Genomic Fingerprinting Research Articles

Next generation drug connectivity mapping for acquiring therapeutic agents to differentially regulate myelination.

Next generation drug connectivity mapping for acquiring therapeutic agents to differentially regulate myelination.

A Personalized Digital Code from Unique Genome Fingerprinting Pattern for Use in Identification and Application on Blockchain

With over 10 million points of genetic variation from person to person, every individual’s genome is unique and provides a highly reliable form of identification. This is because the genetic code is specific to each individual and does not change over time. Genetic information has been used to identify individuals in a variety of contexts, such as criminal investigations, paternity tests, and medical research. In this study, each individual’s genetic makeup has been formatted to create a secure, unique code that incorporates various elements, such as species, gender, and the genetic identification code itself. The combinations of markers required for this code have been derived from common single nucleotide polymorphisms (SNPs), points of variation found in the human genome. The final output is in the form of a 24 numerical code with each number having three possible combinations. The custom code can then be utilized to create various modes of identification on the decentralized blockchain network as well as personalized services and products that offer users a novel way to uniquely identify themselves in ways that were not possible before.

First Report of Potentially Pathogenic Klebsiella pneumoniae from Serotype K2 in Mollusk Tegillarca granosa and Genetic Diversity of Klebsiella pneumoniae in 14 Species of Edible Aquatic Animals.

Klebsiella pneumoniae can cause serious pneumonitis in humans. The bacterium is also the common causative agent of hospital-acquired multidrug-resistant (MDR) infections. Here we for the first time reported the genetic diversity of K. pneumoniae strains in 14 species of edible aquatic animals sampled in the summer of 2018 and 2019 in Shanghai, China. Virulence-related genes were present in the K. pneumoniae strains (n = 94), including the entB (98.9%), mrkD (85.1%), fimH (50.0%), and ybtA (14.9%) strains. Resistance to sulfamethoxazole-trimethoprim was the most prevalent (52.1%), followed by chloramphenicol (31.9%), and tetracycline (27.7%), among the strains, wherein 34.0% had MDR phenotypes. Meanwhile, most strains were tolerant to heavy metals Cu2+ (96.8%), Cr3+ (96.8%), Zn2+ (91.5%), Pb2+ (89.4%), and Hg2+ (81.9%). Remarkably, a higher abundance of the bacterium was found in bottom-dwelling aquatic animals, among which mollusk Tegillarca granosa contained K. pneumoniae 8-2-5-4 isolate from serotype K2 (ST-2026). Genome features of the potentially pathogenic isolate were characterized. The enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR)-based genome fingerprinting classified the 94 K. pneumoniae strains into 76 ERIC genotypes with 63 singletons, demonstrating considerable genetic diversity in the strains. The findings of this study fill the gap in the risk assessment of K. pneumoniae in edible aquatic animals.

Epidemiology, Evolution of Antimicrobial Profile and Genomic Fingerprints of Pseudomonas aeruginosa before and during COVID-19: Transition from Resistance to Susceptibility.

The purpose of the study was to describe the epidemiological implication of Pseudomonas aeruginosa between 2017-2022 in a tertiary hospital from Romania, including the molecular fingerprinting of similar phenotypic strains (multidrug-resistant isolates), which would have an important health impact. The study also describes the resistance profile of P. aeruginosa before and during COVID-19, which might bring new information regarding the management of antibiotic treatments. Information regarding wards, specimen types, species, and antibiotic resistance profile of 1994 strains of Pseudomonas spp. Isolated over a period of 6 years in Mures Clinical County Hospital, Romania, was collected from the WHONET database. From 50 multidrug-resistant isolates, molecular fingerprinting was performed by Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) to prove the potential clonal distribution. A number of 1994 Pseudomonas spp. were isolated between 2017-2022, from which P. aeruginosa was the most frequent species, 97.39% (n = 1942). P. aeruginosa was most frequently isolated in 2017 (n = 538), with the dermatology department as the main source, mainly from pus secretion. A drop in the harvesting rate was noted in 2020 due to COVID-19 restrictions. Regarding the resistance profile, there are a few modifications. The susceptibility of P. aeruginosa to carbapenems, piperacillin-tazobactam, and amikacin suffered alterations before and during COVID-19. The molecular fingerprinting showed three P. aeruginosa clusters, including strains with 80-99% similarity.

Identification and Potential Biotechnological Characterization of Lactic Acid Bacteria Isolated from White Cheese Samples

In this study, the isolation of lactic acid bacteria was carried out from one hundred white cheese samples collected from different regions of Turkey. Subsequently, phenotypic and genotypic characterization of the isolates was performed. Biochemical characteristics of the isolates were determined by API 50CHL. Furthermore, the biotechnological enzyme production potential of the isolates was screened. Genomic fingerprint profiles of the test isolates were detected by using rep-PCR (BOX-PCR), which has been used successfully in the differentiation of microorganisms at the species, subspecies, and even strain levels. The results showed that a total of forty-one bacteria were isolated and seventeen of which are found to be different species. The isolates generally grew at 4-6 pH values, 0-8% NaCl and 30-40°C. Later, isolates thought to be different species were identified by 16S rRNA gene sequence analyses. According to 16S rRNA sequence results, MA56 showed a 96.41% similarity match to Lentilactobacillus buchneri, it is thought to be a new species. In addition, MA19, MA25, MA43, and MA47 were determined to have multi-enzyme production potential. MA43 has a plantaricin gene and it showed a high antagonistic effect on Escherichia coli O157:H7 ATCC 43888 and Pseudomonas aeruginosa ATCC 9027. Inhibition zones were measured at 19 mm and 16 mm respectively.

Multivariate analysis of chemical and genetic diversity of wild Humulus lupulus L. (hop) collected in situ in northern France

The hop plant (Humulus lupulus L.) has been exploited for a long time for both its brewing and medicinal uses, due in particular to its specific chemical composition. These last years, hop cultivation that was in decline has been experiencing a renewal for several reasons, such as a craze for strongly hopped aromatic beers. In this context, the present work aims at investigating the genetic and chemical diversity of fifty wild hops collected from different locations in Northern France. These wild hops were compared to ten commercial varieties and three heirloom varieties cultivated in the same sampled geographical area. Genetic analysis relying on genome fingerprinting using 11 microsatellite markers showed a high level of diversity. A total of 56 alleles were determined with an average of 10.9 alleles per locus and assessed a significant population structure (mean pairwise FST = 0.29). Phytochemical characterization of hops was based on volatile compound analysis by HS-SPME GC-MS, quantification of the main prenylated phenolic compounds by UHPLC-UV as well as untargeted metabolomics by UHPLC-HRMS and revealed a high level of chemical diversity among the assessed wild accessions. In particular, analysis of volatile compounds revealed the presence of some minor but original compounds, such as aromadendrene, allo-aromadendrene, isoledene, β-guaiene, α-ylangene and β-pinene in some wild accessions; while analysis of phenolic compounds showed high content of β-acids in these wild accessions, up to 2.37% of colupulone. Genetic diversity of wild hops previously observed was hence supported by their chemical diversity. Sample soil analysis was also performed to get a pedological classification of these different collection sites. Results of the multivariate statistical analysis suggest that wild hops constitute a huge pool of chemical and genetic diversity of this species.

The Rampancy of Multiple- Drug Resistant Bacteria Isolated from Surface Water of Satow Waterfall, Bau Sarawak

Antimicrobial resistance (AMR) is universally acknowledged as a serious threat to human health, well-being, and prosperity. Many therapeutically important antibiotic resistance genes are thought to have arisen in the environment. Antibiotics are no longer effective in treating infections caused by multi-and pan-resistant bacteria (dubbed "superbugs") that have spread rapidly around the world [1]. Furthermore, AMR bacteria and antimicrobial resistant genes can thrive and proliferate in natural waterbodies because they can act as both recipients and natural reservoirs for the bacteria and genes. Toxic exposure to AMR bacteria and ARGs puts recreationists in danger, reducing their ability to combat illnesses.
 
 Considering Satow Waterfall, Bau is a renowned water-based recreational destination, an assessment of their physiochemical water quality characteristics and the prevalence of AMR bacteria was conducted to corroborate the safety of the water. A total of nine water samples were collected at the surface water (0–30 cm depth) of the upstream, midstream, and downstream. The water quality index (WQI) of the sampling stations ranged from 92.60 to 95, classifying the water under Class I. However, the water is microbiologically polluted. During Trip 2, the highest level of coliform was found in the middle stream at 2.59x105 cfu/mL, which is above the regulatory standard set by the Malaysian Department of Environment (DOE).
 
 A total of 100 isolated bacteria were subjected to (GTG)5- genomic fingerprinting analysis to evaluate the degree of genetic similarity among the samples and thus enable the selection of appropriate clonal representatives. Fifty-four bacterial isolates were chosen as representatives and identified by 16SrDNA sequencing, which confirmed the existence of 21 bacterial genera as shown in Figure 1. All identified isolates were tested against 15 antibiotics of various classes, namely, streptomycin (S), Kanamycin (K), cefotaxime (CTX), cephalothin (KF), tetracycline (TE), doxycycline (DO), carbenicillin (CAR), sulfamethoxazole (SXT), ofloxacin (OFX), levofloxacin (LEV), erythromycin (E), meropenem (MEM), ertapenem (ETP), vancomycin (VA) and chloramphenicol (C) employing the Clinical and Laboratory Standards Institute (CLSI) protocols.
 
 The antibiotic susceptibility test (AST) demonstrated substantial resistance to SXT (41%), E (41%) and CAR (39%), as well as high susceptibility to C (85%), followed by LEV (78%) and OFX (72%). The Multiple Antibiotic Resistance Index (MARI) evaluation demonstrated the MARI varied from 0–0.60, with 33% of the isolates having a MARI greater than 0.20 (Figure 1). 
 In this study, we found that the occurrence of MAR bacteria in water environment where there's an undisclosed source of antibiotic utilisation is still inevitable, highlighting the necessity to constantly monitor the water quality and the pervasiveness of MAR bacteria in recreational water.

Evaluation of ERIC-PCR method for determining genetic diversity among Escherichia coli isolated from human and retail imported frozen shrimp and beef

There is a global concern and increasing reports regarding foodborne disease infections associated with consuming contaminated vegetables, seafood, meat, and poultry products. Among foodborne bacterial pathogens globally, Salmonella, Escherichia coli, and Shigella were the most frequently implicated in causing food poisoning infections in children and adults. In Saudi Arabia, the consumption rates of imported fresh fruits, vegetables, seafood, and meat products are considered high. Therefore, the development of simple PCR based DNA fingerprinting methods is essential to track the source and route of microbial contamination among imported frozen meat and seafood products. A total of 38 E. coli strains were subtyped using ERIC1R, ERIC2, and a pair combination (ERIC1R + ERIC2) to generate genomic fingerprinting. The three Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR)-based primers were generated in 26, 24, and 16 different genotypes while using ERIC1R, ERIC2, and ERIC1R + ERIC2, respectively. The Discrimination Index values obtained by ERIC1R, ERIC2, and ERIC1R + ERIC2 were 0.976, 0.965, and 0.903, respectively. ERIC1R and ERIC2 primers had the best discriminatory ability and typeability value and proved suitable for investigating genetic analysis among the population of E. coli strains. At the same time, the ERIC1R + ERIC2 primer pair has average discriminatory power and typeability value for differentiating E. coli strains. These results suggest that subtyping using ERIC1R and ERIC2 primer is a more reliable and rapid typing strategy for E. coli strains.

Pseudomonas syringae pv. syringae causes bacterial canker on Japanese quince (Chaenomeles japonica)

Japanese quince trees are grown as ornamental plants in Iran, in parks and in orchards close to stone fruit and pome fruit trees. Shoots of Japanese quince (Chaenomeles japonica) showing sunken brown canker symptoms were observed and collected near Sari, the center of Mazandaran province in the North of Iran, during the 2016 growing season. Gram negative bacteria isolated from symptomatic tissues were similar to Pseudomonas syringae pv. syringae (Pss) were pathogenic on Japanese quince and on quince (Cydonia oblonga) seedlings after artificial inoculation, and were re-isolated from diseased hosts. Phylogenetic tree construction using partial sequences of ITS and rpoD genes showed that the Japanese quince isolates were in the same clade as Pss strains. The isolates had ice nucleation activity, and the InaK gene was amplified successfully. According to the results of phenotypic and genotypic characteristics, genomic DNA fingerprinting using REP-PCR, BOX-PCR and IS50-PCR and isolation of total cell proteins, we conclude that Pss is the causal agent of canker of the Japanese quince trees. Therefore, Japanese quince is a new host for Pss causing bacterial canker on many different host plants.

Metagenomic features of bioburden serve as outcome indicators in combat extremity wounds

Battlefield injury management requires specialized care, and wound infection is a frequent complication. Challenges related to characterizing relevant pathogens further complicates treatment. Applying metagenomics to wounds offers a comprehensive path toward assessing microbial genomic fingerprints and could indicate prognostic variables for future decision support tools. Wound specimens from combat-injured U.S. service members, obtained during surgical debridements before delayed wound closure, were subjected to whole metagenome analysis and targeted enrichment of antimicrobial resistance genes. Results did not indicate a singular, common microbial metagenomic profile for wound failure, instead reflecting a complex microenvironment with varying bioburden diversity across outcomes. Genus-level Pseudomonas detection was associated with wound failure at all surgeries. A logistic regression model was fit to the presence and absence of antimicrobial resistance classes to assess associations with nosocomial pathogens. A. baumannii detection was associated with detection of genomic signatures for resistance to trimethoprim, aminoglycosides, bacitracin, and polymyxin. Machine learning classifiers were applied to identify wound and microbial variables associated with outcome. Feature importance rankings averaged across models indicated the variables with the largest effects on predicting wound outcome, including an increase in P. putida sequence reads. These results describe the microbial genomic determinants in combat wound bioburden and demonstrate metagenomic investigation as a comprehensive tool for providing information toward aiding treatment of combat-related injuries.

Bacterial Community with Plant Growth-Promoting Potential Associated to Pioneer Plants from an Active Mexican Volcanic Complex.

Microorganisms in extreme volcanic environments play an important role in the development of plants on newly exposed substrates. In this work, we studied the structure and diversity of a bacterial community associated to Andropogon glomeratus and Cheilanthes aemula at El Chichón volcano. The genetic diversity of the strains was revealed by genomic fingerprints and by 16S rDNA gene sequencing. Furthermore, a metagenomic analysis of the rhizosphere samples was carried out for pioneer plants growing inside and outside the volcano. Multifunctional biochemical tests and plant inoculation assays were evaluated to determine their potential as plant growth-promoting bacteria (PGPB). Through metagenomic analysis, a total of 33 bacterial phyla were identified from A. glomeratus and C. aemula rhizosphere samples collected inside the volcano, and outside the volcano 23 bacterial phyla were identified. For both rhizosphere samples, proteobacteria was the most abundant phylum. With a cultivable approach, 174 bacterial strains were isolated from the rhizosphere and tissue of plants growing outside the volcanic complex. Isolates were classified within the genera Acinetobacter, Arthrobacter, Bacillus, Burkholderia, Cupriavidus, Enterobacter, Klebsiella, Lysinibacillus, Pantoea, Pseudomonas, Serratia, Stenotrophomonas and Pandoraea. The evaluated strains were able to produce indole compounds, solubilize phosphate, synthesize siderophores, showed ACC deaminase and nitrogenase activity, and they had a positive effect on the growth and development of Capsicum chinense. The wide diversity of bacteria associated to pioneer plants at El Chichón volcano with PGPB qualities represent an alternative for the recovery of eroded environments, and they can be used efficiently as biofertilizers for agricultural crops growing under adverse conditions.

Natural Language Processing Approaches for Retrieval of Clinically Relevant Genomic Information in Cancer.

The accelerating impact of genomic data in clinical decision-making has generated a paradigm shift from treatment based on the anatomic origin of the tumor to the incorporation of key genomic features to guide therapy. Assessing the clinical validity and utility of the genomic background of a patient’s cancer represents one of the emerging challenges in oncology practice, demanding the development of automated platforms for extracting clinically relevant genomic information from medical texts. We developed PubMiner, a natural language processing tool to extract and interpret cancer type, therapy, and genomic information from biomedical abstracts. Our initial focus has been the retrieval of gene names, variants, and negations, where PubMiner performed highly in terms of total recall (91.7%) with a precision of 79.7%. Our next steps include developing a web-based interface to promote personalized treatment based on each tumor’s unique genomic fingerprints.

P-481 Characterization of the gene expression profile of breast cancer tumours in patients undergoing ovarian stimulation for fertility preservation purposes

Abstract Study question Could controlled ovarian stimulation (COS) protocols with letrozole supplementation induce changes in the molecular footprints of estrogen receptor-positive (ER+) breast cancer (BC) women? Summary answer Ovarian stimulation with letrozole and gonadotropins does not change the gene expression profile of estrogen receptor-positive (ER+) breast malignant tumours as compared to non-stimulated patients. What is known already Fertility preservation (FP) strategies in ER+ BC patients involve modifications of the classical COS protocols to minimise the impact of estrogen levels during ovarian stimulation. The most common approaches are using the aromatase inhibitor letrozole to block the conversion of testosterone into estradiol in the granulosa cells or the use of tamoxifen to block the ER in the breast tissue. Although retrospective studies suggest that these strategies will have no impact on patient’s survival and disease progression, little is known regarding the effect of the ovarian stimulation on the genomic fingerprints of the exposed cancerous tissue. Study design, size, duration Retrospective, non-randomized, comparative study. Gene expression profiles were compared in biopsies at diagnosis and during excision surgery in two groups of ER+ BC patients: Patients undergoing COS for FP before surgery (COS group, n = 10) and patients not undergoing any type of COS (Control group, n = 11). The former received letrozole (5mg/day) supplements during COS. The latter included 7 patients not doing FP and 4 undergoing ovarian cortex cryopreservation. Patients were recruited between 2009 and 2021. Participants/materials, setting, methods Single 5-µm sections of formalin-fixed, paraffin-embedded (FFPE) needle core biopsy (NCB) and breast surgery (BS) tumour samples were obtained prior to starting gonadotoxic therapies. Differential gene expression and gene set analysis (GSA) was performed in tumour samples before (NCB-sample) and after COS (BS-sample). Simultaneous, quantitative detection of 2549 genes associated with tumour biology was performed with the HTG EdgeSeq Oncology Biomarker Panel. Tumour cell proliferation was also assessed by Ki67 staining. Main results and the role of chance Patients were younger in the COS group (COS: 30.2±3.4 vs Control: 34.5±2.3; p = 0.004). None of the patients experienced any relapse during the observation period. The length of COS was 11.5±3.5 days and 10.6±6.8 MII oocytes were vitrified. The exploratory analysis using principal component analysis revealed no relationships between the two BC biopsy samples and gene expression levels in the experimental groups. The differential expression analysis just revealed 6 genes significantly over-expressed after ovarian stimulation (DUSP1, FOS, EGFR1, NR4A1, JUN and CYR61). From these, DUSP1, FOS, and NR4A1 were also significantly upregulated in the unstimulated group. The GSA showed the cytochrome P450 pathway was significantly enriched after COS. No pathways related to cell proliferation were differentially expressed between groups. However, in unstimulated patients, 6 KEGG pathways were upregulated, with the cytokine-cytokine receptor interaction and the Jak-STAT signalling pathway being the most enriched. Ki67 immunostaining showed no differences in cell proliferation after COS (22.8±7.2 vs 24.0±6.9, p = 0.723). The rate of cell proliferation also remained constant in unstimulated patients (22.9±8.4 vs 25.1±8.1, p = 0.560). Limitations, reasons for caution The main limitations of the present study are the retrospective design and the associated risk of bias, although the population is very homogeneous when it comes to clinical characteristics; and the fact that molecular footprints are only interim markers of survival/response to the treatment. Wider implications of the findings The present study provides molecular evidence supporting the safety of COS using letrozole for oocyte vitrification in young BC patients undergoing ovarian stimulation. This information is crucial to support patient guidance during discussions about fertility preservation. Trial registration number Not applicable

Genomic Diversity of Bradyrhizobium from the Tree Legumes Inga and Lysiloma (Caesalpinioideae-Mimosoid Clade)

We identified diverse bradyrhizobia having distinct ERIC-PCR genomic fingerprints from native American trees Inga vera and Lysiloma spp. In addition, two nodule isolates recovered from Lysiloma divaricatum seedlings inoculated with soil from the tropical house of a UK botanical garden were also identified as Bradyrhizobium. Genomes were obtained (with sizes around 9 Mb each) from nine Inga and Lysiloma bradyrhizobial isolates; plasmids were detected in two of the Inga strains analyzed. Average Nucleotide Identity of whole genomes revealed five novel Bradyrhizobium genomospecies from Mexican trees, while the UK isolates were identified as Bradyrhizobium cajani and Bradyrhizobium brasilense. Inga vera and Lysiloma isolates, despite their genetic distances and different hosts, shared a common set of nod genes that suggested that I. vera and Lysiloma bradyrhizobia produce fucosylated, methylated and carbamylated lipochitooligosaccharides. Uptake hydrogenase hup, hyp and secretion system genes were found in some of the isolates. Lysiloma strains were found to be ineffective on I. vera. Some of the isolates may be used as plant inoculants.

Early Epigenetic Responses in the Genomic DNA Methylation Fingerprints in Cells in Response to Sublethal Exposure of Silver Nanoparticles.

With the rapid development of nanotechnology and nanoscience, nanosafety assessment has raised public concern. Although many studies have illustrated that nanomaterials could lead to genotoxicity, the early alterations of DNA methylation with nanomaterials under low-dose exposure have not been completely clear. In this study, we investigated the potential effect and molecular mechanism of AgNPs on the alternation of DNA methylation fingerprints in HEK293T cells under sublethal exposure. Intriguingly, silver nanoparticle treatment increased 5-mC level and changed methylation-related enzyme contents. Mechanistically, we scrutinized the changes in the molecular signaling and biological functions by means of MeDIP-Seq and RNA-seq. Our results revealed that AgNPs might undermine a number of vital regulatory networks including the metabolic processes, biological regulation and other cellular processes. More specifically at the DNA methylation fingerprints, there were 12 up-regulated and simultaneous hypomethylated genes, and 22 down-regulated and concomitant hypermethylated genes in HEK293T cells responding to AgNPs. Notably, these genes were primarily involved in lipid metabolism and ion metabolism. Together, these responsive genes might be used as early sensitive indicators for the variations of early epigenetic integrity through changing the DNA methylation fingerprints, as reflective of biological risk and toxicity of silver nanoparticles under realistic exposure scenarios.

Profiling the Diversity of Sweet Pepper ‘Peperone Cornetto di Pontecorvo’ PDO (Capsicum annuum) through Multi-Phenomic Approaches and Sequencing-Based Genotyping

The sweet pepper (Capsicum annuum) ‘Peperone Cornetto di Pontecorvo’ is a prominent local variety at risk of genetic erosion cultivated in the Latium region (Italy). This horn-shaped landrace is recognized for its high digestibility due to the thinness of the skin and has been granted the Protected Designation of Origin (PDO) mark since 2010. Nowadays, different accessions are claimed as ‘Peperone Cornetto di Pontecorvo’ and no assay has been conducted to determine authenticity. In this study, 14 ‘Peperone Cornetto di Pontecorvo’ accessions and 7 similar horn-type peppers were investigated for their morpho-agronomic performance and chemical composition. Digital fruit imaging was implemented as a tool to pinpoint with high accuracy the morphometric parameters of berries. In total, 52 traits were scored. The multivariate analysis revealed different clusters that separated ‘Peperone Cornetto di Pontecorvo’ from similar types. The weight and size of fruits, as well as the content of soluble solids, were the most discriminating factors among the cultivars studied. Genomic fingerprinting was performed using ddRAD sequencing, yielding a total of a total of 120 million raw sequences and 2196 high-quality SNPs. Both Bayesian and hierarchical clustering analyses confirmed the existence of two different (K = 2) sub-populations separating ‘Peperone Cornetto di Pontecorvo’ accessions from similar types, thus highlighting a high membership (qi > 0.97) coefficient for accessions cultivated in the Pontecorvo area (Frosinone district). In addition, a direct relationship was found between the genetic diversity of cultivars and their geographical provenance, providing hints on the breeding history of local varieties in diverse rural areas. Genomic markers are revealed as a valuable tool to establish the uniqueness and distinctness of this local variety. This information will be very helpful for recovery, enhancement, and protection from possible imitations.

Quality control of large genome datasets.

SummaryThe 1000 Genomes Project (TGP) is a foundational resource that serves the biomedical community as a standard reference cohort for human genetic variation. There are now seven public versions of these genomes. The TGP Consortium produced the first by mapping its final data release against human reference sequence GRCh37, then “lifted over” these genomes to the improved reference sequence (GRCh38) when it was released, and remapped the original data to GRCh38 with two similar pipelines. As best-practice quality validation, the pipelines that generated these versions were benchmarked against the Genome In A Bottle Consortium’s “platinum quality” genome (NA12878). The New York Genome Center recently released the results of independently resequencing the cohort at greater depth (30×), a phased version informed by the inclusion of related individuals, and independently remapped the original variant calls to GRCh38. We performed a cross-comparison evaluation of all seven versions using genome fingerprinting, which supports ultrafast genome comparison even across reference versions. We noted multiple issues, including discrepancies in cohort membership, disagreement on the overall level of variation, evidence of substandard pipeline performance on specific genomes and in specific regions of the genome, cryptic relationships between individuals, inconsistent phasing, and annotation distortions caused by the history of the reference genome itself. We therefore recommend global quality assessment by rapid genome comparisons, alongside benchmarking as part of best-practice quality assessment of large genome datasets. Our observations also help inform the decision of which version to use, to support analyses by individual researchers.

Isolation of Hermetia illucens larvae core gut microbiota by two different cultivation strategies

Hermetia illucens larvae (black soldier fly larvae, BSFL) convert efficiently organic waste to high quality biomass. To gain knowledge on the specific functions of gut microbes in this process it is a prerequisite to culture members of the core gut microbiota. Two different cultivation strategies were applied here for this purpose, a dilution-to-extinction cultivation and direct plating using six different media to culture aerobic heterotrophic bacteria. A total of 341 isolates were obtained by the dilution-to-extinction cultivation and 138 isolates by direct plating from guts of BSFL reared on chicken feed. Bacterial isolates were phylogenetically identified at the genus level by 16S rRNA gene sequencing (phylotyping) and differentiated at the strain level by genomic fingerprinting (genotyping). The main proportion of isolates was assigned to Proteobacteria, Firmicutes (Bacilli), and Actinobacteria. Predominant genera discussed in literature as member of a potential BSFL core gut microbiota, Providencia, Proteus, Morganella, Enterococcus, Bacillus, and members of the family Enterobacteriaceae, were isolated. A high intra-phylotype diversity was obtained by genomic fingerprinting which was especially enhanced by the dilution-to-extinction cultivation. This study showed that the application of different cultivation strategies including a dilution-to-extinction cultivation helps to culture a higher diversity of the BSFL gut microbiota and that genomic fingerprinting gives a better picture on the genetic diversity of cultured bacteria which cannot be covered by a 16S rRNA gene sequence based identification alone.

RaS-RiPPs in Streptococci and the Human Microbiome.

Radical S-adenosylmethionine (RaS) enzymes have quickly advanced to one of the most abundant and versatile enzyme superfamilies known. Their chemistry is predicated upon reductive homolytic cleavage of a carbon–sulfur bond in cofactor S-adenosylmethionine forming an oxidizing carbon-based radical, which can initiate myriad radical transformations. An emerging role for RaS enzymes is their involvement in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a natural product family that has become known as RaS-RiPPs. These metabolites are especially prevalent in human and mammalian microbiomes because the complex chemistry of RaS enzymes gives rise to correspondingly complex natural products with minimal cellular energy and genomic fingerprint, a feature that is advantageous in microbes with small, host-adapted genomes in competitive environments. Herein, we review the discovery and characterization of RaS-RiPPs from the human microbiome with a focus on streptococcal bacteria. We discuss the varied chemical modifications that RaS enzymes introduce onto their peptide substrates and the diverse natural products that they give rise to. The majority of RaS-RiPPs remain to be discovered, providing an intriguing avenue for future investigations at the intersection of metalloenzymology, chemical ecology, and the human microbiome.

Structural analysis of random transgene integration in CHO manufacturing cell lines by targeted sequencing.

Genetically modified CHO cell lines are traditionally used for the production of biopharmaceuticals. However, an in-depth molecular understanding of the mechanism and exact position of transgene integration into the genome of pharmaceutical manufacturing cell lines is still scarce. Next-generation sequencing (NGS) holds great promise for strongly facilitating the understanding of CHO cell factories, as it has matured to a powerful and affordable technology for cellular genotype analysis. Targeted Locus Amplification (TLA) combined with NGS allows for robust detection of genomic positions of transgene integration and structural genomic changes occurring upon stable integration of expression vectors. TLA was applied to generate comparative genomic fingerprints of several CHO production cell lines expressing different monoclonal antibodies. Moreover, high producers resulting from an additional round of transfection of an existing cell line (supertransfection) were analyzed to investigate the integrity and the number of integration sites. Our analyses enabled detailed genetic characterization of the integration regions with respect to the number of integrates and structural changes of the host cell's genome. Single integration sites per clone with concatenated transgene copies could be detected and were in some cases found to be associated with genomic rearrangements, deletions or translocations. Supertransfection resulted in an increase in titer associated with an additional integration site per clone. Based on the TLA fingerprints, CHO cell lines originating from the same mother clone could clearly be distinguished. Interestingly, two CHO cell lines originating from the same mother clone were shown to differ genetically and phenotypically despite their identical TLA fingerprints. Taken together, TLA provides an accurate genetic characterization with respect to transgene integration sites compared with conventional methods and represents a valuable tool for a comprehensive evaluation of CHO production clones early in cell line development.