Carriage of three plasmids in a single human clinical isolate of Clostridioides difficile.

A subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential importance, C. difficile plasmids remain poorly characterized. Here, we provide the complete genome sequence of a human clinical isolate that carries three high-copy number plasmids from three different plasmid families that are therefore compatible. For two of these, we identify a region capable of sustaining plasmid replication in C. difficile that is also compatible with the plasmid pCD630 that is found in many laboratory strains. Together, our data advance our understanding of C. difficile plasmid biology.

Flexible and Scalable Full-Length CYP2D6 Long Amplicon PacBio Sequencing.

Cytochrome P450 2D6 (CYP2D6) is among the most important genes involved in drug metabolism. Specific variants are associated with changes in the enzyme's amount and activity. Multiple technologies exist to determine these variants, like the AmpliChip CYP450 test, Taqman qPCR, or Second-Generation Sequencing, however, sequence homology between cytochrome P450 genes and pseudogene CYP2D7 impairs reliable CYP2D6 genotyping, and variant phasing cannot accurately be determined using these assays. To circumvent this, we sequenced CYP2D6 using the Pacific Biosciences RSII and obtained high-quality, full-length, phased CYP2D6 sequences, enabling accurate variant calling and haplotyping of the entire gene-locus including exonic, intronic, and upstream and downstream regions. Unphased diplotypes (Roche AmpliChip CYP450 test) were confirmed for 24 of the 25 samples, including gene duplications. Cases with gene deletions required additional specific assays to resolve. In total, 61 unique variants were detected, including variants that had not previously been associated with specific haplotypes. To further aid genomic analysis using standard reference sequences, we have established an LOVD-powered CYP2D6 gene-variant database, and added all reference haplotypes and data reported here. We conclude that our CYP2D6 genotyping approach produces reliable CYP2D6 diplotypes and reveals information about additional variants, including phasing and copy-number variation.

Detecting PKD1 variants in polycystic kidney disease patients by single-molecule long-read sequencing.

A genetic diagnosis of autosomal-dominant polycystic kidney disease (ADPKD) is challenging due to allelic heterogeneity, high GC content, and homology of the PKD1 gene with six pseudogenes. Short-read next-generation sequencing approaches, such as whole-genome sequencing and whole-exome sequencing, often fail at reliably characterizing complex regions such as PKD1. However, long-read single-molecule sequencing has been shown to be an alternative strategy that could overcome PKD1 complexities and discriminate between homologous regions of PKD1 and its pseudogenes. In this study, we present the increased power of resolution for complex regions using long-read sequencing to characterize a cohort of 19 patients with ADPKD. Our approach provided high sensitivity in identifying PKD1 pathogenic variants, diagnosing 94.7% of the patients. We show that reliable screening of ADPKD patients in a single test without interference of PKD1 homologous sequences, commonly introduced by residual amplification of PKD1 pseudogenes, by direct long-read sequencing is now possible. This strategy can be implemented in diagnostics and is highly suitable to sequence and resolve complex genomic regions that are of clinical relevance.

Simultaneous pancreas-kidney transplantation in patients with type 1 diabetes reverses elevated MBL levels in association with MBL2 genotype and VEGF expression.

AIMS/HYPOTHESIS: High levels of circulating mannan-binding lectin (MBL) are associated with the development of diabetic nephropathy and hyperglycaemia-induced vasculopathy. Here, we aimed to assess the effect of glycaemic control on circulating levels of MBL and the relationship of these levels with vascular damage. METHODS: We assessed MBL levels and corresponding MBL2 genotype, together with vascular endothelial growth factor (VEGF) levels as a marker of vascular damage, in type 1 diabetes patients with diabetic nephropathy before and after simultaneous pancreas-kidney (SPK) transplantation. We included diabetic nephropathy patients (n = 21), SPK patients (n = 37), healthy controls (n = 19), type 1 diabetes patients (n = 15) and diabetic nephropathy patients receiving only a kidney transplant (n = 15). Fourteen diabetic nephropathy patients were followed up for 12 months after SPK. RESULTS: We found elevated circulating MBL levels in diabetic nephropathy patients, and a trend towards elevated circulating MBL levels in type 1 diabetes patients, compared with healthy control individuals. MBL levels in SPK patients completely normalised and our data indicate that this predominantly occurs in patients with a polymorphism in the MBL2 gene. By contrast, MBL levels in kidney transplant only patients remained elevated, suggesting that glycaemic control but not reversal of renal failure is associated with decreased MBL levels. In line, levels of glucose and HbA1c, but not creatinine levels and estimated GFR, were correlated with MBL levels. VEGF levels were associated with levels of MBL and HbA1c in an MBL-polymorphism-dependent manner. CONCLUSIONS/INTERPRETATION: Taken together, circulating MBL levels are associated with diabetic nephropathy and are dependent on glycaemic control, possibly in an MBL2-genotype-dependent manner.

TSSV: a tool for characterization of complex allelic variants in pure and mixed genomes.

MOTIVATION: Advances in sequencing technologies and computational algorithms have enabled the study of genomic variants to dissect their functional consequence. Despite this unprecedented progress, current tools fail to reliably detect and characterize more complex allelic variants, such as short tandem repeats (STRs). We developed TSSV as an efficient and sensitive tool to specifically profile all allelic variants present in targeted loci. Based on its design, requiring only two short flanking sequences, TSSV can work without the use of a complete reference sequence to reliably profile highly polymorphic, repetitive or uncharacterized regions. RESULTS: We show that TSSV can accurately determine allelic STR structures in mixtures with 10% representation of minor alleles or complex mixtures in which a single STR allele is shared. Furthermore, we show the universal utility of TSSV in two other independent studies: characterizing de novo mutations introduced by transcription activator-like effector nucleases (TALENs) and profiling the noise and systematic errors in an IonTorrent sequencing experiment. TSSV complements the existing tools by aiding the study of highly polymorphic and complex regions and provides a high-resolution map that can be used in a wide range of applications, from personal genomics to forensic analysis and clinical diagnostics. AVAILABILITY AND IMPLEMENTATION: We have implemented TSSV as a Python package that can be installed through the command-line using pip install TSSV command. Its source code and documentation are available at https://pypi.python.org/pypi/tssv and http://www.lgtc.nl/tssv.

Clostridioides difficile infection with isolates of cryptic clade C-II: a genomic analysis of polymerase chain reaction ribotype 151.

OBJECTIVES: We report a patient case of pseudomembranous colitis associated with a monotoxin-producing Clostridioides difficile belonging to the very rarely diagnosed polymerase chain reaction (PCR) ribotype (RT) 151. To understand why this isolate was not identified using a routine commercial test, we performed a genomic analysis of RT151. METHODS: Illumina short-read sequencing was performed on n = 11 RT151s from various geographical regions to study their genomic characteristics and relatedness. Subsequently, we used PacBio circular consensus sequencing to determine the complete genome sequence of isolates belonging to cryptic clades C-I and C-II, which includes the patient isolate. RESULTS: We found that 1) RT151s are polyphyletic with isolates falling into clades 1 and cryptic clades C-I and C-II; 2) RT151 contains both nontoxigenic and toxigenic isolates and 3) RT151 C-II isolates contained monotoxin pathogenicity loci. The isolate from our patient case report contains a novel-pathogenicity loci insertion site, lacked tcdA and had a divergent tcdB sequence that might explain the failure of the diagnostic test. DISCUSSION: This study shows that RT151 encompasses both typical and cryptic clades and provides conclusive evidence for C. difficile infection due to clade C-II isolates that was hitherto lacking. Vigilance towards C. difficile infection as a result of cryptic clade isolates is warranted.

Combining Protein Expression and Molecular Data Improves Mutation Characterization of Dystrophinopathies.

Duchenne and Becker muscular dystrophy are X-linked recessive inherited disorders characterized by progressive weakness due to skeletal muscle degeneration. Different mutations in the DMD gene, which encodes for dystrophin protein, are responsible for these disorders. The aim of our study was to investigate the relationship between type, size, and location of the mutation that occurs in the DMD gene and their effect on dystrophin protein expression in a cohort of 40 male dystrophinopathy patients and nine females, possible carriers. We evaluated the expression of dystrophin by immunofluorescence and immunoblotting. The mutational spectrum of the DMD gene was established by MLPA for large copy number variants, followed by HRM analysis for point mutations and sequencing of samples with an abnormal melting profile. MLPA revealed 30 deletions (75%) and three duplications (7.5%). HRM analysis accounted for seven-point mutations (17.5%). We also report four novel small mutations (c. 8507G>T, c.3021delG, c.9563_9563+1insAGCATGTTTATGATACAGCA, c.7661-60T>A) in DMD gene. Our work shows that the DNA translational open reading frame and the location of the mutation both influence the expression of dystrophin and disease severity phenotype. The proposed algorithm used in this study demonstrates its accuracy for the characterization of dystrophinopathy patients.

Toward predicting CYP2D6-mediated variable drug response from CYP2D6 gene sequencing data.

Pharmacogenomics is a key component of personalized medicine that promises safer and more effective drug treatment by individualizing drug choice and dose based on genetic profiles. In clinical practice, genetic biomarkers are used to categorize patients into *-alleles to predict CYP450 enzyme activity and adjust drug dosages accordingly. However, this approach leaves a large part of variability in drug response unexplained. Here, we present a proof-of-concept approach that uses continuous-scale (instead of categorical) assignments to predict enzyme activity. We used full CYP2D6 gene sequences obtained with long-read amplicon-based sequencing and cytochrome P450 (CYP) 2D6-mediated tamoxifen metabolism data from a prospective study of 561 patients with breast cancer to train a neural network. The model explained 79% of interindividual variability in CYP2D6 activity compared to 54% with the conventional *-allele approach, assigned enzyme activities to known alleles with previously reported effects, and predicted the activity of previously uncharacterized combinations of variants. The results were replicated in an independent cohort of tamoxifen-treated patients (model R 2 adjusted = 0.66 versus *-allele R 2 adjusted = 0.35) and a cohort of patients treated with the CYP2D6 substrate venlafaxine (model R 2 adjusted = 0.64 versus *-allele R 2 adjusted = 0.55). Human embryonic kidney cells were used to confirm the effect of five genetic variants on metabolism of the CYP2D6 substrate bufuralol in vitro. These results demonstrate the advantage of a continuous scale and a completely phased genotype for prediction of CYP2D6 enzyme activity and could potentially enable more accurate prediction of individual drug response.

High-throughput genotyping of mannose-binding lectin variants using high-resolution DNA-melting analysis.

High Resolution Melting Analysis (HRMA) is a rapid and sensitive method for single nucleotide polymorphism (SNP) analysis. In the present study we present a novel HRMA assay to detect three SNPs in close proximity of each other in the first exon of the gene encoding mannose-binding lectin (MBL), a key molecule of innate immunity. These SNPs have been selected for their known biological and clinical relevance. The three SNPs in MBL2 were simultaneously determined in sixty-nine human DNA samples using HRMA and a single non-fluorescent melting probe, without any post-PCR processing of samples. Combining analyses from amplicon melting and probe melting, we have been able to discriminate ten exon 1 MBL2 genotypes with HRMA, making it a suitable tool for MBL genotyping. A second HRMA assay is presented to detect a relevant polymorphism (Y/X SNP) in the MBL2 promoter region. In conclusion, HRMA is a closed tube assay that is easy to setup and lends itself perfectly for high throughput genotyping of MBL2 variants. The present study thereby facilitates further clinical studies into the role of MBL in inflammatory and infectious disease.

Keratosis Follicularis Spinulosa Decalvans is caused by mutations in MBTPS2.

Keratosis Follicularis Spinulosa Decalvans (KFSD) is a rare genetic disorder characterized by development of hyperkeratotic follicular papules on the scalp followed by progressive alopecia of the scalp, eyelashes, and eyebrows. Associated eye findings include photophobia in childhood and corneal dystrophy. Due to the genetic and clinical heterogeneity of similar disorders, a definitive diagnosis of KFSD is often challenging. Toward identification of the causative gene we reanalyzed a large Dutch KFSD family. SNP arrays (1 M) redefined the locus to a 2.9-Mb region at Xp22.12-Xp22.11. Screening of all 14 genes in the candidate region identified MBTPS2 as the candidate gene carrying a c.1523A>G (p.Asn508Ser) missense mutation. The variant was also identified in two unrelated X-linked KFSD families and cosegregated with KFSD in all families. In symptomatic female carriers, skewed X-inactivation of the normal allele matched with increased severity of symptoms. MBTPS2 is required for cleavage of sterol regulatory element-binding proteins (SREBPs). In vitro functional expression studies of the c.1523A>G mutation showed that sterol responsiveness was reduced by half. Other missense mutations in MBTPS2 have recently been identified in patients with IFAP syndrome. We postulate that both phenotypes are in the spectrum of one genetic disorder with a partially overlapping phenotype.

Accurate quantification of dystrophin mRNA and exon skipping levels in duchenne muscular dystrophy.

Antisense oligonucleotide (AON)-mediated exon skipping aimed at restoring the reading frame is a promising therapeutic approach for Duchenne muscular dystrophy that is currently tested in clinical trials. Numerous AONs have been tested in (patient-derived) cultured muscle cells and the mdx mouse model. The main outcome to measure AON efficiency is usually the exon-skipping percentage, though different groups use different methods to assess these percentages. Here, we compare a series of techniques to quantify exon skipping levels in AON-treated mdx mouse muscle. We compared densitometry of RT-PCR products on ethidium bromide-stained agarose gels, primary and nested RT-PCR followed by bioanalyzer analysis and melting curve analysis. The digital array system (Fluidigm) allows absolute quantification of skipped vs non-skipped transcripts and was used as a reference. Digital array results show that 1 ng of mdx gastrocnemius muscle-derived mRNA contains approximately 1100 dystrophin transcripts and that 665 transcripts are sufficient to determine exon-skipping levels. Quantification using bioanalyzer or densitometric analysis of primary PCR products resulted in values close to those obtained with digital array. The use of the same technique allows comparison between different groups working on exon skipping in the mdx mouse model.

Deep sequencing-based expression analysis shows major advances in robustness, resolution and inter-lab portability over five microarray platforms.

The hippocampal expression profiles of wild-type mice and mice transgenic for deltaC-doublecortin-like kinase were compared with Solexa/Illumina deep sequencing technology and five different microarray platforms. With Illumina's digital gene expression assay, we obtained approximately 2.4 million sequence tags per sample, their abundance spanning four orders of magnitude. Results were highly reproducible, even across laboratories. With a dedicated Bayesian model, we found differential expression of 3179 transcripts with an estimated false-discovery rate of 8.5%. This is a much higher figure than found for microarrays. The overlap in differentially expressed transcripts found with deep sequencing and microarrays was most significant for Affymetrix. The changes in expression observed by deep sequencing were larger than observed by microarrays or quantitative PCR. Relevant processes such as calmodulin-dependent protein kinase activity and vesicle transport along microtubules were found affected by deep sequencing but not by microarrays. While undetectable by microarrays, antisense transcription was found for 51% of all genes and alternative polyadenylation for 47%. We conclude that deep sequencing provides a major advance in robustness, comparability and richness of expression profiling data and is expected to boost collaborative, comparative and integrative genomics studies.

High-resolution melting analysis (HRMA): more than just sequence variant screening.

Transition of the double-stranded DNA molecule to its two single strands, DNA denaturation or melting, has been used for many years to study DNA structure and composition. Recent technological advances have improved the potential of this technology, especially to detect variants in the DNA sequence. Sensitivity and specificity were increased significantly by the development of so-called saturating DNA dyes and by improvements in the instrumentation to measure the melting behavior (improved temperature precision combined with increased measurements per time unit and drop in temperature). Melt analysis using these new instruments has been designated high-resolution melting curve analysis (HRM or HRMA). Based on its ease of use, simplicity, flexibility, low cost, nondestructive nature, superb sensitivity, and specificity, HRMA is quickly becoming the tool of choice to screen patients for pathogenic variants. Here we will briefly discuss the latest developments in HRMA and review in particular other applications that have thus far received less attention, including presequence screening, single nucleotide polymorphism (SNP) typing, methylation analysis, quantification (copy number variants and mosaicism), an alternative to gel-electrophoresis and clone characterization. Together, these diverse applications make HRMA a multipurpose technology and a standard tool that should be present in any laboratory studying nucleic acids.

Resolving the complete genome of Kuenenia stuttgartiensis from a membrane bioreactor enrichment using Single-Molecule Real-Time sequencing.

Anaerobic ammonium-oxidizing (anammox) bacteria are a group of strictly anaerobic chemolithoautotrophic microorganisms. They are capable of oxidizing ammonium to nitrogen gas using nitrite as a terminal electron acceptor, thereby facilitating the release of fixed nitrogen into the atmosphere. The anammox process is thought to exert a profound impact on the global nitrogen cycle and has been harnessed as an environment-friendly method for nitrogen removal from wastewater. In this study, we present the first closed genome sequence of an anammox bacterium, Kuenenia stuttgartiensis MBR1. It was obtained through Single-Molecule Real-Time (SMRT) sequencing of an enrichment culture constituting a mixture of at least two highly similar Kuenenia strains. The genome of the novel MBR1 strain is different from the previously reported Kuenenia KUST reference genome as it contains numerous structural variations and unique genomic regions. We find new proteins, such as a type 3b (sulf)hydrogenase and an additional copy of the hydrazine synthase gene cluster. Moreover, multiple copies of ammonium transporters and proteins regulating nitrogen uptake were identified, suggesting functional differences in metabolism. This assembly, including the genome-wide methylation profile, provides a new foundation for comparative and functional studies aiming to elucidate the biochemical and metabolic processes of these organisms.

Loss of maternal EED results in postnatal overgrowth.

BACKGROUND: Investigating how epigenetic information is transmitted through the mammalian germline is the key to understanding how this information impacts on health and disease susceptibility in offspring. EED is essential for regulating the repressive histone modification, histone 3 lysine 27 tri-methylation (H3K27me3) at many developmental genes. RESULTS: In this study, we used oocyte-specific Zp3-Cre recombinase (Zp3Cre) to delete Eed specifically in mouse growing oocytes, permitting the study of EED function in oocytes and the impact of depleting EED in oocytes on outcomes in offspring. As EED deletion occurred only in growing oocytes and females were mated to normal wild type males, this model allowed the study of oocyte programming without confounding factors such as altered in utero environment. Loss of EED from growing oocytes resulted in a significant overgrowth phenotype that persisted into adult life. Significantly, this involved increased adiposity (total fat) and bone mineral density in offspring. Similar overgrowth occurs in humans with Cohen-Gibson (OMIM 617561) and Weaver (OMIM 277590) syndromes, that result from de novo germline mutations in EED or its co-factor EZH2, respectively. Consistent with a role for EZH2 in human oocytes, we demonstrate that de novo germline mutations in EZH2 occurred in the maternal germline in some cases of Weaver syndrome. However, deletion of Ezh2 in mouse oocytes resulted in a distinct phenotype compared to that resulting from oocyte-specific deletion of Eed. CONCLUSIONS: This study provides novel evidence that altering EED-dependent oocyte programming leads to compromised offspring growth and development in the next generation.

Genotyping DNA Variants with High-Resolution Melting Analysis.

High-resolution melting analysis (HRMA) is a simple, quick, and effective method to scan and screen PCR amplicons for sequence variants. HRMA is a nondestructive closed tube assay; after PCR, DNA melting can directly be performed on the amplified samples without any purification or separation steps. For single SNP genotyping, HRMA is an attractive alternative to Sanger sequencing, restriction enzyme analysis, and hydrolysis probes.

Quantitative DNA Analysis Using Droplet Digital PCR.

Droplet digital PCR (ddPCR) is based on the isolated amplification of thousands of individual DNA molecules simultaneously, with each molecule compartmentalized in a droplet. The presence of amplified product in each droplet is indicated by a fluorescent signal, and the proportion of positive droplets allows the precise quantification of a given sequence. In this chapter we briefly outline the basis of ddPCR, and describe two different applications using the Bio-Rad QX200 system: genotyping copy number variation and quantification of Illumina sequencing libraries.

Full-Length Mitochondrial-DNA Sequencing on the PacBio RSII.

Conventional mitochondrial-DNA (MT DNA) sequencing approaches use Sanger sequencing of 20-40 partially overlapping PCR fragments per individual, which is a time- and resource-consuming process. We have developed a high-throughput, accurate, fast, and cost-effective human MT DNA sequencing approach. In this setup we first generate long-range PCR products for two partially overlapping 7.7 and 9.2 kb MT DNA-specific amplicons, add sample-specific barcodes, and sequence these on the PacBio RSII system to obtain full-length MT DNA sequences for genotyping/haplotyping purposes.

Detection of three single nucleotide polymorphisms in the gene encoding mannose-binding lectin in a single pyrosequencing reaction.

Mannose-binding lectin (MBL) is a key molecule of innate immunity. Binding of MBL to carbohydrates present on pathogens activates the lectin pathway of complement activation, resulting into opsonization and anti-microbial protection. Three frequently occurring single nucleotide polymorphisms (SNPs) are described in the coding region of the MBL2 gene that are associated with abnormal polymerization of the MBL molecule, decreased serum concentrations of high molecular weight MBL, and strongly impaired function. Clinical studies have shown that these MBL SNPs are associated with increased susceptibility to infections, especially in immune-compromised persons, as well as with accelerated progression of chronic diseases. The present study describes a novel method to detect the three major MBL SNPs by pyrosequencing. The close proximity of these SNPs allows their detection in one single pyrosequencing reaction, resulting in clearly distinguishable patterns for each allele combination described until now. This method can be used for the easy and reliable detection of MBL SNPs to identify the basis of functional MBL deficiency in clinical diagnostics and research.

Deletion and duplication screening in the DMD gene using MLPA.

We have designed a multiplex ligation-dependent probe amplification (MLPA) assay to simultaneously screen all 79 DMD gene exons for deletions and duplications in Duchenne and Becker muscular dystrophy (DMD/BMD) patients. We validated the assay by screening 123 unrelated patients from Serbia and Montenegro already screened using multiplex PCR. MLPA screening confirmed the presence of all previously detected deletions. In addition, we detected seven new deletions, nine duplications, one point mutation, and we were able to precisely determine the extent of all rearrangements. To facilitate MLPA-based screening in laboratories lacking specific equipment, we designed the assay such that it can also be performed using agarose gel analysis and ethidium bromide staining. The MLPA assay as described provides a simple and cheap method for deletion and duplication screening in DMD/BMD patients. The assay outperforms the Beggs and Chamberlain multiplex-PCR test, and should be considered as the method of choice for an initial DNA analysis of DMD/BMD patients.