Genomic Library Research Articles

A study on L-threonine and L-serine uptake in Escherichia coli K-12.

In the current study, we report the identification and characterization of the yifK gene product as a novel amino acid carrier in E. coli K-12 cells. Both phenotypic and biochemical analyses showed that YifK acts as a permease specific to L-threonine and, to a lesser extent, L-serine. An assay of the effect of uncouplers and composition of the reaction medium on the transport activity indicates that YifK utilizes a proton motive force to energize substrate uptake. To identify the remaining threonine carriers, we screened a genomic library prepared from the yifK-mutant strain and found that brnQ acts as a multicopy suppressor of the threonine transport defect caused by yifK disruption. Our results indicate that BrnQ is directly involved in threonine uptake as a low-affinity but high-flux transporter, which forms the main entry point when the threonine concentration in the external environment reaches a toxic level. By abolishing YifK and BrnQ activity, we unmasked and quantified the threonine transport activity of the LIV-I branched chain amino acid transport system and demonstrated that LIV-I contributes significantly to total threonine uptake. However, this contribution is likely smaller than that of YifK. We also observed the serine transport activity of LIV-I, which was much lower compared with that of the dedicated SdaC carrier, indicating that LIV-I plays a minor role in the serine uptake. Overall, these findings allow us to propose a comprehensive model of the threonine/serine uptake subsystem in E. coli cells.

A novel nucleic acid-binding protein, Gp49, from mycobacteriophage with mycobactericidal activity has the potential to be a therapeutic agent

The mycobacteriophages encode unique proteins that are potent to be therapeutic agents. We screened several clones with mycobactericidal properties from a genomic library of mycobacteriophages. Here we report the properties of one such clone coding the gene product, Gp49, of the phage Che12. Gp49 is a 16 kD dimeric protein having an HTH motif at its C-terminal and is highly conserved among mycobacteriophages and likely to be part of phage DNA replication machinery. Alphafold predicts it to be an α-helical protein. However, its CD spectrum showed it to be predominantly β-sheeted. It is a high-affinity heparin-binding protein having similarities with the macrophage protein Azurocidin. Its β-sheeted apo-structure gets transformed into α-helix upon binding to heparin. It binds to linear dsDNA as well as ssDNA and RNA cooperatively in a sequence non-specific manner. This DNA binding property enables it to inhibit both in vitro and in vivo transcription. The c-terminal HTH motif is responsible for binding to both heparin and nucleic acids. Its in vivo localization on DNA could cause displacements of many DNA-binding proteins from the bacterial chromosome. We surmised that the bactericidal activity of Gp49 arises from its non-specific DNA binding leading to the inhibition of many host-DNA-dependent processes. Its heparin-binding ability could have therapeutic/diagnostic usages in bacterial sepsis treatment.

CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death

Accumulation of aggregated and misfolded proteins, leading to endoplasmic reticulum stress and activation of the unfolded protein response, is a hallmark of several neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease. Genetic screens are powerful tools that are proving invaluable in identifying novel modulators of disease associated processes. Here, we performed a loss-of-function genetic screen using a human druggable genome library, followed by an arrayed-screen validation, in human iPSC-derived cortical neurons. We identified and genetically validated 13 genes, whose knockout was neuroprotective against Tunicamycin, a glycoprotein synthesis inhibitor widely used to induce endoplasmic reticulum stress. We also demonstrated that pharmacological inhibition of KAT2B, a lysine acetyltransferase identified by our genetic screens, by L-Moses, attenuates Tunicamycin-mediated neuronal cell death and activation of CHOP, a key pro-apoptotic member of the unfolded protein response in both cortical and dopaminergic neurons. Follow-up transcriptional analysis suggested that L-Moses provided neuroprotection by partly reversing the transcriptional changes caused by Tunicamycin. Finally, L-Moses treatment attenuated total protein levels affected by Tunicamycin, without affecting their acetylation profile. In summary, using an unbiased approach, we identified KAT2B and its inhibitor, L-Moses, as potential therapeutic targets for neurodegenerative diseases.

Cloning of Three Cytokinin Oxidase/Dehydrogenase Genes in Bambusa oldhamii.

Cytokinin oxidase/dehydrogenase (CKX) catalyzes the irreversible breakdown of active cytokinins, which are a class of plant hormones that regulate cell division. According to conserved sequences of CKX genes from monocotyledons, PCR primers were designed to synthesize a probe for screening a bamboo genomic library. Cloned results of three genes encoding cytokinin oxidase were named as follows: BoCKX1, BoCKX2, and BoCKX3. In comparing the exon-intron structures among the above three genes, there are three exons and two introns in BoCKX1 and BoCKX3 genes, whereas BoCKX2 contains four exons and three introns. The amino acid sequence of BoCKX2 protein shares 78% and 79% identity with BoCKX1 and BoCKX3 proteins, respectively. BoCKX1 and BoCKX3 genes are particularly closely related given that the amino acid and nucleotide sequence identities are more than 90%. These three BoCKX proteins carried putative signal peptide sequences typical of secretion pathway, and a GHS-motif was found at N-terminal flavin adenine dinucleotide (FAD) binding domain, suggesting that BoCKX proteins might covalently conjugate with an FAD cofactor through a predicted histidine residue.

Microsatellite DNA markers for the commercially important fish Hypophthalmus donascimientoi (Pisces: Siluriformes: Pimelodidae) in the Amazon basin: isolation, characterization and amplification in congeneric species.

The genus Hypophthalmus comprises six species (H. edentatus, H. marginatus, H. fimbriatus, and H. oremaculatus), and the recently described: H. donascimientoi and H. celiae. The popular name for Hypophthalmus spp. in Brazil is mapará, this name refers to the six species. This group of fish has commercial importance for the states of Amazonas and Pará and, for this reason, requires studies to identify fish stocks. One approach is to use molecular markers, which have been very useful in studies with identification and population analysis of fish. Microsatellite molecular markers (SSRs) are one of the most informative markers for this purpose. There is little populations study of Hypophthalmus using SSRs, and there are less than six loci for the species Hypophthalmus marginatus available in the literature. With the construction of a genomic library of H. donascimientoi, we aimed to isolate and characterize SSRS markers and evaluate the extent of interspecific amplification. A genomic library was constructed with regions enriched of microsatellite for Hypophthalmus donascimientoi. A total of 126 contigs with 42 SSRs were used to design flanking primers for 39 microsatellites. Fifteen loci were characterized in three locations of the Solimões/Amazonas Rivers. The number of alleles ranged from one to 17 with a total of 126 alleles. The mean observed heterozygosity (HO) and expected heterozygosity (HE) were 0.721-0.692, respectively (S.d. HO 0.061 and HE 0.060). Two loci showed significant deviation in the HWE. The PIC ranged from 0.375 to 0.908. Such identified, 12 highly informative loci, and two moderately informative loci. Among the fifteen loci characterized, seven were successfully amplified in four other species of the genus. The microsatellite showed promise for estimating the genetic variability of H. donascimientoi and can be used as an efficient tool in population analyses of this species and in congeneric species analyzed.

Mitochondrial single-cell ATAC-seq for high-throughput multi-omic detection of mitochondrial genotypes and chromatin accessibility.

Natural sequence variation within mitochondrial DNA (mtDNA) contributes to human phenotypes and may serve as natural genetic markers in human cells for clonal and lineage tracing. We recently developed a single-cell multi-omic approach, called 'mitochondrial single-cell assay for transposase-accessible chromatin with sequencing' (mtscATAC-seq), enabling concomitant high-throughput mtDNA genotyping and accessible chromatin profiling. Specifically, our technique allows the mitochondrial genome-wide inference of mtDNA variant heteroplasmy along with information on cell state and accessible chromatin variation in individual cells. Leveraging somatic mtDNA mutations, our method further enables inference of clonal relationships among native ex vivo-derived human cells not amenable to genetic engineering-based clonal tracing approaches. Here, we provide a step-by-step protocol for the use of mtscATAC-seq, including various cell-processing and flow cytometry workflows, by using primary hematopoietic cells, subsequent single-cell genomic library preparation and sequencing that collectively take ~3-4 days to complete. We discuss experimental and computational data quality control metrics and considerations for the extension to other mammalian tissues. Overall, mtscATAC-seq provides a broadly applicable platform to map clonal relationships between cells in human tissues, investigate fundamental aspects of mitochondrial genetics and enable additional modes of multi-omic discovery.

Serial Recombineering Cloning to Build Selectable and Tagged Genomic P[acman] BAC Clones for Selection Transgenesis and Functional Gene Analysis using Drosophila melanogaster.

Transgenes with genomic DNA fragments that encompass genes of interest are the gold standard for complementing null alleles in rescue experiments in the fruit fly Drosophila melanogaster. Of particular interest are genomic DNA clones available as bacterial artificial chromosomes (BACs) or fosmids from publicly available genomic DNA libraries. Genes contained within BAC and fosmid clones can be easily modified by recombineering cloning to insert peptide or protein tags to localize, visualize, or manipulate gene products, and to create point mutations or deletions for structure-function analysis of the inserted genes. However, since transgenesis efficiency is inversely correlated with transgene size, obtaining transgenic animals for increasingly larger BAC and fosmid clones requires increasingly laborious screening efforts using the transgenesis marker commonly used for these transgenes, the dominant eye color marker white+ . We recently described a drug-based selectable genetic platform for Drosophila melanogaster, which included four resistance markers that allow direct selection of transgenic animals, eliminating the need to identify transgenic progeny by laborious phenotypic screening. By integrating these resistance markers into BAC transgenes, we were able to isolate animals containing large transgenes by direct selection, avoiding laborious screening. Here we present procedures on how to upgrade BAC clones by serial recombineering cloning to build both selectable and tagged BAC transgenes, for selection transgenesis and functional gene analysis, respectively. We illustrate these procedures using a BAC clone encompassing the gene encoding the synaptic vesicle protein, cysteine string protein. We demonstrate that the modified BAC clone, serially recombineered with a selectable marker for selection transgenesis and an N-terminal green fluorescent protein tag for gene expression analysis, is functional by showing the expression pattern obtained after successful selection transgenesis. The protocols cover: (1) cloning and preparation of the recombineering templates needed for serial recombineering cloning to incorporate selectable markers and protein tags; (2) preparing electrocompetent cells needed to perform serial recombineering cloning; and (3) the serial recombineering workflow to generate both selectable and tagged genomic BAC reporter transgenes for selection transgenesis and functional gene analysis in Drosophila melanogaster. The protocols we describe can be easily adapted to incorporate any of four selectable markers, protein tags, or any other modification for structure-function analysis of the genes present within any of the BAC or fosmid clones. A protocol for generating transgenic animals using serially recombineered BAC clones is presented in an accompanying Current Protocols article (Venken, Matinyan, Gonzalez, & Dierick, 2023a). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cloning and preparation of recombineering templates used for serial recombineering cloning. Basic Protocol 2: Making electrocompetent cells of the bacterial strains used to perform serial recombineering cloning or induction of plasmid copy number. Basic Protocol 3: Serial recombineering cloning to generate both selectable and tagged genomic P[acman] BAC reporter transgenes for selection transgenesis and gene expression analysis in Drosophila melanogaster.

Hypertension, type 2 diabetes, obesity, and p53 mutations negatively correlate with metastatic colorectal cancer patients' survival.

We studied the predictive and prognostic influences of hypertension (HT), type 2 diabetes (T2D), weight, and p53 mutations in metastatic colorectal cancer (CRC) patients. T2D was diagnosed according to the ADA criteria. HT was classified according to the ACC/AHA guidelines. BMI (body-mass index) was calculated and classified according to the WHO criteria. TruSigt™Oncology 500 kit was applied to construct the genomic libraries for Next Generation Sequencing (NGS) analysis. The Illumina NovaSeq 6000 technological platform and the Illumina TruSight Oncology 500 bioinformatics pipeline were applied to analyze results. Overall survival (OS) was calculated through Kaplan-Meier curves. Univariate and multivariate analyses were performed to assess the relationships between clinical and/or molecular covariates. Associations between HT, T2D, BMI, p53, and clinical variables were evaluated by the χ2 test. P < 0.05 were considered statistically significant. Two-hundred-forty-four patients were enrolled. One-hundred-twenty (49.2%), 110 (45.1%), and 50 (20.5%) patients were affected by overweight, HT, and T2D, respectively. DC (disease control) was achieved more frequently in patients without T2D (83.1%) compared to the diabetic ones (16.9%) (P = 0.0246). DC, KRAS mutational status, T2D, BMI, and concomitant presence of T2D, BMI, and HT associated with survival (P < 0.05). At multivariate analysis, age (≥65 vs. <65 years), response to first-line chemotherapy (DC vs. no DC), and concomitant presence of T2D, BMI, and HT (HR: 4.56; 95% CI: 2.40-8.67; P = 0.0217) emerged as independent prognostic variables. P53 was mutated in 31/53 analyzed cases (60.4%). The most frequent gene variants were p.Arg175His and p.Cys135Tyr. High BMI (>25 kg/m2) associated with occurrence of p53 mutations (P < 0.0001). P53 mutated patients presented a worse prognosis compared to the wild-type ones (HR: 3.21; 95% CI: 1.43-7.23; P = 0.0047). Diabetic, hypertensive and overweight metastatic CRC patients are a negative prognostic subgroup deserving specific therapeutic strategies. P53 mutations associate with prognosis and BMI unrevealing complex and unexplored connections between metabolism and cancer occurrence.

Combining methods for non-invasive fecal DNA enables whole genome and metagenomic analyses in wildlife biology.

Non-invasive biological samples benefit studies that investigate rare, elusive, endangered, or dangerous species. Integrating genomic techniques that use non-invasive biological sampling with advances in computational approaches can benefit and inform wildlife conservation and management. Here, we used non-invasive fecal DNA samples to generate low- to medium-coverage genomes (e.g., >90% of the complete nuclear genome at six X-fold coverage) and metagenomic sequences, combining widely available and accessible DNA collection cards with commonly used DNA extraction and library building approaches. DNA preservation cards are easy to transport and can be stored non-refrigerated, avoiding cumbersome or costly sample methods. The genomic library construction and shotgun sequencing approach did not require enrichment or targeted DNA amplification. The utility and potential of the data generated was demonstrated through genome scale and metagenomic analyses of zoo and free-ranging African savanna elephants (Loxodonta africana). Fecal samples collected from free-ranging individuals contained an average of 12.41% (5.54-21.65%) endogenous elephant DNA. Clustering of these elephants with others from the same geographic region was demonstrated by a principal component analysis of genetic variation using nuclear genome-wide SNPs. Metagenomic analyses identified taxa that included Loxodonta, green plants, fungi, arthropods, bacteria, viruses and archaea, showcasing the utility of this approach for addressing complementary questions based on host-associated DNA, e.g., pathogen and parasite identification. The molecular and bioinformatic analyses presented here contributes towards the expansion and application of genomic techniques to conservation science and practice.

Whole-genome sequencing of SARS-CoV-2 isolates from symptomatic and asymptomatic individuals in Tanzania.

Coronavirus Disease-2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accounts for considerable morbidity and mortality globally. Paucity of SARS-CoV-2 genetic data from Tanzania challenges in-country tracking of the pandemic. We sequenced SARS-CoV-2 isolated in the country to determine circulating strains, mutations and phylogenies and finally enrich international genetic databases especially with sequences from Africa. This cross-sectional study utilized nasopharyngeal swabs of symptomatic and asymptomatic adults with positive polymerase chain reaction tests for COVID-19 from January to May 2021. Viral genomic libraries were prepared using ARTIC nCoV-2019 sequencing protocol version three. Whole-genome sequencing (WGS) was performed using Oxford Nanopore Technologies MinION device. In silico genomic data analysis was done on ARTIC pipeline version 1.2.1 using ARTIC nCoV-2019 bioinformatics protocol version 1.1.0. Twenty-nine (42%) out of 69 samples qualified for sequencing based on gel electrophoretic band intensity of multiplex PCR amplicons. Out of 29 isolates, 26 were variants of concern [Beta (n = 22); and Delta (n = 4)]. Other variants included Eta (n = 2) and B.1.530 (n = 1). We found combination of mutations (S: D80A, S: D215G, S: K417N, ORF3a: Q57H, E: P71L) in all Beta variants and absent in other lineages. The B.1.530 lineage carried mutations with very low cumulative global prevalence, these were nsp13:M233I, nsp14:S434G, ORF3a:A99S, S: T22I and S: N164H. The B.1.530 lineage clustered phylogenetically with isolates first reported in south-east Kenya, suggesting regional evolution of SARS-CoV-2. We provide evidence of existence of Beta, Delta, Eta variants and a locally evolving lineage (B.1.530) from samples collected in early 2021 in Tanzania. This work provides a model for ongoing WGS surveillance that will be required to inform on emerging and circulating SARS-CoV-2 diversity in Tanzania and East Africa.

Development of novel microsatellite markers for population differentiation and detection of natural selection in wild populations of butter catfish, Ompok bimaculatus (Bloch, 1794).

Butter catfish (Ompok bimaculatus) is a preferred species in South East Asia, with huge aquaculture potential. However, there is limited information about genetic stock composition due to insufficient markers. The goal of this study was to develop de novo microsatellite markers. For sequencing, genomic SMRT bell libraries (1.5 Kbp size) were prepared for O. bimaculatus. A total of 114 SSR containing sequences were used for primer designing. Polymorphic loci were validated by genotyping 83 individuals from four distant riverine populations, viz., Brahmaputra, Bichiya, Gomti and Kaveri. A total of 30 microsatellite loci were polymorphic, of which five were found to be associated with functional genes and eight (four positive and four negative) loci were found to be under selection pressure. A total of 115 alleles were detected in all loci and PIC ranged from 0.539 to 0.927 and pair-wise FST values from 0.1267 to 0.26002 (p < 0.001), with an overall FST value of 0.17047, indicating the presence of population sub-structure. Cross-species transferability of 29 loci (96.67%) was successful in congener species, Ompok pabda. The novel SSR markers developed in this study would facilitate stock characterization of natural populations, to be used in future selection breeding programs and planning conservation strategies in these species. Identified non-neutral markers will give insights into the effect of local adaptation on genetic differentiation in the natural population of this species.

The Creation of a Gene Library of Valuable Plant Species in Armenia

Using DNA banks or gene libraries is the most efficient method of protecting and managing valuable, endangered, and rare gene pools. Genomic libraries are collections of bacteria colonies with restriction sites on their genomic DNA that contain desired genes from a specific organism. The “Scientific Center of AgroBiotechnology” branch of ANAU has created gene libraries of 34 plant species, which are preserved in the National Genebank of Crops and their Wild Relatives, which forms a basis for genomic selection. This allows genetic identification, barcoding, DNA formula decoding, and, most importantly, is the basis for genome identification. Based on the results, plasmids can incorporate restriction segments of genomic DNA and be efficiently transformed. The high level of correlation between the number of necessary clones and the efficiency of transformation of all studied plant species proves the completeness of the gene pool.

Ultralow-Input Genome Library Preparation for Nanopore Sequencing with Droplet MDA.

Genome sequencing of small species, such as those of meinofauna, can be challenging due to the extremely low input of genomic DNA. While nanopore sequencing is a promising technology for genome assembly due to its limitless long reads, recommended input of 1μg for the Ligation Sequencing Kit often precludes the use of this technology. Here, I detail an unbiased droplet-based multiple displacement amplification of picogram order of DNA to realize nanopore sequencing with ultralow input of genomic DNA. For this purpose, a microfluidic chip of 10X Genomics Chromium Controller is utilized. With this method, over 10μg of unbiased amplicons around 10 kbp in length can be obtainedfrom as low as 50 μg of input DNA, which is enough for the construction of multiple sequencing libraries, or for the size selection of longer DNA fragments.

Molecular insight for baru Dipteryx alata (Fabaceae) populations based on novel SSRs

Baru tree (Dipteryx alata) is an arboreal, fruitful plant native to the Cerrado biome with an important socioeconomic impact. Populations of this species are a good model to study anthropogenic disturbances on the biome through the genetic information. In this study, we developed seven new polymorphic microsatellite markers for D. alata, using an enriched genomic library. We performed loci characterization in three populations, obtaining a total of 49 alleles, with an average of 5 to 5.57 alleles per locus. A significant content of polymorphic information was obtained, as indicated by the average expected heterozygosity (uHE), with a total average of 0.58 to 0.65 per locus. The average value of the observed heterozygosity (Ho) was also high, with a total average of 0.73 to 0.85 per locus. Some of the loci are in linkage disequilibrium, such as DalatG6 with DalatB3, DalatH3 and DalatB4. The estimate of the combined loci for the probability of paternity exclusion obtained an average value of 1.00 for all loci, and the average combined probability of identity, the values were (1.210^-5) to (4.410^-6). All markers are informative and suitable for studies on genetic diversity and population structure, aiming at the conservation and management of the species.

142. Evaluation of Meropenem-Vaborbactam Susceptibility and Underlying Resistance Mechanisms among Clinical KPC-producing Klebsiella pneumoniae

Abstract Background Meropenem-vaborbactam (MV) is the first carbapenem/β-lactamase inhibitor combination developed to restore meropenem susceptibility against KPC-producing carbapenem-resistant Enterobacterales(CRE). Vaborbactam (VAB) potently inhibits Ambler class A and C β-lactamases by reversible covalent binding of boronate to serine side chains of β-lactamases. Resistance to MV in non-metallo-β-lactamase (MBL) producing Klebsiella pneumoniae (KP) isolates has been described but remains rare. We sought to identify the major molecular mechanisms associated with MV resistance in KPC-producing KP (KPC-KP) isolates. Methods Clinical isolates with elevated MV MICs were identified by the consult service. Additional clinical isolates with mutations in ompK35 or ompK36 genes were selected from a historic database. Isolates with MBL or OXA-48-like genes were excluded. Controls were comprised of MV susceptible KPC-KP isolates. MICs determination was done using Sensititre automated broth microdilution (BMD) according to CLSI. VAB and avibactam concentrations were held at 8 µg/ml and 4 µg/ml, respectively. Whole genome sequencing (WGS) was performed on all isolates. Genome libraries were prepared using Illumina Nextera XT and sequencing was performed on MiSeq and MinION. Results A total 119 KPC-KP isolates were included in the study. All isolates were resistant to meropenem. Twenty-one KPC-KP with elevated MV MICs were identified. All MV resistant isolates harbored mutations in ompK36 genes. Glycine/aspartate (GD 134-135) insertion, premature stop codon in ompK36 genes, and concomitantly elevated blaKPC copy number were predominant among MV resistant isolates. No insertion elements in ompK36 gene promoter region were found. Two MV resistant isolates exhibited unique mutations in blaKPC and envZ genes. See table for WGS and MIC results. Table 1.Whole genome sequencing and MICs of MV resistant KPC-KP isolatesα: Truncated at nodes 14 and 76, partial genotype consistent with blaSHV-12WT: Wild type*: Premature stop codonGD: Duplication of Glycine (G134) and Aspartate (D135)FS: Frameshift mutationins: insertionMEM: meropenemMVB: meropenem-vaborbactamCZA: ceftazidime-avibactamCFD: cefiderocolN/A: not availableTable 2.Whole genome sequencing and MICs of MV susceptible KPC-KP isolates Conclusion MV resistant KPC-KP isolates were reliably analyzed using WGS to reveal the contribution of omp gene mutations and blaKPC copy number to this phenotype. Elevated MV MICs were additionally recognized among clinical isolates from a historic database preceding MV availability. In the absence of MBL production, caution remains warranted with the use of MV empirically against KPC-KP due to non-β-lactamase mediated resistance mechanisms. Disclosures Daniel D. Rhoads, M.D. PhD, Luminex: Advisor/Consultant|Talis Biomedical: Advisor/Consultant|Thermo Fisher: Advisor/Consultant Federico Perez, M.D., Accelerate: Grant/Research Support|Merck: Grant/Research Support|Pfizer: Grant/Research Support Robert A. Bonomo, MD, NIH VA: Grant/Research Support|VenatoRx Merck Wockhardt Cystic Fibrosis Foundation: Grant/Research Support.

BAC-FISH Based Physical Map of Endangered Catfish Clarias magur for Chromosome Cataloguing and Gene Isolation through Positional Cloning.

Construction of a physical chromosome map of a species is important for positional cloning, targeted marker development, fine mapping of genes, selection of candidate genes for molecular breeding, as well as understanding the genome organization. The genomic libraries in the form of bacterial artificial chromosome (BAC) clones are also a very useful resource for physical mapping and identification and isolation of full-length genes and the related cis acting elements. Some BAC-FISH based studies reported in the past were gene based physical chromosome maps of Clarias magur (magur) to understand the genome organization of the species and to establish the relationships with other species in respect to genes' organization and evolution in the past. In the present study, we generated end sequences of the BAC clones and analyzed those end sequences within the scaffolds of the draft genome of magur to identify and map the genes bioinformatically for each clone. A total of 36 clones mostly possessing genes were identified and used in probe construction and their subsequent hybridization on the metaphase chromosomes of magur. This study successfully mapped all 36 specific clones on 16 chromosome pairs, out of 25 pairs of magur chromosomes. These clones are now recognized as chromosome-specific makers, which are an aid in individual chromosome identification and fine assembly of the genome sequence, and will ultimately help in developing anchored genes' map on the chromosomes of C. magur for understanding their organization, inheritance of important fishery traits and evolution of magur with respect to channel catfish, zebrafish and other species.

IE1 of Autographa californica Multiple Nucleopolyhedrovirus Activates Low Levels of Late Gene Expression in the Absence of Virus RNA Polymerase.

Early and late gene expressions of baculoviruses have been known to rely on host RNA polymerase II and a virus-encoded RNA polymerase, separately. In this study, we found that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) recombinant bacmids with the individual RNA polymerase subunit genes deleted could support low levels of expression of a reporter gene under the control of the promoter of a typical late gene, vp39, in transfected Sf9 cells. Through multistep subcloning of a genomic library of the virus and transient expression assay analysis, ie1 was identified to be the only viral gene that was responsible for activation of late gene expression in the absence of the viral RNA polymerase. Furthermore, IE1 was found to be capable of activating reporter gene expression from the promoters of additional late genes polh, p6.9, odv-e18, odv-e25, and gp41, independent of any additional viral factors. Deletion of ie1 from the virus genome eliminated late gene expression. The IE1-activated late gene expression was enhanced by the viral hr4b. It was shown to be insensitive to inhibition of α-amanitin and did not appear to have stable transcription start sites. It is proposed that IE1 may serve to recruit newly synthesized viral RNA polymerase to viral DNA by activating low levels of pretranscription of the late genes to create an appropriate DNA conformation. IMPORTANCE The late gene expression of baculovirus has been known to depend on the virus-encoded RNA polymerase, which consists of four subunits. The immediate-early gene ie1 was found to be required for viral early gene expression, late gene expression, and DNA replication. How it functions in late gene expression remains unclear. In this study, we found that AcMNPV IE1 could activate low levels of gene expression from late gene promoters independently of any additional viral factors, with nonspecific transcription start sites. This new finding will shed light on the role of IE1 in the regulation of late gene expression and the understanding of the mechanism of late gene transcription initiation.

Application status of metagenomics in quality research of traditional Chinese medicinal materials

Metagenomics constructs genomic libraries by directly extracting DNA from all microorganisms from environmental samples, and studies microbial diversity and functional composition by microbiology and genomics. The metagenomic technology and genome sequencing can be used to discover the unculturable environmental microorganisms and study the natural products in the medicinal materials related to them, which overcome the limitations of microbial isolation and culture technology and is of great significance for the discovery of new genes or genomes. The quality and safety of traditional Chinese medicine materials(TCMMs) have always been a hot topic in the research on TCMMs. In addition to the intrinsic characteristics, environmental factors and human intervention also affect the quality and clinical efficacy of TCMMs. Environmental microorganisms, rhizosphere microorganisms, and endophytes play an essential role in the growth, secondary metabolism, processing, and storage of medicinal plants. As an emerging discipline, metagenomics has been used in a variety of fields. It can not only avoid the problems caused by the limited technical conditions or strict experimental requirements but also evaluate the quality of TCMMs from a new perspective. This paper introduced the application status of metagenomics in the research of TCMMs and proposed the theoretical basis and research direction for the application of metagenomics in quality research and identification of TCMMs.

Optimized conditions for Listeria, Salmonella and Escherichia whole genome sequencing using the Illumina iSeq100 platform with point-and-click bioinformatic analysis.

Whole-genome sequencing (WGS) data have become an integral component of public health investigations and clinical diagnostics. Still, many veterinary diagnostic laboratories cannot afford to implement next generation sequencing (NGS) due to its high cost and the lack of bioinformatic knowledge of the personnel to analyze NGS data. Trying to overcome these problems, and make NGS accessible to every diagnostic laboratory, thirteen veterinary diagnostic laboratories across the United States (US) initiated the assessment of Illumina iSeq100 sequencing platform for whole genome sequencing of important zoonotic foodborne pathogens Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The work presented in this manuscript is a continuation of this multi-laboratory effort. Here, seven AAVLD accredited diagnostic laboratories explored a further reduction in sequencing costs and the usage of user-friendly platforms for genomic data analysis. Our investigation showed that the same genomic library quality could be achieved by using a quarter of the recommended reagent volume and, therefore a fraction of the actual price, and confirmed that Illumina iSeq100 is the most affordable sequencing technology for laboratories with low WGS demand. Furthermore, we prepared step-by-step protocols for genomic data analysis in three popular user-friendly software (BaseSpace, Geneious, and GalaxyTrakr), and we compared the outcomes in terms of genome assembly quality, and species and antimicrobial resistance gene (AMR) identification. No significant differences were found in assembly quality, and the three analysis methods could identify the target bacteria species. However, antimicrobial resistance genes were only identified using BaseSpace and GalaxyTrakr; and GalaxyTrakr was the best tool for this task.

Profiling of mitochondrial heteroplasmy in single human oocytes by next-generation sequencing.

Mitochondrial DNA (mtDNA) plays a crucial role in the development of a competent oocyte. Indeed, mtDNA alterations may predispose to chromosome nondisjunction, resulting in infertility due to a reduced vitality and quality of oocytes and embryos. In this methods paper, the multiple displacement amplification approach was applied in combination with next-generation sequencing (NGS) to amplify and sequence, in single-end, the entire mtDNA of single human oocytes to directly construct genomic NGS libraries, and subsequently, to highlight and quantify the mutations they presented. The bioinformatic workflow was carried out with a specific ad hoc developed in-house software. This approach proved to be sensitive and specific, also highlighting the mutations present in heteroplasmy, showing deletion, insertion or substitution mutations in the genes involved in the respiratory chain, even if the found variants were benign or of uncertain meaning. The analysis of mtDNA mutations in the oocyte could provide a better understanding of specific genetic abnormalities and of their possible effect on oocyte developmental competence. This study shows how this approach, based on a massive parallel sequencing of clonally amplified DNA molecules, allows to sequence the entire mitochondrial genome of single oocytes in a short time and with a single analytical run and to verify mtDNA mutations.