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Asset Tag: CCS

September 22, 2019

Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance.

Antibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. Standard laboratory evolution approaches explore only a small fraction of the sequence space and fail to identify exceedingly rare resistance mutations and combinations thereof. Therefore, new rapid and exhaustive methods are needed to accurately assess the potential of resistance evolution and uncover the underlying mutational mechanisms. Here, we introduce directed evolution with random genomic mutations (DIvERGE), a method that allows an up to million-fold increase in mutation rate along the full lengths of multiple predefined loci in a range of bacterial species. In a single day, DIvERGE generated specific mutation combinations, yielding clinically significant resistance against trimethoprim and ciprofloxacin. Many of these mutations have remained previously undetected or provide resistance in a species-specific manner. These results indicate pathogen-specific resistance mechanisms and the necessity of future narrow-spectrum antibacterial treatments. In contrast to prior claims, we detected the rapid emergence of resistance against gepotidacin, a novel antibiotic currently in clinical trials. Based on these properties, DIvERGE could be applicable to identify less resistance-prone antibiotics at an early stage of drug development. Finally, we discuss potential future applications of DIvERGE in synthetic and evolutionary biology. Copyright © 2018 the Author(s). Published by PNAS.

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September 22, 2019

A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining.

DNA ligases are key enzymes in molecular and synthetic biology that catalyze the joining of breaks in duplex DNA and the end-joining of DNA fragments. Ligation fidelity (discrimination against the ligation of substrates containing mismatched base pairs) and bias (preferential ligation of particular sequences over others) have been well-studied in the context of nick ligation. However, almost no data exist for fidelity and bias in end-joining ligation contexts. In this study, we applied Pacific Biosciences Single-Molecule Real-Time sequencing technology to directly sequence the products of a highly multiplexed ligation reaction. This method has been used to profile the ligation of all three-base 5′-overhangs by T4 DNA ligase under typical ligation conditions in a single experiment. We report the relative frequency of all ligation products with or without mismatches, the position-dependent frequency of each mismatch, and the surprising observation that 5′-TNA overhangs ligate extremely inefficiently compared to all other Watson-Crick pairings. The method can easily be extended to profile other ligases, end-types (e.g. blunt ends and overhangs of different lengths), and the effect of adjacent sequence on the ligation results. Further, the method has the potential to provide new insights into the thermodynamics of annealing and the kinetics of end-joining reactions.

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September 22, 2019

Tumor-specific mitochondrial DNA variants are rarely detected in cell-free DNA.

The use of blood-circulating cell-free DNA (cfDNA) as a “liquid biopsy” in oncology is being explored for its potential as a cancer biomarker. Mitochondria contain their own circular genomic entity (mitochondrial DNA, mtDNA), up to even thousands of copies per cell. The mutation rate of mtDNA is several orders of magnitude higher than that of the nuclear DNA. Tumor-specific variants have been identified in tumors along the entire mtDNA, and their number varies among and within tumors. The high mtDNA copy number per cell and the high mtDNA mutation rate make it worthwhile to explore the potential of tumor-specific cf-mtDNA variants as cancer marker in the blood of cancer patients. We used single-molecule real-time (SMRT) sequencing to profile the entire mtDNA of 19 tissue specimens (primary tumor and/or metastatic sites, and tumor-adjacent normal tissue) and 9 cfDNA samples, originating from 8 cancer patients (5 breast, 3 colon). For each patient, tumor-specific mtDNA variants were detected and traced in cfDNA by SMRT sequencing and/or digital PCR to explore their feasibility as cancer biomarker. As a reference, we measured other blood-circulating biomarkers for these patients, including driver mutations in nuclear-encoded cfDNA and cancer-antigen levels or circulating tumor cells. Four of the 24 (17%) tumor-specific mtDNA variants were detected in cfDNA, however at much lower allele frequencies compared to mutations in nuclear-encoded driver genes in the same samples. Also, extensive heterogeneity was observed among the heteroplasmic mtDNA variants present in an individual. We conclude that there is limited value in tracing tumor-specific mtDNA variants in blood-circulating cfDNA with the current methods available. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

A rapid method for directed gene knockout for screening in G0 zebrafish.

Zebrafish is a powerful model for forward genetics. Reverse genetic approaches are limited by the time required to generate stable mutant lines. We describe a system for gene knockout that consistently produces null phenotypes in G0 zebrafish. Yolk injection of sets of four CRISPR/Cas9 ribonucleoprotein complexes redundantly targeting a single gene recapitulated germline-transmitted knockout phenotypes in >90% of G0 embryos for each of 8 test genes. Early embryonic (6 hpf) and stable adult phenotypes were produced. Simultaneous multi-gene knockout was feasible but associated with toxicity in some cases. To facilitate use, we generated a lookup table of four-guide sets for 21,386 zebrafish genes and validated several. Using this resource, we targeted 50 cardiomyocyte transcriptional regulators and uncovered a role of zbtb16a in cardiac development. This system provides a platform for rapid screening of genes of interest in development, physiology, and disease models in zebrafish. Copyright © 2018 Elsevier Inc. All rights reserved.

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September 22, 2019

Catching up with Lyme disease antigenic variation computationally.

The spirochetes that cause Lyme disease have an elaborate antigenic variation system that produces millions of variants, thus evading the immune response. Verhey et al. have applied next-generation sequencing and computational analysis to gain new insights into how these bacteria keep ‘one step ahead’ of elimination by the host. Copyright © 2018 Elsevier Ltd. All rights reserved.

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September 22, 2019

Hepacivirus A infection in horses defines distinct envelope hypervariable regions and elucidates potential roles of viral strain and adaptive immune status in determining envelope diversity and infection outcome.

Hepacivirus A (also known as nonprimate hepacivirus and equine hepacivirus) is a hepatotropic virus that can cause both transient and persistent infections in horses. The evolution of intrahost viral populations (quasispecies) has not been studied in detail for hepacivirus A, and its roles in immune evasion and persistence are unknown. To address these knowledge gaps, we first evaluated the envelope gene (E1 and E2) diversity of two different hepacivirus A strains (WSU and CU) in longitudinal blood samples from experimentally infected adult horses, juvenile horses (foals), and foals with severe combined immunodeficiency (SCID). Persistent infection with the WSU strain was associated with significantly greater quasispecies diversity than that observed in horses who spontaneously cleared infection (P = 0.0002) or in SCID foals (P < 0.0001). In contrast, the CU strain was able to persist despite significantly lower (P < 0.0001) and relatively static envelope diversity. These findings indicate that envelope diversity is a poor predictor of hepacivirus A infection outcomes and could be dependent on strain-specific factors. Next, entropy analysis was performed on all E1/E2 genes entered into GenBank. This analysis defined three novel hypervariable regions (HVRs) in E2, at residues 391 to 402 (HVR1), 450 to 461 (HVR2), and 550 to 562 (HVR3). For the experimentally infected horses, entropy analysis focusing on the HVRs demonstrated that these regions were under increased selective pressure during persistent infection. Increased diversity in the HVRs was also temporally associated with seroconversion in some horses, suggesting that these regions may be targets of neutralizing antibody and may play a role in immune evasion.IMPORTANCE Hepacivirus C (hepatitis C virus) is estimated to infect 150 million people worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. In contrast, its closest relative, hepacivirus A, causes relatively mild disease in horses and is frequently cleared. The relationship between quasispecies evolution and infection outcome has not been explored for hepacivirus A. To address this knowledge gap, we examined envelope gene diversity in horses with resolving and persistent infections. Interestingly, two strain-specific patterns of quasispecies diversity emerged. Persistence of the WSU strain was associated with increased quasispecies diversity and the accumulation of amino acid changes within three novel hypervariable regions following seroconversion. These findings provided evidence that envelope gene mutation is influenced by adaptive immune pressure and may contribute to hepacivirus persistence. However, the CU strain persisted despite relative evolutionary stasis, suggesting that some hepacivirus strains may use alternative mechanisms to persist in the host. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Methylation of the reelin gene promoter in peripheral blood and its relationship with the cognitive function of schizophrenia patients.

There is a decrease in the expression of the reelin gene (RELN) in the brain of schizophrenia patients, which can underlie observed cognitive abnormalities. It is suggested that this decrease is caused by the hypermethylation of the RELN promoter. The aim of the study was to investigate methylation of the RELN promoter in the peripheral blood of schizophrenia patients and its association with their cognitive deficits. A modified SMRT-BS (single-molecule real-time bisulfite sequencing) was used. We determined the methylation rate of 170 CpG sites within a 1465 bp DNA region containing the entire CpG island in the RELN promoter in 51 schizophrenia patients and 52 healthy controls. All subjects completed a battery of neuropsychological tests. There were no DNA methylation changes associated with schizophrenia. Most CpGs sites were unmethylated in both groups. At the same time, there was a variability in the methylation level of different regions within the promoter. The methylation level in the area from -258 to -151 bp relative to RELN transcription start site was a significant predictor of the index of patients’ cognitive functioning if sex, age, smoking, education, and polymorphism rsl858815 had been considered. The positive correlation between the methylation rate in this region and cognitive index suggests that the hypomethylation of the RELN promoter could contribute to the development of cognitive deficits in schizophrenia.

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September 22, 2019

Identification of the KPC plasmid pCT-KPC334: New insights on the evolutionary pathway of epidemic plasmids harboring fosA3-blaKPC-2 genes.

A novel, non-conjugative plasmid pKP1034 isolated from a fosfomycin-resistant, carbapenemase-producing Klebsiella pneumonia strain KP1034 was recently reported to carry fosA3, blaKPC-2, blaCTX-M-65, blaSHV-12 and rmtB genes, and was hypothesized to evolve from several recombination events of two closely related plasmids, pHN7A8 and pKPC-LK30 [1]. In this study, a plasmid pCT-KPC334 carrying fosA3, blaKPC-2, blaCTX-M-65, blaSHV-12, blaTEM-1, and rmtB genes was identified, providing evidence on the evolutionary pathway of plasmids harboring fosA3-blaKPC-2 genes.

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September 22, 2019

A multiplex homology-directed DNA repair assay reveals the impact of more than 1,000 BRCA1 missense substitution variants on protein function.

Loss-of-function pathogenic variants in BRCA1 confer a predisposition to breast and ovarian cancer. Genetic testing for sequence changes in BRCA1 frequently reveals a missense variant for which the impact on cancer risk and on the molecular function of BRCA1 is unknown. Functional BRCA1 is required for the homology-directed repair (HDR) of double-strand DNA breaks, a critical activity for maintaining genome integrity and tumor suppression. Here, we describe a multiplex HDR reporter assay for concurrently measuring the effects of hundreds of variants of BRCA1 for their role in DNA repair. Using this assay, we characterized the effects of 1,056 amino acid substitutions in the first 192 residues of BRCA1. Benchmarking these results against variants with known effects on DNA repair function or on cancer predisposition, we demonstrate accurate discrimination of loss-of-function versus benign missense variants. We anticipate that this assay can be used to functionally characterize BRCA1 missense variants at scale, even before the variants are observed in results from genetic testing. Copyright © 2018 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Comprehensive profiling of four base overhang ligation fidelity by T4 DNA Ligase and application to DNA assembly.

Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzyme-dependent DNA assembly methods enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined by the ligation of Watson-Crick base-paired overhangs. However, ligation of mismatched overhangs leads to erroneous assembly, and low-efficiency Watson Crick pairings can lead to truncated assemblies. Using sets of empirically vetted, high-accuracy junction pairs avoids this issue but limits the number of parts that can be joined in a single reaction. Here, we report the use of comprehensive end-joining ligation fidelity and bias data to predict high accuracy junction sets for Golden Gate assembly. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions and enabled accurate and efficient assembly of a lac cassette from up to 24-fragments in a single reaction.

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September 22, 2019

Loss of Rap1 supports recombination-based telomere maintenance independent of RNA-DNA hybrids in fission yeast

To investigate the molecular changes needed for cells to maintain their telomeres by recombination, we monitored telomere appearance during serial culture of fission yeast cells lacking the telomerase recruitment factor Ccq1. Rad52 is loaded onto critically short telomeres shortly after germination despite continued telomere erosion, suggesting that recruitment of recombination factors is not sufficient to maintain telomeres in the absence of telomerase function. Instead, survivor formation coincides with the derepression of telomeric repeat-containing RNA (TERRA). Degradation of telomere-associated TERRA in this context drives a severe growth crisis, ultimately leading to a distinct type of linear survivor with altered cytological telomere characteristics and the eviction of the shelterin component Rap1 (but not the TRF1/TRF2 orthologue, Taz1) from the telomere. We demonstrate that deletion of Rap1 is protective, preventing the growth crisis that is otherwise triggered by degradation of telomere-engaged TERRA in survivors with linear chromosomes. Thus, modulating the stoichiometry of shelterin components appears to support recombination-dependent survivors to persist in the absence of telomere-engaged TERRA.

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September 22, 2019

Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep.

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in?~?50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.

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September 22, 2019

Biparental Inheritance of Mitochondrial DNA in Humans.

Although there has been considerable debate about whether paternal mitochondrial DNA (mtDNA) transmission may coexist with maternal transmission of mtDNA, it is generally believed that mitochondria and mtDNA are exclusively maternally inherited in humans. Here, we identified three unrelated multigeneration families with a high level of mtDNA heteroplasmy (ranging from 24 to 76%) in a total of 17 individuals. Heteroplasmy of mtDNA was independently examined by high-depth whole mtDNA sequencing analysis in our research laboratory and in two Clinical Laboratory Improvement Amendments and College of American Pathologists-accredited laboratories using multiple approaches. A comprehensive exploration of mtDNA segregation in these families shows biparental mtDNA transmission with an autosomal dominantlike inheritance mode. Our results suggest that, although the central dogma of maternal inheritance of mtDNA remains valid, there are some exceptional cases where paternal mtDNA could be passed to the offspring. Elucidating the molecular mechanism for this unusual mode of inheritance will provide new insights into how mtDNA is passed on from parent to offspring and may even lead to the development of new avenues for the therapeutic treatment for pathogenic mtDNA transmission.

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September 22, 2019

Relationship between Alzheimer’s disease-associated SNPs within the CLU gene, local DNA methylation and episodic verbal memory in healthy and schizophrenia subjects.

Genetic variation may impact on local DNA methylation patterns. Therefore, information about allele-specific DNA methylation (ASM) within disease-related loci has been proposed to be useful for the interpretation of GWAS results. To explore mechanisms that may underlie associations between Alzheimer’s disease (AD) and schizophrenia risk CLU gene and verbal memory, one of the most affected cognitive domains in both conditions, we studied DNA methylation in a region between AD-associated SNPs rs9331888 and rs9331896 in 72 healthy individuals and 73 schizophrenia patients. Using single-molecule real-time bisulfite sequencing we assessed the haplotype-dependent ASM in this region. We then investigated whether its methylation could influence episodic verbal memory measured with the Rey Auditory Verbal Learning Test in these two cohorts. The region showed a complex methylation pattern, which was similar in healthy and schizophrenia individuals and unrelated to haplotypes. The pattern predicted memory scores in controls. The results suggest that epigenetic modifications within the CLU locus may play a role in memory variation, independent of ASM. Copyright © 2018 Elsevier B.V. All rights reserved.

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September 21, 2019

Towards Personalized Medicine: An Improved De Novo Assembly Procedure for Early Detection of Drug Resistant HIV Minor Quasispecies in Patient Samples.

The third-generation sequencing technology, PacBio, has shown an ability to sequence the HIV virus amplicons in their full length. The long read of PaBio offers a distinct advantage to comprehensively understand the virus evolution complexity at quasispecies level (i.e. maintaining linkage information of variants) comparing to the short reads from Illumina shotgun sequencing. However, due to the highnoise nature of the PacBio reads, it is still a challenge to build accurate contigs at high sensitivity. Most of previously developed NGS assembly tools work with the assumption that the input reads are fairly accurate, which is largely true for the data derived from Sanger or Illumina technologies. When applying these tools on PacBio high-noise reads, they are largely driven by noise rather than true signal eventually leading to poor results in most cases. In this study, we propose the de novo assembly procedure, which comprises a positivefocused strategy, and linkage-frequency noise reduction so that it is more suitable for PacBio high-noise reads. We further tested the unique de novo assembly procedure on HIV PacBio benchmark data and clinical samples, which accurately assembled dominant and minor populations of HIV quasispecies as expected. The improved de novo assembly procedure shows potential ability to promote PacBio technology in the field of HIV drug-resistance clinical detection, as well as in broad HIV phylogenetic studies.

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