Big data in genomic research for big questions with examples from covid-19 and other zoonoses.

Omics research inevitably involves the collection and analysis of big data, which can only be handled by automated approaches. Here we point out that the analysis of big data in the field of genomics dictates certain requirements, such as specialized software, quality control of input data, and simplification for visualization of the results. The latter results in a loss of information, as is exemplified for phylogenetic trees. Clear communication of big data analyses can be enhanced by novel visualization strategies. The interpretation of findings is sometimes hampered when dedicated analytical tools are not fully understood by microbiologists, while the researchers performing these analyses may not have a full overview of the biology of the microbes under study. These issues are illustrated here, using SARS-Cov-2 and Salmonella enterica as zoonotic examples. Whereas in scientific communications jargon should be avoided or explained, nomenclature to group similar organisms and distinguish these from more distant relatives is not only essential, but also influences the interpretation of results. Unfortunately, changes in taxonomically accepted names are now so frequent that they hamper rather than assist research, as is illustrated with difficulties of microbiome studies. Nomenclature to group viral isolates, as is done for SARS-Cov2, is also not without difficulties. Some weaknesses in current omics research stem from poor quality of data or biased databases, and problems can be magnified by machine learning approaches. Moreover, the overall opus of scientific publications can now be considered "big data", as is illustrated by the avalanche of COVID-19-related publications. The peer-review model of scientific publishing is only barely coping with this novel situation, resulting in retractions and the publication of bogus works. The avalanche of scientific publications that originated from the current pandemic can obstruct literature searches, and this will unfortunately continue over time.

Decoding the epitranscriptional landscape from native RNA sequences.

Traditional epitranscriptomics relies on capturing a single RNA modification by antibody or chemical treatment, combined with short-read sequencing to identify its transcriptomic location. This approach is labor-intensive and may introduce experimental artifacts. Direct sequencing of native RNA using Oxford Nanopore Technologies (ONT) can allow for directly detecting the RNA base modifications, although these modifications might appear as sequencing errors. The percent Error of Specific Bases (%ESB) was higher for native RNA than unmodified RNA, which enabled the detection of ribonucleotide modification sites. Based on the %ESB differences, we developed a bioinformatic tool, epitranscriptional landscape inferring from glitches of ONT signals (ELIGOS), that is based on various types of synthetic modified RNA and applied to rRNA and mRNA. ELIGOS is able to accurately predict known classes of RNA methylation sites (AUC > 0.93) in rRNAs from Escherichiacoli, yeast, and human cells, using either unmodified in vitro transcription RNA or a background error model, which mimics the systematic error of direct RNA sequencing as the reference. The well-known DRACH/RRACH motif was localized and identified, consistent with previous studies, using differential analysis of ELIGOS to study the impact of RNA m6A methyltransferase by comparing wild type and knockouts in yeast and mouse cells. Lastly, the DRACH motif could also be identified in the mRNA of three human cell lines. The mRNA modification identified by ELIGOS is at the level of individual base resolution. In summary, we have developed a bioinformatic software package to uncover native RNA modifications.

Comparison of Monkeypox virus genomes from the 2017 Nigeria outbreak and the 2022 outbreak.

AIMS: The current Monkeypox virus (MPX) outbreak is not only the largest known outbreak to date caused by a strain belonging to the West-African clade, but also results in remarkably different clinical and epidemiological features compared to previous outbreaks of this virus. Here, we consider the possibility that mutations in the viral genome may be responsible for its changed characteristics. METHODS AND RESULTS: Six genome sequences of isolates from the current outbreak were compared to five genomes of isolates from the 2017 outbreak in Nigeria and to two historic genomes, all belonging to the West-African clade. We report differences that are consistently present in the 2022 isolates but not in the others. Although some variation in repeat units was observed, only two were consistently found in the 2022 genomes only, and these were located in intergenic regions. A total of 55 single nucleotide polymorphisms were consistently present in the 2022 isolates compared to the 2017 isolates. Of these, 25 caused an amino acid substitution in a predicted protein. CONCLUSIONS: The nature of the substitution and the annotation of the affected protein identified potential candidates that might affect the virulence of the virus. These included the viral DNA helicase and transcription factors. SIGNIFICANCE: This bioinformatic analysis provides guidance for wet-lab research to identify changed properties of the MPX.

Interactions between the Gut Microbiome, Lung Conditions, and Coronary Heart Disease and How Probiotics Affect These.

The importance of a healthy microbiome cannot be overemphasized. Disturbances in its composition can lead to a variety of symptoms that can extend to other organs. Likewise, acute or chronic conditions in other organs can affect the composition and physiology of the gut microbiome. Here, we discuss interorgan communication along the gut-lung axis, as well as interactions between lung and coronary heart diseases and between cardiovascular disease and the gut microbiome. This triangle of organs, which also affects the clinical outcome of COVID-19 infections, is connected by means of numerous receptors and effectors, including immune cells and immune-modulating factors such as short chain fatty acids (SCFA) and trimethlamine-N-oxide (TMAO). The gut microbiome plays an important role in each of these, thus affecting the health of the lungs and the heart, and this interplay occurs in both directions. The gut microbiome can be influenced by the oral uptake of probiotics. With an improved understanding of the mechanisms responsible for interorgan communication, we can start to define what requirements an 'ideal' probiotic should have and its role in this triangle.

Mechanisms linking preterm birth to onset of cardiovascular disease later in adulthood.

Cardiovascular disease (CVD) rates in adulthood are high in premature infants; unfortunately, the underlying mechanisms are not well defined. In this review, we discuss potential pathways that could lead to CVD in premature babies. Studies show intense oxidant stress and inflammation at tissue levels in these neonates. Alterations in lipid profile, foetal epigenomics, and gut microbiota in these infants may also underlie the development of CVD. Recently, probiotic bacteria, such as the mucin-degrading bacterium Akkermansia muciniphila have been shown to reduce inflammation and prevent heart disease in animal models. All this information might enable scientists and clinicians to target pathways to act early to curtail the adverse effects of prematurity on the cardiovascular system. This could lead to primary and secondary prevention of CVD and improve survival among preterm neonates later in adult life.

Case of Microcephaly after Congenital Infection with Asian Lineage Zika Virus, Thailand.

We sequenced the virus genomes from 3 pregnant women in Thailand with Zika virus diagnoses. All had infections with the Asian lineage. The woman infected at gestational week 9, and not those infected at weeks 20 and 24, had a fetus with microcephaly. Asian lineage Zika viruses can cause microcephaly.

E. coli and colorectal cancer: a complex relationship that deserves a critical mindset.

To the multiple factors that may eventually result in colorectal cancer (CRC), strains of E. coli have now been added, in particular strains producing colibactin from their polyketide synthesis (pks) locus. The evidence and mechanistic explanations for this unfortunate effect of what is in most cases a harmless commensal are discussed in the first part of this review. In the second part, observations are presented and discussed that do not fit with the hypothesis that colibactin-producing E. coli produce CRC. The last part of this review is reserved for an alternative explanation of the function of this enigmatic colibactin, a toxin that has not yet been isolated. It is hypothesized that E. coli preferentially colonizes cancerous lesions as an effect rather than a cause and that colibactin production provides a selective advantage to compete with other bacteria.

Genome-Based Comparison of Clostridioides difficile: Average Amino Acid Identity Analysis of Core Genomes.

Infections due to Clostridioides difficile (previously known as Clostridium difficile) are a major problem in hospitals, where cases can be caused by community-acquired strains as well as by nosocomial spread. Whole genome sequences from clinical samples contain a lot of information but that needs to be analyzed and compared in such a way that the outcome is useful for clinicians or epidemiologists. Here, we compare 663 public available complete genome sequences of C. difficile using average amino acid identity (AAI) scores. This analysis revealed that most of these genomes (640, 96.5%) clearly belong to the same species, while the remaining 23 genomes produce four distinct clusters within the Clostridioides genus. The main C. difficile cluster can be further divided into sub-clusters, depending on the chosen cutoff. We demonstrate that MLST, either based on partial or full gene-length, results in biased estimates of genetic differences and does not capture the true degree of similarity or differences of complete genomes. Presence of genes coding for C. difficile toxins A and B (ToxA/B), as well as the binary C. difficile toxin (CDT), was deduced from their unique PfamA domain architectures. Out of the 663 C. difficile genomes, 535 (80.7%) contained at least one copy of ToxA or ToxB, while these genes were missing from 128 genomes. Although some clusters were enriched for toxin presence, these genes are variably present in a given genetic background. The CDT genes were found in 191 genomes, which were restricted to a few clusters only, and only one cluster lacked the toxin A/B genes consistently. A total of 310 genomes contained ToxA/B without CDT (47%). Further, published metagenomic data from stools were used to assess the presence of C. difficile sequences in blinded cases of C. difficile infection (CDI) and controls, to test if metagenomic analysis is sensitive enough to detect the pathogen, and to establish strain relationships between cases from the same hospital. We conclude that metagenomics can contribute to the identification of CDI and can assist in characterization of the most probable causative strain in CDI patients.

Suggested mechanisms for Zika virus causing microcephaly: what do the genomes tell us?

BACKGROUND: Zika virus (ZIKV) is an emerging human pathogen. Since its arrival in the Western hemisphere, from Africa via Asia, it has become a serious threat to pregnant women, causing microcephaly and other neuropathies in developing fetuses. The mechanisms behind these teratogenic effects are unknown, although epidemiological evidence suggests that microcephaly is not associated with the original, African lineage of ZIKV. The sequences of 196 published ZIKV genomes were used to assess whether recently proposed mechanistic explanations for microcephaly are supported by molecular level changes that may have increased its virulence since the virus left Africa. For this we performed phylogenetic, recombination, adaptive evolution and tetramer frequency analyses, and compared protein sequences for the presence of protease cleavage sites, Pfam domains, glycosylation sites, signal peptides, trans-membrane protein domains, and phosphorylation sites. RESULTS: Recombination events within or between Asian and Brazilian lineages were not observed, and likewise there were no differences in protease cleavage, glycosylation sites, signal peptides or trans-membrane domains between African and Brazilian strains. The frequency of Retinoic Acid Response Element (RARE) sequences was increased in Brazilian strains. Genetic adaptation was also apparent by tetramer signatures that had undergone major changes in the past but has stabilized in the Brazilian lineage despite subsequent geographic spread, suggesting the viral population presently propagates in the same host species in various regions. Evidence for selection pressure was recognized for several amino acid sites in the Brazilian lineage compared to the African lineage, mainly in nonstructural proteins, especially protein NS4B. A number of these positively selected mutations resulted in an increased potential to be phosphorylated in the Brazilian lineage compared to the African linage, which may have increased their potential to interfere with neural fetal development. CONCLUSIONS: ZIKV seems to have adapted to a limited number of hosts, including humans, during which its virulence increased. Its protein NS4B, together with NS4A, has recently been shown to inhibit Akt-mTOR signaling in human fetal neural stem cells, a key pathway for brain development. We hypothesize that positive selection of novel phosphorylation sites in the protein NS4B of the Brazilian lineage could interfere with phosphorylation of Akt and mTOR, impairing Akt-mTOR signaling and this may result in an increased risk for developmental neuropathies.

Diversity of Pseudomonas Genomes, Including Populus-Associated Isolates, as Revealed by Comparative Genome Analysis.

The Pseudomonas genus contains a metabolically versatile group of organisms that are known to occupy numerous ecological niches, including the rhizosphere and endosphere of many plants. Their diversity influences the phylogenetic diversity and heterogeneity of these communities. On the basis of average amino acid identity, comparative genome analysis of >1,000 Pseudomonas genomes, including 21 Pseudomonas strains isolated from the roots of native Populus deltoides (eastern cottonwood) trees resulted in consistent and robust genomic clusters with phylogenetic homogeneity. All Pseudomonas aeruginosa genomes clustered together, and these were clearly distinct from other Pseudomonas species groups on the basis of pangenome and core genome analyses. In contrast, the genomes of Pseudomonas fluorescens were organized into 20 distinct genomic clusters, representing enormous diversity and heterogeneity. Most of our 21 Populus-associated isolates formed three distinct subgroups within the major P. fluorescens group, supported by pathway profile analysis, while two isolates were more closely related to Pseudomonas chlororaphis and Pseudomonas putida. Genes specific to Populus-associated subgroups were identified. Genes specific to subgroup 1 include several sensory systems that act in two-component signal transduction, a TonB-dependent receptor, and a phosphorelay sensor. Genes specific to subgroup 2 contain hypothetical genes, and genes specific to subgroup 3 were annotated with hydrolase activity. This study justifies the need to sequence multiple isolates, especially from P. fluorescens, which displays the most genetic variation, in order to study functional capabilities from a pangenomic perspective. This information will prove useful when choosing Pseudomonas strains for use to promote growth and increase disease resistance in plants.

Comparative genomics of Bifidobacterium, Lactobacillus and related probiotic genera.

Six bacterial genera containing species commonly used as probiotics for human consumption or starter cultures for food fermentation were compared and contrasted, based on publicly available complete genome sequences. The analysis included 19 Bifidobacterium genomes, 21 Lactobacillus genomes, 4 Lactococcus and 3 Leuconostoc genomes, as well as a selection of Enterococcus (11) and Streptococcus (23) genomes. The latter two genera included genomes from probiotic or commensal as well as pathogenic organisms to investigate if their non-pathogenic members shared more genes with the other probiotic genomes than their pathogenic members. The pan- and core genome of each genus was defined. Pairwise BLASTP genome comparison was performed within and between genera. It turned out that pathogenic Streptococcus and Enterococcus shared more gene families than did the non-pathogenic genomes. In silico multilocus sequence typing was carried out for all genomes per genus, and the variable gene content of genomes was compared within the genera. Informative BLAST Atlases were constructed to visualize genomic variation within genera. The clusters of orthologous groups (COG) classes of all genes in the pan- and core genome of each genus were compared. In addition, it was investigated whether pathogenic genomes contain different COG classes compared to the probiotic or fermentative organisms, again comparing their pan- and core genomes. The obtained results were compared with published data from the literature. This study illustrates how over 80 genomes can be broadly compared using simple bioinformatic tools, leading to both confirmation of known information as well as novel observations.

The first three waves of the Covid-19 pandemic hint at a limited genetic repertoire for SARS-CoV-2.

The genomic diversity of SARS-CoV-2 is the result of a relatively low level of spontaneous mutations introduced during viral replication. With millions of SARS-CoV-2 genome sequences now available, we can begin to assess the overall genetic repertoire of this virus. We find that during 2020, there was a global wave of one variant that went largely unnoticed, possibly because its members were divided over several sublineages (B.1.177 and sublineages B.1.177.XX). We collectively call this Janus, and it was eventually replaced by the Alpha (B.1.1.7) variant of concern (VoC), next replaced by Delta (B.1.617.2), which itself might soon be replaced by a fourth pandemic wave consisting of Omicron (B.1.1.529). We observe that splitting up and redefining variant lineages over time, as was the case with Janus and is now happening with Alpha, Delta and Omicron, is not helpful to describe the epidemic waves spreading globally. Only ∼5% of the 30 000 nucleotides of the SARS-CoV-2 genome are found to be variable. We conclude that a fourth wave of the pandemic with the Omicron variant might not be that different from other VoCs, and that we may already have the tools in hand to effectively deal with this new VoC.

Mash-based analyses of Escherichia coli genomes reveal 14 distinct phylogroups.

In this study, more than one hundred thousand Escherichia coli and Shigella genomes were examined and classified. This is, to our knowledge, the largest E. coli genome dataset analyzed to date. A Mash-based analysis of a cleaned set of 10,667 E. coli genomes from GenBank revealed 14 distinct phylogroups. A representative genome or medoid identified for each phylogroup was used as a proxy to classify 95,525 unassembled genomes from the Sequence Read Archive (SRA). We find that most of the sequenced E. coli genomes belong to four phylogroups (A, C, B1 and E2(O157)). Authenticity of the 14 phylogroups is supported by several different lines of evidence: phylogroup-specific core genes, a phylogenetic tree constructed with 2613 single copy core genes, and differences in the rates of gene gain/loss/duplication. The methodology used in this work is able to reproduce known phylogroups, as well as to identify previously uncharacterized phylogroups in E. coli species.

Genome update: the 1000th genome--a cautionary tale.

There are now more than 1000 sequenced prokaryotic genomes deposited in public databases and available for analysis. Currently, although the sequence databases GenBank, DNA Database of Japan and EMBL are synchronized continually, there are slight differences in content at the genomes level for a variety of logistical reasons, including differences in format and loading errors, such as those caused by file transfer protocol interruptions. This means that the 1000th genome will be different in the various databases. Some of the data on the highly accessed web pages are inaccurate, leading to false conclusions for example about the largest bacterial genome sequenced. Biological diversity is far greater than many have thought. For example, analysis of multiple Escherichia coli genomes has led to an estimate of around 45 000 gene families - more genes than are recognized in the human genome. Moreover, of the 1000 genomes available, not a single protein is conserved across all genomes. Excluding the members of the Archaea, only a total of four genes are conserved in all bacteria: two protein genes and two RNA genes.

Comparison of 61 sequenced Escherichia coli genomes.

Escherichia coli is an important component of the biosphere and is an ideal model for studies of processes involved in bacterial genome evolution. Sixty-one publically available E. coli and Shigella spp. sequenced genomes are compared, using basic methods to produce phylogenetic and proteomics trees, and to identify the pan- and core genomes of this set of sequenced strains. A hierarchical clustering of variable genes allowed clear separation of the strains into clusters, including known pathotypes; clinically relevant serotypes can also be resolved in this way. In contrast, when in silico MLST was performed, many of the various strains appear jumbled and less well resolved. The predicted pan-genome comprises 15,741 gene families, and only 993 (6%) of the families are represented in every genome, comprising the core genome. The variable or 'accessory' genes thus make up more than 90% of the pan-genome and about 80% of a typical genome; some of these variable genes tend to be co-localized on genomic islands. The diversity within the species E. coli, and the overlap in gene content between this and related species, suggests a continuum rather than sharp species borders in this group of Enterobacteriaceae.

On the origins of a Vibrio species.

Thirty-two genome sequences of various Vibrionaceae members are compared, with emphasis on what makes V. cholerae unique. As few as 1,000 gene families are conserved across all the Vibrionaceae genomes analysed; this fraction roughly doubles for gene families conserved within the species V. cholerae. Of these, approximately 200 gene families that cluster on various locations of the genome are not found in other sequenced Vibrionaceae; these are possibly unique to the V. cholerae species. By comparing gene family content of the analysed genomes, the relatedness to a particular species is identified for two unspeciated genomes. Conversely, two genomes presumably belonging to the same species have suspiciously dissimilar gene family content. We are able to identify a number of genes that are conserved in, and unique to, V. cholerae. Some of these genes may be crucial to the niche adaptation of this species.

Comment on "Enumeration of Escherichia coli in Probiotic Products. Microorganisms 2019, 7, 437".

Recently, Zimmer and Dorea published a communication on the enumeration of Escherichia coli in probiotic products containing this species [...].

No Genotoxicity Is Detectable for Escherichia coli Strain Nissle 1917 by Standard In Vitro and In Vivo Tests.

Probiotic Escherichia coli strain Nissle 1917 (EcN) has a long history of safe use. However, the recently discovered presence of a pks locus in its genome presumably producing colibactin has questioned its safety, as colibactin has been implicated in genotoxicity. Here, we assess the genotoxic potential of EcN. Metabolic products were tested in vitro by the Ames test, a mutagenicity assay developed to detect point mutation-inducing activity. Live EcN were tested by an adapted Ames test. Neither the standard nor the adapted Ames test resulted in increased numbers of revertant colonies, indicating that EcN metabolites or viable cells lacked mutagenic activity. The in vivo Mammalian Alkaline Comet Assay (the gold standard for detecting DNA-strand breaks) was used to determine potentially induced DNA-strand breaks in cells of the gastro-intestinal tract of rats orally administered with viable EcN. Bacteria were given at 109-1011 colony forming units (CFU) per animal by oral gavage on 2 consecutive days and daily for a period of 28 days to 5 rats per group. No significant differences compared to negative controls were found. These results demonstrate that EcN does not induce DNA-strand breaks and does not have any detectable genotoxic potential in the test animals.

Comparative genomics of hepatitis A virus, hepatitis C virus, and hepatitis E virus provides insights into the evolutionary history of Hepatovirus species.

The intraspecies genomic diversity of the single-strand RNA (+) virus species hepatitis A virus (Hepatovirus), hepatitis C virus (Hepacivirus), and hepatitis E virus (Orthohepevirus) was compared. These viral species all can cause liver inflammation (hepatitis), but share no gene similarity. The codon usage of human hepatitis A virus (HAV) is suboptimal for replication in its host, a characteristic it shares with taxonomically related rodent, simian, and bat hepatitis A virus species. We found this codon usage to be strikingly similar to that of Triatoma virus that infects blood-sucking kissing bugs. The codon usage of that virus is well adapted to its insect host. The codon usage of HAV is also similar to other invertebrate viruses of various taxonomic families. An evolutionary ancestor of HAV and related virus species is hypothesized to be an insect virus that underwent a host jump to infect mammals. The similarity between HAV and invertebrate viruses goes beyond codon usage, as they also share amino acid composition characteristics, while not sharing direct sequence homology. In contrast, hepatitis C virus and hepatitis E virus are highly similar in codon usage preference, nucleotide composition, and amino acid composition, and share these characteristics with Human pegivirus A, West Nile virus, and Zika virus. We present evidence that these observations are only partly explained by differences in nucleotide composition of the complete viral codon regions. We consider the combination of nucleotide composition, amino acid composition, and codon usage preference suitable to provide information on possible evolutionary similarities between distant virus species that cannot be investigated by phylogeny.

In silico Selection of Amplification Targets for Rapid Polymorphism Screening in Ebola Virus Outbreaks.

To achieve maximum transmission chain tracking in the current Ebola outbreak, whole genome sequencing (WGS) has been proposed to provide optimal information. However, WGS remains a costly and time-intensive procedure that is poorly suited for the large numbers of samples being generated, especially under severe time and work-environment constraints as in the present DRC outbreak. To better prepare for future outbreaks, where an apparent single outbreak may actually represent overlapping outbreaks caused by independent variants, and where rapid identification of emerging new transmission chains will be essential, a more practical method would be to amplify and sequence genomic areas that reveal the highest information to differentiate EBOV variants. We have identified four highly informative polymorphism PCR sequencing targets, suitable for rapid tracing of transmission chains and identification of new sources of Ebola outbreaks, an approach which will be far more practical in the field than WGS.