Wochenende - modular and flexible alignment-based shotgun metagenome analysis.

BACKGROUND: Shotgun metagenome analysis provides a robust and verifiable method for comprehensive microbiome analysis of fungal, viral, archaeal and bacterial taxonomy, particularly with regard to visualization of read mapping location, normalization options, growth dynamics and functional gene repertoires. Current read classification tools use non-standard output formats, or do not fully show information on mapping location. As reference datasets are not perfect, portrayal of mapping information is critical for judging results effectively. RESULTS: Our alignment-based pipeline, Wochenende, incorporates flexible quality control, trimming, mapping, various filters and normalization. Results are completely transparent and filters can be adjusted by the user. We observe stringent filtering of mismatches and use of mapping quality sharply reduces the number of false positives. Further modules allow genomic visualization and the calculation of growth rates, as well as integration and subsequent plotting of pipeline results as heatmaps or heat trees. Our novel normalization approach additionally allows calculation of absolute abundance profiles by comparison with reads assigned to the human host genome. CONCLUSION: Wochenende has the ability to find and filter alignments to all kingdoms of life using both short and long reads, and requires only good quality reference genomes. Wochenende automatically combines multiple available modules ranging from quality control and normalization to taxonomic visualization. Wochenende is available at https://github.com/MHH-RCUG/nf_wochenende .

Genome-Wide Methylation Mapping Using Nanopore Sequencing Technology Identifies Novel Tumor Suppressor Genes in Hepatocellular Carcinoma.

Downregulation of multiple tumor suppressor genes (TSGs) plays an important role in cancer formation. Recent evidence has accumulated that cancer progression involves genome-wide alteration of epigenetic modifications, which may cause downregulation of the tumor suppressor gene. Using hepatocellular carcinoma (HCC) as a system, we mapped 5-methylcytosine signal at a genome-wide scale using nanopore sequencing technology to identify novel TSGs. Integration of methylation data with gene transcription profile of regenerated liver and primary HCCs allowed us to identify 10 potential tumor suppressor gene candidates. Subsequent validation led us to focus on functionally characterizing one candidate-glucokinase (GCK). We show here that overexpression of GCK inhibits the proliferation of HCC cells via induction of intracellular lactate accumulation and subsequently causes energy crisis due to NAD+ depletion. This suggests GCK functions as a tumor suppressor gene and may be involved in HCC development. In conclusion, these data provide valuable clues for further investigations of the process of tumorigenesis in human cancer.

Implementation of RNA sequencing and array CGH in the diagnostic workflow of the AIEOP-BFM ALL 2017 trial on acute lymphoblastic leukemia.

Risk-adapted therapy has significantly contributed to improved survival rates in pediatric acute lymphoblastic leukemia (ALL) and reliable detection of chromosomal aberrations is mandatory for risk group stratification. This study evaluated the applicability of panel-based RNA sequencing and array CGH within the diagnostic workflow of the German study group of the international AIEOP-BFM ALL 2017 trial. In a consecutive cohort of 117 children with B cell precursor (BCP) ALL, array analysis identified twelve cases with an IKZF1plus profile of gene deletions and one case of masked hypodiploidy. Genetic markers BCR-ABL1 (n = 1), ETV6-RUNX1 (n = 25), and rearrangements involving KMT2A (n = 3) or TCF3 (n = 3) were assessed by established conventional techniques such as karyotyping, FISH, and RT-PCR. Comparison of these results with RNA sequencing analysis revealed overall consistency in n=115/117 cases, albeit with one undetected AFF1-KMT2A fusion in RNA sequencing and one undetected ETV6-RUNX1 fusion in conventional analyses. The combined application of RNA sequencing, FISH, and CGH+SNP array reliably detected all genetic markers necessary for risk stratification and will be used as the diagnostic standard workflow for BCP-ALL patients enrolled in the AIEOP-BFM ALL 2017 study. Prospectively, consistent collection of genome-wide CGH+SNP array as well as RNA sequencing data will be a valuable source to elucidate new prognostic lesions beyond established markers of pediatric ALL. In this respect, RNA sequencing identified various gene fusions in up to half of the IKZF1plus (n = 6/12) and B-other (n = 19/36) cases but not in cases with hyperdiploid karyotypes (n = 35). Among these fusions, this study reports several previously undescribed in frame PAX5 fusions, including PAX5-MYO1G and PAX5-NCOA6.

The identification of pathogenic variants in BRCA1/2 negative, high risk, hereditary breast and/or ovarian cancer patients: High frequency of FANCM pathogenic variants.

NGS-based multiple gene panel resequencing in combination with a high resolution CGH-array was used to identify genetic risk factors for hereditary breast and/or ovarian cancer in 237 high risk patients who were previously tested negative for pathogenic BRCA1/2 variants. All patients were screened for pathogenic variants in 94 different cancer predisposing genes. We identified 32 pathogenic variants in 14 different genes (ATM, BLM, BRCA1, CDH1, CHEK2, FANCG, FANCM, FH, HRAS, PALB2, PMS2, PTEN, RAD51C and NBN) in 30 patients (12.7%). Two pathogenic BRCA1 variants that were previously undetected due to less comprehensive and sensitive methods were found. Five pathogenic variants are novel, three of which occur in genes yet unrelated to hereditary breast and/or ovarian cancer (FANCG, FH and HRAS). In our cohort we discovered a remarkably high frequency of truncating variants in FANCM (2.1%), which has recently been suggested as a susceptibility gene for hereditary breast cancer. Two patients of our cohort carried two different pathogenic variants each and 10 other patients in whom a pathogenic variant was confirmed also harbored a variant of unknown significance in a breast and ovarian cancer susceptibility gene. We were able to identify pathogenic variants predisposing for tumor formation in 12.3% of BRCA1/2 negative breast and/or ovarian cancer patients.

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase.

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a ß-bulge at the C-terminus of ß-strand 3, which is a feature observed in many proteins of this superfamily.

Interclonal gradient of virulence in the Pseudomonas aeruginosa pangenome from disease and environment.

The population genomics of Pseudomonas aeruginosa was analysed by genome sequencing of representative strains of the 15 most frequent clonal complexes in the P. aeruginosa population and of the five most common clones from the environment of which so far no isolate from a human infection has been detected. Gene annotation identified 5892-7187 open reading frame (ORFs; median 6381 ORFs) in the 20 6.4-7.4 Mbp large genomes. The P. aeruginosa pangenome consists of a conserved core of at least 4000 genes, a combinatorial accessory genome of a further 10 000 genes and 30 000 or more rare genes that are present in only a few strains or clonal complexes. Whole genome comparisons of single nucleotide polymorphism synteny indicated unrestricted gene flow between clonal complexes by recombination. Using standardized acute lettuce, Galleria mellonella and murine airway infection models the full spectrum of possible host responses to P. aeruginosa was observed with the 20 strains ranging from unimpaired health following infection to 100% lethality. Genome comparisons indicate that the differential genetic repertoire of clones maintains a habitat-independent gradient of virulence in the P. aeruginosa population.

Sputum metagenomics of people with bronchiectasis.

Background: The microbiota in the sputum of people with bronchiectasis has repeatedly been investigated in cohorts of different geographic origin, but so far has not been studied to the species level in comparison to control populations including healthy adults and smokers without lung disease. Methods: The microbial metagenome from sputa of 101 European Bronchiectasis Registry (EMBARC) study participants was examined by using whole-genome shotgun sequencing. Results: Our analysis of the metagenome of people with bronchiectasis revealed four clusters characterised by a predominance of Haemophilus influenzae, Pseudomonas aeruginosa or polymicrobial communities with varying compositions of nonpathogenic commensals and opportunistic pathogens. The metagenomes of the severely affected patients showed individual profiles characterised by low alpha diversity. Importantly, nearly 50% of patients with severe disease were grouped in a cluster characterised by commensals. Comparisons with the sputum metagenomes of healthy smokers and healthy nonsmokers revealed a gradient of depletion of taxa in bronchiectasis, most often Neisseria subflava, Fusobacterium periodonticum and Eubacterium sulci. Conclusion: The gradient of depletion of commensal taxa found in healthy airways is a key feature of bronchiectasis associated with disease severity.

Intraclonal genome diversity of Pseudomonas aeruginosa clones CHA and TB.

BACKGROUND: Adaptation of Pseudomonas aeruginosa to different living conditions is accompanied by microevolution resulting in genomic diversity between strains of the same clonal lineage. In order to detect the impact of colonized habitats on P. aeruginosa microevolution we determined the genomic diversity between the highly virulent cystic fibrosis (CF) isolate CHA and two temporally and geographically unrelated clonal variants. The outcome was compared with the intraclonal genome diversity between three more closely related isolates of another clonal complex. RESULTS: The three clone CHA isolates differed in their core genome in several dozen strain specific nucleotide exchanges and small deletions from each other. Loss of function mutations and non-conservative amino acid replacements affected several habitat- and lifestyle-associated traits, for example, the key regulator GacS of the switch between acute and chronic disease phenotypes was disrupted in strain CHA. Intraclonal genome diversity manifested in an individual composition of the respective accessory genome whereby the highest number of accessory DNA elements was observed for isolate PT22 from a polluted aquatic habitat. Little intraclonal diversity was observed between three spatiotemporally related outbreak isolates of clone TB. Although phenotypically different, only a few individual SNPs and deletions were detected in the clone TB isolates. Their accessory genome mainly differed in prophage-like DNA elements taken up by one of the strains. CONCLUSIONS: The higher geographical and temporal distance of the clone CHA isolates was associated with an increased intraclonal genome diversity compared to the more closely related clone TB isolates derived from a common source demonstrating the impact of habitat adaptation on the microevolution of P. aeruginosa. However, even short-term habitat differentiation can cause major phenotypic diversification driven by single genomic variation events and uptake of phage DNA.

Advances in computational analysis of metagenome sequences.

Second-generation sequencing technologies are revolutionizing the study of metagenomes. Whole-genome shotgun sequencing of metagenomic DNA may become an attractive alternative to the current widely used ribosomal RNA gene studies. Large data sets of short sequence reads are mapped onto a custom microbial reference sequence. If a bacterial pangenome of completely sequenced genomes is taken as a reference, the output consists of the distribution of bacterial taxa in and bacterial gene contents of the metagenome. The relative abundance of functional categories and of individual pathways and fitness traits encoded by the metagenomic gene pool provides insight into habitat-specific features of the microbial community. Polymorphic sites in sequence reads may resolve the number and abundance of individual clonal complexes of dominant species in the polymicrobial community. These SNPs and de novo mutations may be exploited to trace the spatiotemporal spread of clones and the emergence of novel traits such as fitness or resistance determinants. In conclusion, massively parallel sequencing of metagenomic DNA allows deep insights into the composition and the genetic repertoire of polymicrobial communities.

Intraclonal diversity of the Pseudomonas aeruginosa cystic fibrosis airway isolates TBCF10839 and TBCF121838: distinct signatures of transcriptome, proteome, metabolome, adherence and pathogenicity despite an almost identical genome sequence.

Microevolution of closely related Pseudomonas aeruginosa was compared in the clone TB strains TBCF10839 and TBCF121838 which had been isolated from two unrelated individuals with cystic fibrosis who had acquired clone TB during a local outbreak. Compared with the strain PAO1 reference sequence the two clone TB genomes shared 23 155 nucleotide exchanges, 32 out-of-frame indels in the coding region and another repertoire of replacement and genomic islands such as PAGI-1, PAGI-2, PAGI-5, LESGI-1 and LES-prophage 4. Only TBCF121838 carried a genomic island known from Ralstonia pickettii. Six of the seven strain-specific sequence variations in the core genome were detected in genes affecting motility, biofilm formation or virulence, i.e. non-synonymous nucleotide substitutions in mexS, PA3729, PA5017, mifR, a frameshift mutation in pilF (TBCF121838) and an intragenic deletion in pilQ (TBCF10839). Despite their almost identical genome sequence the two strains differed strongly from each other in transcriptome and metabolome profiles, mucin adherence and phagocytosis assays. TBCF121838 was susceptible to killing by neutrophils, but TBCF10839 could grow in leucocytes. Microevolution in P. aeruginosa apparently can generate novel complex traits by few or even single mutations provided that predisposing mutational events had occurred before in the clonal lineage.

Structural genome variants of Pseudomonas aeruginosa clone C and PA14 strains.

Plasticity of Pseudomonas aeruginosa chromosomes is mainly driven by an extended accessory genome that is shaped by insertion and deletion events. Further modification of the genome composition can be induced by chromosomal inversion events which lead to relocation of genes in the affected genomic DNA segments, modify the otherwise highly conserved core genome synteny and could even alter the location of the replication terminus. Although the genome of the first sequenced strain, PAO1, displayed such a large genomic inversion, knowledge on such recombination events in the P. aeruginosa population is limited. Several large inversions had been discovered in the late 1990s in cystic fibrosis isolates of the major clonal lineage C by physical genome mapping, and subsequent work on these examples led to the characterization of the DNA at the recombination breakpoints and a presumed recombination mechanism. Since then, the topic was barely addressed in spite of the compilation of thousands of P. aeruginosa genome sequences that are deposited in databases. Due to the use of second-generation sequencing, genome contig assembly had usually followed synteny blueprints provided by the existing reference genome sequences. Inversion detection was not feasible by these approaches, as the respective read lengths did not allow reliable resolution of sequence repeats that are typically found at the borders of inverted segments. In this study, we applied PacBio and MinION long-read sequencing to isolates of the mentioned clone C collection. Confirmation of inversions predicted from the physical mapping data demonstrated that unbiased sequence assembly of such read datasets allows the detection of genomic inversions and the resolution of the recombination breakpoint regions. Additional long-read sequencing of representatives of the other major clonal lineage, PA14, revealed large inversions in several isolates, from cystic fibrosis origin as well as from other sources. These findings indicated that inversion events are not restricted to strains from chronic infection background, but could be widespread in the P. aeruginosa population and contribute to genome plasticity. Moreover, the monitored examples emphasized the role of small mobile DNA units, such as IS elements or transposons, and accessory DNA elements in the inversion-related recombination processes.

Longitudinal development of the airway metagenome of preterm very low birth weight infants during the first two years of life.

Preterm birth is accompanied with many complications and requires severe therapeutic regimens at the neonatal intensive care unit. The influence of the above-mentioned factors on the premature-born infants' respiratory metagenome or more generally its maturation is unknown. We therefore applied shotgun metagenome sequencing of oropharyngeal swabs to analyze the airway metagenome development of 24 preterm infants from one week postpartum to 15 months of age. Beta diversity analysis revealed a distinct clustering of airway microbial communities from hospitalized preterms and samples after hospital discharge. At nine and 15 months of age, the preterm infants lost their hospital-acquired individual metagenome signatures towards a common taxonomic structure. However, ecological network analysis and Random Forest classification of cross-sectional data revealed that by this age the preterm infants did not succeed in establishing the uniform and stable bacterial community structures that are characteristic for healthy full-term infants.

Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication.

To identify cellular mechanisms responsible for pressure overload triggered heart failure, we isolated cardiomyocytes, endothelial cells, and fibroblasts as most abundant cell types from mouse hearts in the subacute and chronic stages after transverse aortic constriction (TAC) and performed RNA-sequencing. We detected highly cell-type specific transcriptional responses with characteristic time courses and active intercellular communication. Cardiomyocytes after TAC exerted an early and sustained upregulation of inflammatory and matrix genes and a concomitant suppression of metabolic and ion channel genes. Fibroblasts, in contrast, showed transient early upregulation of inflammatory and matrix genes and downregulation of angiogenesis genes, but sustained induction of cell cycle and ion channel genes during TAC. Endothelial cells transiently induced cell cycle and extracellular matrix genes early after TAC, but exerted a long-lasting upregulation of inflammatory genes. As we found that matrix production by multiple cell types triggers pathological cellular responses, it might serve as a future therapeutic target.

Microevolution of the major common Pseudomonas aeruginosa clones C and PA14 in cystic fibrosis lungs.

Clones C and PA14 are the worldwide most abundant clonal complexes in the Pseudomonas aeruginosa population. The microevolution of clones C and PA14 was investigated in serial cystic fibrosis (CF) airway isolates collected over 20 years since the onset of colonization. Intraclonal evolution in CF lungs was resolved by genome sequencing of first, intermediate and late isolates and subsequent multimarker SNP genotyping of the whole strain panel. Mapping of sequence reads onto the P. aeruginosa PA14 reference genome unravelled an intraclonal and interclonal sequence diversity of 0.0035% and 0.68% respectively. Clone PA14 diversified into three branches in the patient's lungs, and the PA14 population acquired 15 nucleotide substitutions and a large deletion during the observation period. The clone C genome remained invariant during the first 3 years in CF lungs; however, 15 years later 947 transitions and 12 transversions were detected in a clone C mutL mutant strain. Key mutations occurred in retS, RNA polymerase, multidrug transporter, virulence and denitrification genes. Late clone C and PA14 persistors in the CF lungs were compromised in growth and cytotoxicity, but their mutation frequency was normal even in mutL mutant clades.

Pseudomonas putida KT2440 genome update by cDNA sequencing and microarray transcriptomics.

Pseudomonas putida KT2440 is a completely sequenced biosafety strain that has retained its capability to survive and function in the environment. The global mRNA expression profiles of the KT2440 strain grown at 10°C and 30°C were determined by deep cDNA sequencing to refine the genome annotation. Transcriptome sequencing identified 36 yet unknown small non-coding RNAs, 143 novel ORFs in 106 intergenic regions, 42 unclassified genes and eight highly expressed leaderless mRNA transcripts. The genome coordinates of eight genes and the organization of 57 operons were corrected. No overrepresented sequence motifs were detected in the 5'-untranslated regions. The 50 most highly expressed genes made up 60% of the total mRNA pool. Comparison of cDNA sequencing, Affymetrix and Progenika microarray data from the same mRNA preparation revealed a higher sensitivity and specificity of cDNA sequencing, a relatively poor correlation between the normalized cDNA reads and microarray signal intensities, and a systematic signal-dependent bias of microarrays in the detection of differentially regulated genes. The study demonstrates the power of next-generation cDNA sequencing for the quantitation of mRNA transcripts and the refinement of bacterial genome annotation.

Genome diversity of Pseudomonas aeruginosa PAO1 laboratory strains.

Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Taking the existing PAO1-UW genome sequence (named after the University of Washington, which led the sequencing project) as a blueprint, the genome sequences of reference strains MPAO1 and PAO1-DSM (stored at the German Collection for Microorganisms and Cell Cultures [DSMZ]) were resolved by physical mapping and deep short read sequencing-by-synthesis. MPAO1 has been the source of near-saturation libraries of transposon insertion mutants, and PAO1-DSM is identical in its SpeI-DpnI restriction map with the original isolate. The major genomic differences of MPAO1 and PAO1-DSM in comparison to PAO1-UW are the lack of a large inversion, a duplication of a mobile 12-kb prophage region carrying a distinct integrase and protein phosphatases or kinases, deletions of 3 to 1,006 bp in size, and at least 39 single-nucleotide substitutions, 17 of which affect protein sequences. The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Subline PAO1-DSM outnumbered the two other sublines in late stationary growth phase. In conclusion, P. aeruginosa PAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. High-throughput genome resequencing will resolve more cases and could become a proper quality control for strain collections.

Novel candidate genes for ECT response prediction-a pilot study analyzing the DNA methylome of depressed patients receiving electroconvulsive therapy.

BACKGROUND: Major depressive disorder (MDD) represents a serious global health concern. The urge for efficient MDD treatment strategies is presently hindered by the incomplete knowledge of its underlying pathomechanism. Despite recent progress (highlighting both genetics and the environment, and thus DNA methylation, to be relevant for its development), 30-50% of MDD patients still fail to reach remission with standard treatment approaches. Electroconvulsive therapy (ECT) is one of the most powerful options for the treatment of pharmacoresistant depression; nevertheless, ECT remission rates barely reach 50% in large-scale naturalistic population-based studies. To optimize MDD treatment strategies and enable personalized medicine in the long- term, prospective indicators of ECT response are thus in great need. Because recent target-driven analyses revealed DNA methylation baseline differences between ECT responder groups, we analyzed the DNA methylome of depressed ECT patients using next-generation sequencing. In this pilot study, we did not only aim to find novel targets for ECT response prediction but also to get a deeper insight into its possible mechanism of action. RESULTS: Longitudinal DNA methylation analysis of peripheral blood mononuclear cells isolated from a cohort of treatment-resistant MDD patients (n = 12; time points: before and after 1st and last ECT, respectively) using a TruSeq-Methyl Capture EPIC Kit for library preparation, led to the following results: (1) The global DNA methylation differed neither between the four measured time points nor between ECT responders (n = 8) and non-responders (n = 4). (2) Analyzing the DNA methylation variance for every probe (=1476812 single CpG sites) revealed eight novel candidate genes to be implicated in ECT response (protein-coding genes: RNF175, RNF213, TBC1D14, TMC5, WSCD1; genes encoding for putative long non-coding RNA transcripts: AC018685.2, AC098617.1, CLCN3P1). (3) In addition, DNA methylation of two CpG sites (located within AQP10 and TRERF1) was found to change during the treatment course. CONCLUSIONS: We suggest ten novel candidate genes to be implicated in either ECT response or its possible mechanism. Because of the small sample size of our pilot study, our findings must be regarded as preliminary.

Visualization of Pseudomonas genomic structure by abundant 8-14mer oligonucleotides.

Under- and over-represented mono- to hexanucleotides are signatures of bacterial genomes, but the compositional biases of octa- to tetradecanucleotides have not yet been explored. Thirteen completely sequenced genomes of the Pseudomonas genus were searched for highly overrepresented 8-14mers. Between 59-989 overrepresented 8-14mers were found to exceed the applied threshold value. All genomic data sets of the 13 strains showed a consistent pattern, with individual oligomers clustering in either non-coding or coding regions. Non-coding oligonucleotides were typically part of longer repeats. Coding oligonucleotides were evenly distributed in the core genome, preferred one reading frame and matched with the local tetranucleotide usage patterns. Genomic islands were recognized by the depletion of overrepresented oligonucleotides. Several mainly coding 8-14mers occurred in genomes on average every 10 000 bp or less. Such frequently occurring 8-14mers could become useful markers for species identification. In the future of next-generation ultra-high throughput DNA sequencing, the composition of bacterial metagenomes may be quantified by scanning the primary sequence reads for these 8-14mer markers.

The SeqWord Genome Browser: an online tool for the identification and visualization of atypical regions of bacterial genomes through oligonucleotide usage.

BACKGROUND: Data mining in large DNA sequences is a major challenge in microbial genomics and bioinformatics. Oligonucleotide usage (OU) patterns provide a wealth of information for large scale sequence analysis and visualization. The purpose of this research was to make OU statistical analysis available as a novel web-based tool for functional genomics and annotation. The tool is also available as a downloadable package. RESULTS: The SeqWord Genome Browser (SWGB) was developed to visualize the natural compositional variation of DNA sequences. The applet is also used for identification of divergent genomic regions both in annotated sequences of bacterial chromosomes, plasmids, phages and viruses, and in raw DNA sequences prior to annotation by comparing local and global OU patterns. The applet allows fast and reliable identification of clusters of horizontally transferred genomic islands, large multi-domain genes and genes for ribosomal RNA. Within the majority of genomic fragments (also termed genomic core sequence), regions enriched with housekeeping genes, ribosomal proteins and the regions rich in pseudogenes or genetic vestiges may be contrasted. CONCLUSION: The SWGB applet presents a range of comprehensive OU statistical parameters calculated for a range of bacterial species, plasmids and phages. It is available on the Internet at http://www.bi.up.ac.za/SeqWord/mhhapplet.php.

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase. Corrigendum.

A statement is amended in the article by Isupov et al. [(2015). Acta Cryst. D71, 2344-2353].