Construction Of DNA Libraries Research Articles

Abstract 320: Development and automation of a streamlined targeted enrichment method for cancer mutation detection

Abstract Next Generation Sequencing (NGS) of DNA is a powerful tool for the detection of genetic variations, including single nucleotide polymorphisms, copy number variation, and small insertions/deletions. Target enrichment technologies enhance DNA sequencing by enabling users to analyze specific regions of interest only, which helps to effectively increase sequencing depth and sample throughput while minimizing cost. However, many targeted DNA library construction workflows are not optimal for routine genetic testing needs due to inconsistent, lengthy workflow. This poses significant challenge to application like cancer mutation profiling that requires detection sensitivity down to 1% or below due to heterogeneous nature of tumor tissue. In addition, high throughput testing with minimal hands on time is desirable with the rapid advance of precision medicine. Magnetic bead separations are commonly used between the individual reactions of targeted DNA library preparation workflows for cleanup and size selection. These bead clean up reactions are not only labor-intensive and tedious procedures but can also greatly contribute to sample-to sample variation especially when employing automated library preparation procedures. With the sophisticated QIAseq Targeted DNA Pro, we developed a novel streamlined targeted DNA workflow that greatly reduces handling variation by replacing the majority of bead clean up steps with enzymatic reactions, which now can be finished in as little as six hours. In combination with the newly developed QIAseq Normalizer kit, the protocol has been successfully implemented on the Hamilton NGS Star liquid handling platform resulting in up to 96 highly uniform, complex and ready-to-sequence libraries that can be effortless generated in one run. This complete automated solution saves researcher up to four hours of hands-on time. When using commercial reference samples, cancer mutations down to 1% were consistently detected with this automated workflow. The presented data demonstrate that this integrated and streamlined workflow does not only provide an automation friendly workflow but also allows to reliably identify low frequent cancer mutations. The applications presented here are for research use only. Not for use in diagnostic procedures. Citation Format: Zhong Wu, Michelle Baird, Cornelia Mechlen, Peter Hahn, Jonathan M. Shaffer. Development and automation of a streamlined targeted enrichment method for cancer mutation detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 320.

Identification of bacteria associated with canine otitis externa based on 16S rDNA high-throughput sequencing.

Bacteria are regarded as predisposing and perpetuating factors causing otitis externa (OE), whereas auricular anatomy is a predisposing factor. This study aims to investigate bacterial populations in the external auditory canals of healthy dogs and dogs with OE. Four categories of ear swabs included healthy erect-ear dogs, erect-ear dogs with OE, healthy pendulous-ear dogs and pendulous-ear dogs with OE. After bacterial DNA extraction, 16S rDNAs were amplified using specific primers within a V3/V4 region. Following DNA library construction, high-throughput sequencing was performed on MiSeq (Illumina). CLC Microbial Genomics Module was used to determine the rarefaction curve, bacterial classification, relative abundance, richness and diversity index. The results demonstrated that healthy dogs had higher bacterial richness and diversity than the dogs with OE. Comparable with culture-dependent methods described previously, this study revealed predominant Corynebacterium spp., Pseudomonas spp., Staphylococcus spp., and Proteus spp. in OE cases. Furthermore, high-throughput sequencing might disclose some potential emerging pathogens including Tissierella spp., Acinetobacter spp., and Achromobacter spp., which have not been reported in previous canine OE cases. Nevertheless, larger sample sizes are further required for an extensive evidence-based investigation.

Metagenome Investigation of Ocular Microbiota of Cataract Patients With and Without Type 2 Diabetes.

Our objective was to investigate differences in the ocular surface bacterial composition in cataract patients with and without type 2 diabetes (T2D). Twenty-four diabetic patients with cataracts (group D) and 14 sex- and age-matched patients with age-related cataracts (group N) were recruited for this study. All samples underwent DNA extraction, fragmentation, purification, library construction, and metagenomic sequencing. The overall conjunctival sac bacterial composition was similar between group D and group N, as determined by alpha diversity and beta diversity. Nevertheless, significant differences were observed in the relative abundance of specific bacteria. At the phylum level, group D had a significantly lower abundance of Chlamydiae, Tenericutes, Chloroflexi, Cyanobacteria, Cossaviricota, Chytridiomycota, Artverviricota, Zoopagomycota, Peploviricota, Deinococcus-Thermus, Preplasmiviricota, and Nucleocytoviricota. At the genus level, group D had a significantly lower abundance of Chlamydia, Mycoplasma, Salmonella, Chryseobacterium, Roseovarius, Desulfococcus, Kangiella, Anaerococcus, and Idiomarina but a significantly higher abundance of Parabacteroides, Phocaeicola, and Sphingomonas. Bacteria such as Aquificae, Parabacteroides, Flavobacterium, Austwickia, Aquifex, Tenacibaculum, and Chryseobacterium in group D and Tenericutes, Chlamydiae, Porphyromonas, Mycoplasma, Chlamydia, Kangiella, Idiomarina, Roseovarius, Aliiroseovarius, and Desulfococcus in group N could be used as conjunctival sac biomarkers, according to the linear discriminant analysis effect size. Gene Ontology functional annotation indicated that bacterial catalytic activity, metabolic processes, locomotion, virion, and reproduction were enriched in group D, while immune system processes were enriched in group N. In addition, the top 30 differentially expressed virulence factors (VFs) were all more enriched in group D. The bacterial composition was similar between the two groups. Several bacterial strains that were reported beneficial in gut were decreased, and pathogenic bacteria were increased in T2D. Furthermore, group D had more active bacterial terms and increased VF expression, suggesting that the susceptibility of diabetic patients to infection is closely related to functional changes in the ocular surface flora. Our conjunctival microbiota atlas provides a reference for investigating ocular complications related to diabetes. The altered composition and functional profile of the ocular microbial community in diabetic patients offer evidence for the need to prevent infection during cataract surgery.

Distortion of Population Statistics due to the Use of Different Methodological Approaches to the Construction of Genomic DNA Libraries.

Several different methods of DNA library preparation for paleogenetic studies are now available. However, the chemical reactions underlying each of them can affect the primary sequence of ancient DNA (aDNA) in the libraries and taint the results of a statistical analysis. In this paper, we compare the results of a sequencing of the aDNA libraries of a Bronze Age sample from burials of the Caucasian burial ground Klady, prepared using three different approaches: (1) shotgun sequencing, (2) strategies for selecting target genomic regions, and (3) strategies for selecting target genomic regions, including DNA pre-treatment with a mixture of uracil-DNA glycosylase (UDG) and endonuclease VIII. The impact of the studied approaches to genomic library preparation on the results of a secondary analysis of the statistical data, namely F4 statistics, ADMIXTURE, and principal component analysis (PCA), was analyzed. It was shown that preparation of genomic libraries without the use of UDG can result in distorted statistical data due to postmortem chemical modifications of the aDNA. This distortion can be alleviated by analyzing only the single nucleotide polymorphisms caused by transversions in the genome.

Mammalian Chromosome Analysis and Sorting by Flow Cytometry.

The analysis of chromosomes by flow cytometry is termed flow cytogenetics, and it involves the analysis and sorting of single mitotic chromosomes in suspension. The study of flow karyograms provides insight into chromosome number and structure to provide information on chromosomal DNA content and can enable the detection of deletions, translocations, or any forms of aneuploidy. Beyond its clinical applications, flow cytogenetics greatly contributed to the Human Genome Project through the ability to sort pure populations of chromosomes for gene mapping, cloning, and the construction of DNA libraries. Maximizing the potential of these important applications of flow cytogenetics relies on precise instrument setup and optimal sample processing, both of which impact the accuracy and quality of the data that are generated. This article is a compilation of the existing protocols that describe the stepwise methodology of accumulating, isolating, and staining metaphase chromosomes to prepare single-chromosome suspensions for flow cytometric analysis and sorting. Although the chromosome preparation protocols have remained largely unchanged, cytometer technology has advanced dramatically since these protocols were originally developed. Advances in cytometry technologies offer new and exciting approaches for understanding and monitoring chromosomal aberrations, but the hallmark of these protocols remains their simplicity in methodologies and reagent requirements and the accuracy of data resolvable to every chromosome of the cell. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mitotic block and cell harvesting Basic Protocol 2: Propidium iodide isolation Support Protocol 1: Swelling test Basic Protocol 3: MgSO4 low-molecular-weight isolation Basic Protocol 4: Polyamine high-molecular-weight isolation Support Protocol 2: Molecular-weight determination of chromosomal DNA Basic Protocol 5: Chromosome analysis and sorting.

PANDORA-Seq unveils the hidden small noncoding RNA landscape in atherosclerosis of LDL receptor-deficient mice

Small noncoding RNAs (sncRNAs) play diverse roles in numerous biological processes. While the widely used RNA sequencing (RNA-Seq) method has advanced sncRNA discovery, RNA modifications can interfere with the complementary DNA library construction process, preventing the discovery of highly modified sncRNAs including transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) that may have important functions in disease development. To address this technical obstacle, we recently developed a novel PANDORA-Seq (Panoramic RNA Display by Overcoming RNA Modification Aborted Sequencing) method to overcome RNA modification-elicited sequence interferences. To identify novel sncRNAs associated with atherosclerosis development, LDL receptor-deficient (LDLR−/−) mice were fed a low-cholesterol diet or high-cholesterol diet (HCD) for 9 weeks. Total RNAs isolated from the intima were subjected to PANDORA-Seq and traditional RNA-Seq. By overcoming RNA modification-elicited limitations, PANDORA-Seq unveiled an rsRNA/tsRNA-enriched sncRNA landscape in the atherosclerotic intima of LDLR−/− mice, which was strikingly different from that detected by traditional RNA-Seq. While microRNAs were the dominant sncRNAs detected by traditional RNA-Seq, PANDORA-Seq substantially increased the reads of rsRNAs and tsRNAs. PANDORA-Seq also detected 1,383 differentially expressed sncRNAs induced by HCD feeding, including 1,160 rsRNAs and 195 tsRNAs. One of HCD-induced intimal tsRNAs, tsRNA-Arg-CCG, may contribute to atherosclerosis development by regulating the proatherogenic gene expression in endothelial cells. Overall, PANDORA-Seq revealed a hidden rsRNA and tsRNA population associated with atherosclerosis development. These understudied tsRNAs and rsRNAs, which are much more abundant than microRNAs in the atherosclerotic intima of LDLR−/− mice, warrant further investigations.

Cre/lox-Mediated CRISPRi Library Reveals Core Genome of a Type I Methanotroph Methylotuvimicrobium buryatense 5GB1C.

Methanotrophs play key roles in global methane cycling and are promising platforms for methane bioconversion. However, major gaps existing in fundamental knowledge undermines understanding of these methane-consuming microorganisms. To associate genes with a phenotype at the genome-wide level, we developed a Cre/lox-mediated method for constructing a large-scale CRISPRi library in a model methanotroph Methylotuvimicrobium buryatense 5GB1C. The efficiency of this Cre mediated integration method was up to a level of 105 CFU/μg DNA. Targeting 4,100 predicted protein-coding genes, our CRISPRi pooled screening uncovered 788 core genes for the growth of strain 5GB1C using methane. The core genes are highly consistent with the gene knockout results, indicating the reliability of the CRISPRi screen. Insights from the core genes include that annotated isozymes generally exist in metabolic pathways and many core genes are hypothetical genes. This work not only provides functional genomic data for both fundamental research and metabolic engineering of methanotrophs, but also offers a method for CRISPRi library construction. IMPORTANCE Due to their key role in methane cycling and their industrial potential, methanotrophs have drawn increasing attention. Genome-wide experimental approaches for gene-phenotype mapping accelerate our understanding and engineering of a bacterium. However, these approaches are still unavailable in methanotrophs. This work has two significant implications. First, the core genes identified here provide functional genetic basics for complete reconstruction of the metabolic network and afford more clues for knowledge gaps. Second, the Cre-mediated knock-in method developed in this work enables large-scale DNA library construction in methanotrophs; the CRISPRi library can be used to screen the genes associated with special culture conditions.

Reconstructing the Genomic History of Multiple Myeloma Precursor Disease: Novel Insights and Clinical Applicability

Introduction Early indolent stages of Multiple Myeloma (MM), which include monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), are heterogeneous diseases with overall rates of progression of 1% to 10% per year. Mutations present in MGUS and SMM have been characterized by our group and others and improve current risk models. However, whether any genomic events are sufficient for an MGUS or SMM to transform to MM remains an open fundamental question. To address this, we sequenced the largest cohort of MGUS and SMM genomes to date starting with a low-input DNA template, enriching for low burden of disease compared to previous studies. We report the translational implications of a large-scale characterization of indolent stages of MM after 2 years of follow-up. Methods Bone marrow (BM) samples were collected from 130 patients with MGUS and SMM after informed consent for research use (IRB #14-174). Plasma cells were selected from BM samples using CD138+ magnetic beads before DNA purification and library construction. Tumor and matched germline libraries were whole-genome sequenced (WGS) to 60X and 30X coverage respectively on NovaSeq 6000 S4 flowcells at the Genomics Platform of the Broad Institute. Analysis was performed with methods from the Cancer Program of the Broad Institute. All structural variants were manually reviewed and compared to fluorescence in situ hybridization (FISH) clinical laboratory reports. Results The median age of participants was 62 (range, 37 to 87), with 52% female. At the time of BM sampling, all patients were untreated; 24 patients diagnosed with MGUS (18%) and 106 diagnosed with SMM (82%) were homogeneously distributed across the IMWG 2/20/20 risk stratification stages (Low-risk: 32%, Intermediate-risk: 34%, High-risk: 34%). Somatic mutation burden increased with progressive disease staging, with a median of 2,666 single-nucleotide variants in MGUS (range 429-6546), and 3,233, 4,080, and 5,111 in low, intermediate, and high-risk SMM, respectively (range 463-11083, 161-8408, and 2549-7218, respectively, P=0.007). Compared to non-progressors, progressors were found to have an increase of molecular clock signature, symbolizing continuous accumulation of mutations over time (Fold change 2.40, P=0.002), and this difference was independent of patient age and disease stage. Confirming previous studies, APOBEC signature was strongly associated with patients with MAF alterations (t(14;16), t(14;20), median in MAF, 24%, non-MAF, 5.8%, P=3E-5), and uniquely identified in one patient with MMSET t(4;14) translocation. Catastrophic structural variants (SV) such as chromothripsis and chromoplexy were found in 7% of patients across 2/20/20 stages. Complex SVs were strongly associated with clinical progression to symptomatic MM (Odds Ratio (OR) 9.83, CI95%[1.91, 53]). Being statistically independent of risk staging, these events are a potential new risk factor not captured by current clinical models, but successfully characterized by an unbiased genomic assay. In our cohort, MYC was found rearranged with IgL, IgH, and other partners in 9% of patients. Strikingly, all but one case also harbored a RAS hotspot mutation, showing a strong non-random cooperation between RAS and MYC (P=0.005), not previously observed, which translated into increased risk of clinical progression (OR 7.75, CI95%[1.26, 48.8]). Moreover, most MYC-abnormal tumors (translocation or complex rearrangement) were missed by clinical FISH testing. WGS detected >99% of translocations and copy number abnormalities reported by FISH. In clinical reports with unknown translocations, WGS could identify translocation partners, including IgH-MYC and rare IgH-MAFA. In 82% of patients, an increased diagnostic yield was found by WGS compared to FISH using existing models, showing clinical applicability to replace molecular cytogenetics. Conclusion Analysis of the largest cohort of MGUS and SMM genomes to date was able to decipher the genomic history of MM, unraveling events including molecular clock signature, APOBEC, complex SVs, and MYC/RAS cooperation. This can further define a genomic risk model of disease progression independent of clinical tumor burden markers such as 2/20/20. This highlights the potential of WGS to solve the "missing predictability" of current clinical models of progression.

Robotic Construction and Screening of Lanthipeptide Variant Libraries in Escherichia coli.

Lanthipeptides are a major class of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by thioether cross-links called lanthionine (Lan) and methyllanthionine (MeLan). Previously, we developed a method to produce mature lanthipeptides in recombinant Escherichia coli, but manual steps hinder large-scale analogue screening. Here we devised an automated workflow for creating and screening variant libraries of haloduracin, a two-component class II lanthipeptide. An integrated work cell of a synthetic biology foundry was programmed to robotically execute DNA library construction, host transformation, peptide production, mass spectrometry analysis, and activity screening by agar diffusion assay. For recombinantly produced Halα peptides, the sequence-activity relationship of 380 single-residue variants and >1300 triple-residue combinatorial variants were rapidly analyzed in microplates within weeks. The peptide expression levels in E.coli were also visualized via robotic creation and analysis of GFP-lanthipeptide fusions for select peptide mutants. Following shake-flask fermentation and purification, one Halα mutant was confirmed with enhanced specific antimicrobial activity relative to the wild-type peptide. Overall, this approach may be generally applicable for the high-throughput characterization and engineering of RiPP natural products.

Clinical and genetic characteristics of the patients with hypertension and hypokalemia carrying a novel SCNN1A mutation

The objective of this study was to clinically and genetically characterize a pedigree with Liddle syndrome (LS). A LS pedigree comprising with one proband and seven family members was enrolled. The subjects’ symptoms, laboratory results and genotypes were analyzed. Peripheral venous samples were collected from the subjects, and genomic DNA was extracted. DNA library construction and exome capture were performed on an Illumina HiSeq 4000 platform. The selected variant sites were validated using Sanger sequencing. The mutation effects were investigated using prediction tools. The proband and her paternal male family members had mild hypertension, hypokalemia and muscle weakness, including the absence of low renin and low aldosterone. Genetic analysis revealed that the proband carried a compound heterozygous mutation in SCNN1A, a novel heterozygous mutation, c.1130T > G (p.Ile377Ser) and a previously characterized polymorphism, c.1987A > G (p.Thr633Ala). The novel mutation site was inherited in an autosomal dominant manner and was predicted by in silico tools to exert a damaging effect. Alterations in the SCNN1A domain were also predicted by protein structure modeling. After six months of follow-up, treatment had significantly improved the patient’s limb weakness and electrolyte levels. The novel mutation c.1130T > G of the SCNN1A gene was detected in the pedigree with LS. The clinical manifestations of the pedigree were described, which expand the phenotypic spectrum of LS. This result of this study also emphasizes the value of genetic testing for diagnosing LS.

An Optimized Transformation Protocol for Escherichia coli BW3KD with Supreme DNA Assembly Efficiency.

DNA cloning requires two steps: the assembly of recombinant DNA molecules and the transformation of the product into a host organism for replication. High efficiencies in both processes can increase the success rate. Recently, we developed an Escherichia coli BW3KD strain with higher transformation efficiency than commonly used cloning strains. Here, we further developed a simple method named TSS-HI (transformation storage solution optimized by Hannahan and Inoue method) for competent cell preparation, which combined the advantages of three common methods for operational simplicity and high transformation efficiency. When competent BW3KD cells were prepared using this developed method, the transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/μg DNA, which exceeded the levels of commercial chemically competent cells and homemade electrocompetent cells. BW3KD cells formed colonies within 7 h on lysogeny broth agar plates, quicker than the well-known fast-growing E. coli cloning strain Mach1. The competent cells worked effectively for the transformation of assembled DNA of 1 to 7 fragments in one step and promoted efficiencies of transformation or cloning with large plasmids. The cloning efficiency of BW3KD cells prepared by this method increased up to 828-fold over that of E. coli XL1-Blue MRF' cells prepared by a common method. Thus, competent cells are suitable for different cloning jobs and should help with the increased demand for DNA assembly in biological studies and biotechnology. IMPORTANCE DNA transformation is commonly used in cloning; however, high transformation efficiency becomes a limiting factor in many applications, such as the construction of CRISPR and DNA libraries, the assembly of multiple fragments, and the transformation of large plasmids. We developed a new competent cell preparation method with unmatched transformation efficiency. When the BW3KD strain, derived from Escherichia coli BW25113 cells, was prepared by this method, its transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/μg DNA, which broke the record for chemically prepared competent cells. Routine cloning could be completed in 1 day due to the high growth rate of this strain. The competent cells were shown to be highly efficient for transformation or cloning with large plasmids and for the assembly of multiple fragments. The results highlight the effectiveness of the new protocol and the usefulness of the BW3KD strain as the host.

Effect of feeding raw potato starch on the composition dynamics of the piglet intestinal microbiome.

Raw potato starch (RPS) is resistant to digestion, escapes absorption, and is metabolized by intestinal microflora in the large intestine and acts as their energy source. In this study, we compared the effect of different concentrations of RPS on the intestinal bacterial community of weaned piglets. Male weaned piglets (25-days-old, 7.03±0.49 kg) were either fed a corn/soybean-based control diet (CON, n = 6) or two treatment diets supplemented with 5% RPS (RPS5, n = 4) or 10% RPS (RPS10, n = 4) for 20 days and their fecal samples were collected. The day 0 and 20 samples were analyzed using a 16S rRNA gene sequencing technology, followed by total genomic DNA extraction, library construction, and high-throughput sequencing. After statistical analysis, five phyla and 45 genera accounting for over 0.5% of the reads in any of the three groups were further analyzed. Furthermore, short-chain fatty acids (SCFAs) in the day 20 fecal samples were analyzed using gas chromatography. Significant changes were not observed in the bacterial composition at the phylum level even after 20 d post feeding (dpf); however, the abundance of Intestinimonas and Barnesiella decreased in both RPS treatment groups compared to the CON group. Consumption of 5% RPS increased the abundance of Roseburia (p<0.05) and decreased the abundance of Clostridium (p<0.01) and Mediterraneibacter (p< 0.05). In contrast, consumption of 10% RPS increased the abundance of Olsenella (p<0.05) and decreased the abundance of Campylobacter (p<0.05), Kineothrix (p<0.05), Paraprevotella (p<0.05), and Vallitalea (p<0.05). Additionally, acetate (p<0.01), butyrate (p<0.05), valerate (p = 0.01), and total SCFAs (p = 0.01) were upregulated in the RPS5 treatment group. Feeding 5% RPS altered bacterial community composition and promoted gut health in weaned piglets. Thus, resistant starch as a feed additive may prevent diarrhea in piglets during weaning.

Noninvasive prenatal prediction of fetal haplotype with Spearman rank correlation analysis model.

BackgroundNoninvasive prenatal testing (NIPT) has been widely used clinically to detect fetal chromosomal aneuploidy with high accuracy rates, gradually replacing traditional serological screening. However, the application of NIPT for monogenic diseases is still in an immature stage of exploration. The detection of mutations in peripheral blood of pregnant women requires precise qualitative and quantitative techniques, which limits its application. The bioinformatic strategies based on the SNP (single nucleotide polymorphism) linkage analysis are more practical, which can be divided into two types depending on whether proband information is needed. Hidden Markov Mode (HMM) and Sequential probability ratio test (SPRT) are suitable for families with probands. In contrast, methods based on databases and population demographic information are suitable for families without probands.MethodsIn this study, we proposed a Spearman rank correlation analysis method to infer the fetal haplotypes based on core family information. Allele frequencies of SNPs that were used to construct parental haplotypes were calculated as sets of nonparametric variables, in contrast to their theoretical values represented by a fetal fraction (FF). The effects on the calculation of the fetal concentration of two DNA enrichment methods, multiple‐PCR amplification, and targeted hybrid capture, were compared, and the heterozygosity distribution of SNPs within pedigrees was analyzed to reveal the best conditions for the model application.ResultsPredictions of the paternal haplotype inheritance were in line with expectations for both DNA library construction methods, while for maternal haplotype inheritance prediction, the rates were 96.55% for method multiple‐PCR amplification and 95.8% for method targeted hybrid capture.ConclusionPositive prediction rates showed that the maternal haplotype prediction was not as accurate as paternal one, due to the large amount of maternal noise in the mother's peripheral blood. Although this model is relatively immature, it provides a new perspective for noninvasive prenatal clinical tests of monogenic diseases.

Phage Enzyme-Assisted Direct In Vivo DNA Assembly in Multiple Microorganisms

The assembly of DNA fragments is extremely important for molecular biology. Increasing numbers of studies have focused on streamlining the laborious and costly protocols via in vivo DNA assembly. However, the existing methods were mainly developed for Escherichia coli or Saccharomyces cerevisiae, whereas there are few direct in vivo DNA assembly methods for other microorganisms. The use of shuttle vectors and tedious plasmid extraction and transformation procedures make DNA cloning in other microorganisms laborious and inefficient, especially for DNA library construction. In this study, we developed a "phage enzyme-assisted in vivo DNA assembly" (PEDA) method via combinatorial expression of T5 exonuclease and T4 DNA ligase. PEDA facilitated the in vivo assembly of DNA fragments with homologous sequences as short as 5 bp, and it is applicable to multiple microorganisms, such as Ralstonia eutropha, Pseudomonas putida, Lactobacillus plantarum, and Yarrowia lipolytica. The cloning efficiency of optimized PEDA is much higher than that of the existing in vivo DNA assembly methods and comparable to that of in vitro DNA assembly, making it extremely suitable for DNA library cloning. Collectively, PEDA will boost the application of in vivo DNA assembly in various microorganisms.

Construction of Aerobic/Anaerobic-Substrate-Induced Gene Expression Procedure for Exploration of Metagenomes From Subseafloor Sediments.

Substrate-induced gene expression (SIGEX) is a high-throughput promoter-trap method. It is a function-based metagenomic screening tool that relies on transcriptional activation of a reporter gene green fluorescence protein (gfp) by a metagenomic DNA library upon induction with a substrate. However, its use is limited because of the relatively small size of metagenomic DNA libraries and incompatibility with screening metagenomes from anaerobic environments. In this study, these limitations of SIGEX were addressed by fine-tuning metagenome DNA library construction protocol and by using Evoglow, a green fluorescent protein that forms a chromophore even under anaerobic conditions. Two metagenomic libraries were constructed for subseafloor sediments offshore Shimokita Peninsula (Pacific Ocean) and offshore Joetsu (Japan Sea). The library construction protocol was improved by (a) eliminating short DNA fragments, (b) applying topoisomerase-based high-efficiency ligation, (c) optimizing insert DNA concentration, and (d) column-based DNA enrichment. This led to a successful construction of metagenome DNA libraries of approximately 6 Gbp for both samples. SIGEX screening using five aromatic compounds (benzoate, 3-chlorobenzoate, 3-hydroxybenzoate, phenol, and 2,4-dichlorophenol) under aerobic and anaerobic conditions revealed significant differences in the inducible clone ratios under these conditions. 3-Chlorobenzoate and 2,4-dichlorophenol led to a higher induction ratio than that for the other non-chlorinated aromatic compounds under both aerobic and anaerobic conditions. After the further screening of induced clones, a clone induced by 3-chlorobenzoate only under anaerobic conditions was isolated and characterized. The clone harbors a DNA insert that encodes putative open reading frames of unknown function. Previous aerobic SIGEX attempts succeeded in the isolation of gene fragments from anaerobes. This study demonstrated that some gene fragments require a strict in vivo reducing environment to function and may be potentially missed when screened by aerobic induction. The newly developed anaerobic SIGEX scheme will facilitate functional exploration of metagenomes from the anaerobic biosphere.

Application of nanopore sequencing in diagnosis of secondary infections in patients with severe COVID-19.

To explore the application value of nanopore sequencing technique in the diagnosis and treatment of secondary infections in patients with severe coronavirus disease 2019 (COVID-19). A total of 77 clinical specimens from 3 patients with severe COVID-19 were collected. After heat inactivation, all samples were subjected to total nucleic acid extraction based on magnetic bead enrichment. The extracted DNA was used for DNA library construction, then nanopore real-time sequencing detection was performed. The sequencing data were subjected to Centrifuge software database species matching and R program differential analysis to obtain potential pathogen identification. Nanopore sequencing results were compared with respiratory pathogen qPCR panel screening and conventional microbiological testing results to verify the effectiveness of nanopore sequencing detection. Nanopore sequencing results showed that positive pathogen were obtained in 44 specimens (57.1%). The potential pathogens identified by nanopore sequencing included , , and , et al. , , were also detected in clinical microbiological culture-based detection; was detected in respiratory pathogen screening qPCR panel; was only detected by the nanopore sequencing technique. Comprehensive considerations with the clinical symptoms, the patient was treated with antibiotics against , and the infection was controlled. Nanopore sequencing may assist the diagnosis and treatment of severe COVID-19 patients through rapid identification of potential pathogens.

Efficient Linear dsDNA Tagging Using Deoxyuridine Excision*.

Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3' ssDNA overhangs in gene assembly and molecular cloning procedures. Unlike approaches that use a multi-base pair motif to specify a DNA cut site, dU excision requires only a dT→dU substitution. Consequently, excision sites can be embedded in biologically active DNA sequences by placing dU substitutions at non-perturbative positions. In this work, I describe a molecular tagging method that uses dU excision to exchange a segment of a dsDNA strand with a long synthetic oligonucleotide. The core workflow of this method, called deoxyUridine eXcision-tagging (dUX-tagging), is an efficient one-pot reaction: strategically positioned dU nucleotides are excised from dsDNA to generate a 3' overhang so that additional sequence can be appended by annealing and ligating a tagging oligonucleotide. The tagged DNA is then processed by one of two procedures to fill the 5' overhang and remove excess tagging oligo. To facilitate its widespread use, all dUX-tagging procedures exclusively use commercially available reagents. As a result, dUX-tagging is a concise and easily implemented approach for high-efficiency linear dsDNA tagging.

The diagnostic yield of whole exome sequencing as a first approach in consanguineous Omani renal ciliopathy syndrome patients.

Background: Whole exome sequencing (WES) is becoming part of routine clinical and diagnostic practice. In the investigation of inherited cystic kidney disease and renal ciliopathy syndromes, WES has been extensively applied in research studies as well as for diagnostic utility to detect various novel genes and variants. The yield of WES critically depends on the characteristics of the patient population. Methods: In this study, we selected 8 unrelated Omani children, presenting with renal ciliopathy syndromes with a positive family history and originating from consanguineous families. We performed WES in affected children to determine the genetic cause of disease and to test the yield of this approach, coupled with homozygosity mapping, in this highly selected population. DNA library construction and WES was carried out using SureSelect Human All Exon V6 Enrichment Kit and Illumina HiSeq platform. For variants filtering and annotation Qiagen Variant Ingenuity tool was used. Nexus copy number software from BioDiscovery was used for evaluation of copy number variants and whole gene deletions. Patient and parental DNA was used to confirm mutations and the segregation of alleles using Sanger sequencing. Results: Genetic analysis identified 4 potential causative homozygous variants each confirmed by Sanger sequencing in 4 clinically relevant ciliopathy syndrome genes, ( TMEM231, TMEM138, WDR19 and BBS9), leading to an overall diagnostic yield of 50%. Conclusions: WES coupled with homozygosity mapping provided a diagnostic yield of 50% in this selected population. This genetic approach needs to be embedded into clinical practise to allow confirmation of clinical diagnosis, to inform genetic screening as well as family planning decisions. Half of the patients remain without diagnosis highlighting the technical and interpretational hurdles that need to be overcome in the future.

The diagnostic yield of whole exome sequencing as a first approach in consanguineous Omani renal ciliopathy syndrome patients

Background: Whole exome sequencing (WES) is becoming part of routine clinical and diagnostic practice. In the investigation of inherited cystic kidney disease and renal ciliopathy syndromes, WES has been extensively applied in research studies as well as for diagnostic utility to detect various novel genes and variants. The yield of WES critically depends on the characteristics of the patient population. Methods: In this study, we selected 8 unrelated Omani children, presenting with renal ciliopathy syndromes with a positive family history and originating from consanguineous families. We performed WES in affected children to determine the genetic cause of disease and to test the yield of this approach, coupled with homozygosity mapping, in this highly selected population. DNA library construction and WES was carried out using SureSelect Human All Exon V6 Enrichment Kit and Illumina HiSeq platform. For variants filtering and annotation Qiagen Variant Ingenuity tool was used. Nexus copy number software from BioDiscovery was used for evaluation of copy number variants and whole gene deletions. Patient and parental DNA was used to confirm mutations and the segregation of alleles using Sanger sequencing. Results: Genetic analysis identified 4 potential causative homozygous variants each confirmed by Sanger sequencing in 4 clinically relevant ciliopathy syndrome genes, ( TMEM231, TMEM138, WDR19 and BBS9), leading to an overall diagnostic yield of 50%. Conclusions: WES coupled with homozygosity mapping provided a diagnostic yield of 50% in this selected population. This genetic approach needs to be embedded into clinical practise to allow confirmation of clinical diagnosis, to inform genetic screening as well as family planning decisions. Half of the patients remain without diagnosis highlighting the technical and interpretational hurdles that need to be overcome in the future.

Feasibility of applying shotgun metagenomic analyses to grapevine leaf, rhizosphere and soil microbiome characterisation

Background and Aims The characterisation of plant microbiomes using metabarcoding strategies is expected to be progressively replaced by shotgun metagenomic (SMg) approaches. In the present report we explore the potential of applying SMg to grapevine leaf, rhizosphere and soil samples. Methods and Results Our strategy involved combining a single method for column-based DNA extraction of multiple tissues, with sequencing using the BGISEQ short-read next-generation sequencing platform. This study details aspects of DNA isolation, library construction, sequencing and early bioinformatics treatment of read data, including the use of subsampling as a proxy for detection of sample microbial proportions. The combination of an innovative sequencing platform with recent mapping tools allowed for the characterisation of the microbiome associated with the grapevine leaf, rhizosphere and adjacent soil. Conclusions We coupled a robust single extraction/library preparation protocol suitable for contrasting samples, with cost-effective BGISEQ short-read next-generation sequencing technology, to generate a landscape of grapevine-associated microbial diversity. Significance of the Study Shotgun metagenomics is emerging as a state-of-the-art approach to grapevine microbiome characterisation. The present report provides a detailed and technical roadmap targeting multiple aspects of this important approach.