2/28, 3.5%)] were the other detected variants. Two pathogenic variants, C.595_597delGGA and c.356C > T, were detected for the first time. The c.769 G > A variant detected in 6 patients from 5 families was evaluated in terms of phenotype-genotype correlation. There was no definite genotype – phenotype correlation. Conclusions: To date, more than 120 patients of LS with SURF1 pathogenic variants have been reported. We shared the clinical, molecular data and natural course of 16 new SURF1 defect patients from our country. This study is the first comprehensive research from Turkey that provides information about disease-causing variants in the SURF1 gene. The identification of common variants and phenotype of the SURF1 gene is important for understanding SURF1 related LS. Synopsis: SURF1 gene defects are one of the most important causes of LS; patients have a homogeneous clinical and biochemical phenotype. © 2020 The Authors, G0800674 Biotechnology and Biological Sciences Research Council, BBSRC: L016354 European Molecular Biology Organization, EMBO EMBO Türkiye Bilimsel ve Teknolojik Araştirma Kurumu, TÜBITAK Bilim Akademisi Newcastle upon Tyne Hospitals NHS Foundation Trust NIHR Bristol Biomedical Research Centre Lily Mae Foundation Medical Research Council, MRC, RM and RWT are supported by the Wellcome Centre for Mitochondrial Research ( 203105/Z/16/Z ), the Medical Research Council (MRC) International Centre for Genomic Medicine in Neuromuscular Disease, the Mitochondrial Disease Patient Cohort (UK) ( G0800674 ), Newcastle University Centre for Ageing and Vitality (supported by the Biotechnology and Biological Sciences Research Council and Medical Research Council L016354 ), UK NIHR Biomedical Research Centre for Ageing and Age-related disease award to the Newcastle upon Tyne Hospitals NHS Foundation Trust , the Lily Foundation and the UK NHS Specialist Commissioners which funds the “Rare Mitochondrial Disorders of Adults and Children” Diagnostic Service in Newcastle upon Tyne ( http://www.newcastle-mitochondria.com . OA is supported by the European Molecular Biology Organization (EMBO) Installation grant funded by the Scientific and Technological Research Council of Turkey (TUBITAK); Science Academy, Turkey; TUBITAK International Fellowship for Outstanding Researchers Programme.

Summary Decades of artificial selection have significantly improved performance and efficiency of animal production systems. However, little is known about the microevolution of genomes due to intensive breeding. Using whole-genome sequencing, we document dynamic changes of chicken genomes under divergent selection on adiposity over 19 generations. Directional selection reduced within-line but increased between-line genomic differences. We observed that artificial selection tended to result in recruitment of preexisting variations of genes related to adipose tissue growth. In addition, novel mutations contributed to divergence of phenotypes under selection but contributed significantly less than preexisting genomic variants. Integration of 15 generations genome sequencing, genome-wide association study, and multi-omics data further identified that genes involved in signaling pathways important to adipogenesis, such as autophagy and lysosome (URI1, MBL2), neural system (CHAT), and endocrine (PCSK1) pathways, were under strong selection. Our study provides insights into the microevolutionary dynamics of domestic animal genomes under artificial selection., Graphical Abstract, Highlights • Directional selection reduces within-line but increases between-line genomic difference • Artificial selection tends to recruit preexisting variations of genes for fatness • Novel mutations contribute to the divergence of fatness under artificial selection • Genes involved in signaling pathways important to adipogenesis are under selection, Biological Sciences; Evolutionary Biology; Genetics; Genomics

Strain Marseille-Q0835T is an aerobic, non-motile and non-spore-forming Gram-positive coccus isolated from the stools of a Burkinabe woman. In this report, we present its phenotypic description including MALDI-TOF mass spectrometry analysis and genome sequencing. Strain Marseille-Q0835T; 2.9768-Mb genome exhibited a 41.9 mol% G+C content and 2699 predicted genes. Considering phenotypic features and comparative genome studies, we propose the strain Marseille-Q0835T as the type strain of Enterococcus burkinafasonensis sp. nov., a new species within the family Enterococcaceae.

Summary Kingdonia uniflora, an alpine herb, has an extremely narrow distribution and represents a model for studying evolutionary mechanisms of species that have adapted to undisturbed environments for evolutionarily long periods of time. We assembled a 1,004.7-Mb draft genome (encoding 43,301 genes) of K. uniflora and found significant overrepresentation in gene families associated with DNA repair, underrepresentation in gene families associated with stress response, and loss of most plastid ndh genes. During the evolutionary process, the overrepresentation of gene families involved in DNA repair could help asexual K. uniflora reduce the accumulation of deleterious mutations, while reducing genetic diversity, which is important in responding to environment fluctuations. The underrepresentation of gene families related to stress response and functional loss of ndh genes could be due to lack or loss of ability to respond to environmental changes caused by long-term adaptation to a relatively stable ecological environment., Graphical Abstract, Highlights • Provides a high-quality assembly for K. uniflora genome • K. uniflora shows overrepresentation in gene families associated with DNA repair • K. uniflora shows underrepresentation in gene families associated with stress response • The current endangered status of K. uniflora is related with adaptive degeneration, Biological Sciences; Plant Genetics; Plant Ecology; Plant Evolution

Vigna nakashimae is one of the closely related species of Vigna angularis (Adzuki bean). Two strain of ‘Ukushima’ and ‘G418’ were identified as salt tolerance strains in Vigna nakashimae from gene bank collection. F2 populations from an inter- or intra-specific cross between the sensitive and tolerant strains are useful for the detection of salt tolerance QTL in Vigna nakashimae. Although Vigna angularis reference genome is available and useful for genetic analysis by genotyping-by-sequencing/RADseq in closely related species, it is not enough for isolation of responsible genes. To reveal sequence variation in Vigna nakashimae “Ukushima” and “G418”, the whole genome sequencing was performed using Illumina HiSeq X Ten system (411,174,986 and 478,116,282 read). NGS data was deposited in the DNA Data Bank of Japan (DDBJ) under accession number DRA009307. Keywords: Vigna nakashimae, Closely wild relatives of Adzuki bean, Next-generation sequencing, HiSeq, Whole genome sequencing

Cancer is a sickness, initiated and driven via accumulation and interaction of genetic and epigenetic modification of genes involved in regulation of cell communication, signaling and growth division. Epigenetics is known as heritable changes in the expression of genes which are different from mutations that are not attributable to modification of DNA sequence. There are two leading epigenetic mechanisms; histone modification and DNA methylation. The acts of epigenetics in controlling of gene expression have been considered as elementary process in the pathogenesis of a variety of malignancies, for instance reproductive organs cancers. Recently, numerous exciting discoveries in epigenetics field have been focused in the study of reproductive cancers organs in females. Epigenetic modifications in the DNA of cancer patients and precancerous lesions provide the promise of novel biomarkers for early cancer prediction, diagnosis, prognosis, and treatment response. In addition, various epigenetic modifications represent potential targets of novel therapeutic strategies and treatments of cancer. In the future, pioneering diagnostic methodologies, technologies and treatment regimens will be based on epigenetic mechanisms and may be integrated into the gynecologist's practice. Keywords: Cancer, DNA methylation, Epigenetics, Histones modifications, Female, Urogenital organs

The brain contains immense cellular diversity. Early neuroanatomists identified broad structural brain features and used a variety of stains to characterize, draw, and name a number of constituent cell types. However, our granular understanding of what defines cellular identity, and how this relates to human neurogenesis, has been transformed by the sequencing of the human genome and subsequent technological advances. The genome enabled a translation of single gene level studies to genome-wide, unbiased characterization of gene expression patterns. Rapidly developing gene sequencing technology has revolutionized our understanding of cellular diversity, first based on microarray technology, then RNA-sequencing of 3′ fragments, to more recent whole-transcriptome sequencing, and ultimately single-cell sequencing. Single-cell sequencing allows for a heterogeneous tissue, like the brain, to be broken down into its component cell types based on fundamentally relevant single cell level gene programs. The advent of single-cell sequencing has been transformative for many fields, but using single-cell sequencing in the study of the brain and specifically brain development has enabled a convergence of the understanding of gene expression with features such as area, layer, age, connectivity, morphology, and ultimately cellular function, to achieve a core foundational understanding of cell type identity. While a number of these approaches are now being applied to human development, they have already been widely applied to rodent models, especially the mouse. Due to limitations of accessibility of human brain tissue, in vitro model systems such as the cerebral organoid have been developed to further our understanding of cellular and molecular developmental programs and to functionally test hypotheses gathered from sequencing studies. Here, we focus on established and emerging single-cell technologies, how they have enabled discovery in human brain development, and how organoid models recapitulate cellular developmental trajectories.

The decreased cost of human exome and genome sequencing provides new opportunities for diagnosing genetic disorders. In order to utilize this information, we need better and more robust methods for interpreting sequencing results, including determining whether specific missense variants are likely to be pathogenic. Using the protein PTEN (phosphatase and tensin homolog) as an example, we show how recent developments in both experiments and computational modeling can be used to determine whether a missense variant is likely to be pathogenic. Missense variants in PTEN can cause both autism spectrum disorder and increased risk of different forms of cancer, yet distinguishing such disease-associated variants from more harmless genetic variation is difficult. One approach relies on multiplexed experiments that enable determination of the effect of all possible missense variants in a cellular assay. Another approach is to use computational methods to predict variant effects. We compared two different multiplexed experiments and two computational methods to classify variant effects in PTEN. We distinguished between methods that focus on effects on protein stability and protein-specific methods that are more directly related to enzyme activity. Our results on PTEN suggest that ∼60% of pathogenic variants cause loss of function because they destabilize the folded protein, which is subsequently degraded. Methods that quantify a broader range of effects on PTEN activity perform better at predicting variant effects. Either experimental method performs better than the corresponding computational predictions, so that, for example, experiments that probe cellular abundance perform better at identifying pathogenic variants than predictions of thermodynamic stability. Our results suggest that loss of stability of PTEN is a key driver for disease, and we hypothesize that experiments and prediction methods that probe protein stability can be used to find variants with similar mechanisms in other genes.

With the development of next-generation sequencing (NGS) technologies, genome sequencing process has become cheaper and faster, making it possible the use of the technology in daily routine and, as a result, the number of registered genome projects is increasing rapidly. The pan-omics approaches have been applied for comparison of several genomes in a multipronged strategy. The pan-genome is composed of the core genome, shared genome, and singleton subsets, where the core genome is composed of all the commonly shared genes by all strains of the species; shared genome contains genes that are present in two or more, but not all strains from a species; and the singletons are strain-specific genes. The subsets from pan-transcriptomics and pan-proteomics may be classified in the same way. The concept of pan-genomics is so deep that it has been perfectly applied in the studies of several organisms and diseases. It has great potential which may bring a closer understanding and combating with prokaryotic and eukaryotic diseases. In this chapter, we review the concepts of omics, pan-omics, and the areas of applications of these methods.

Introduction: Squamous cell carcinomas (SqCCs) are the most common solid tumors in humans and are found across multiple organ systems. Although, integrated analysis of genetic alterations divulge similarities between SqCCs from various body sites, certain genes appear to be more frequently mutated in a given SqCC. These subtle differences may hold the key to determining the differentiation characteristics and predicting aggressiveness of tumors. Materials and Method: Fifty-four cases of SqCCs, in which the primary location of the tumor could be ascertained by clinical and radiological findings, were included in this study. Next generation sequencing data was analyzed for recurrent genetic abnormalities. Results: Genetic alterations were found in 219 genes in the 54 cases studied. TP53 mutations were found to be more frequent in pulmonary SqCCs (86.5%) as compared to non-pulmonary SqCCs (58.8%) (p

Highlights • This novel m.5860delTA variant in the MT-TY gene that caused mitochondrial disorder can manifest clinical features suggestive of myasthenic syndromes. • This variant has been demonstrated to be pathogenic by our histochemical, immunohistochemical and protein studies. • Muscle biopsy remains an important diagnostic investigation in the era of next generation sequencing., Mitochondrial DNA (mtDNA)-related diseases often pose a diagnostic challenge and require rigorous clinical and laboratory investigation. Pathogenic variants in the mitochondrial tRNA gene MT-TY, which encodes the tRNATyr, are a rare cause of mitochondrial disease. Here we describe a novel m.5860delTA anticodon variant in the MT-TY gene in a patient who initially presented with features akin to a childhood onset myasthenic syndrome. Using histochemical, immunohistochemical and protein studies we demonstrate that this mutation leads to severe biochemical defects of mitochondrial translation, which is reflected in the early onset and progressive phenotype. This case highlights the clinical overlap between mtDNA-related diseases and other neuromuscular disorders, and demonstrates the potential pitfalls in analysis of next generation sequencing results, given whole exome sequencing of a blood DNA sample failed to make a genetics diagnosis. Muscle biopsy remains an important requirement in the diagnosis of mitochondrial disease and in establishing the pathogenicity of novel mtDNA variants.

Humans can now decipher the letters of the DNA alphabet at a breathtaking pace. Genes, defined as specific DNA sequences, act as instructions that determine the characteristics of all cells and their viruses. The discovery and adaptation of precise molecular scissors has created new possibilities to modify genes at an unprecedented precision. In order to do so, the scissors are first equipped with a tracking device to target a well-defined region within the vast genome. Next, they are introduced into the cell nucleus where they induce a precise DNA break in the targeted gene or genome area. The DNA damage is then repaired by the cellular machinery that can lead to an intended mutation affecting the desired trait of an organism. This directed approach of genome editing has led to countless breakthroughs in applications across biology, including agricultural sciences. The tool receiving the most attention in this genome editing revolution is CRISPR-Cas9.