Laurence EttwillerNew England Biolabs · Research Department
Laurence Ettwiller
Doctor of Philosophy
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185
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Introduction
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Publications
Publications (185)
Methylation patterns in bacteria can be used to study Restriction-Modification (RM) or other defense systems with novel properties. While m4C and m6A methylation is well characterized mainly through PacBio sequencing, the landscape of m5C methylation is under-characterized. To bridge this gap, we performed RIMS-seq2 on microbiomes composed of resol...
Multiomics requires concerted recording of independent information, ideally from a single experiment. In this study, we introduce RIMS-seq2, a high-throughput technique to simultaneously sequence genomes and overlay methylation information while requiring only a small modification of the experimental protocol for high throughput DNA sequencing to i...
Winged helix (wH) domains, also termed winged helix-turn-helix (wHTH) domains, are widespread in all kingdoms of life and have diverse roles. In the context of DNA binding and DNA modification sensing, some eukaryotic wH domains are known as sensors of non-methylated CpG. In contrast, the prokaryotic wH domains in DpnI and HhiV4I act as sensors of...
The modified DNA base 2,6 aminopurine (2-aminoadenine, (d)Z base) was originally found in phages to counteract host encoded restriction systems. However, only a limited number of restriction endonucleases (REases) have been tested on dZ-modified DNA. Herein we report the results of 147 REases activity on dZ-modified PCR DNA. Among the enzymes teste...
Multi-omics requires concerted recording of independent information, ideally from a single experiment. In this study, we introduce RIMS-seq2, a high-throughput technique to simultaneously sequence genomes and overlay methylation information while requiring only a small modification of the experimental protocol for high throughput DNA sequencing to...
Winged helix (wH) domains, also termed winged helix-turn-helix (wHTH) domains, are widespread in all kingdoms of life, and have diverse roles. In the context of DNA binding and DNA modification sensing, some eukaryotic wH domains are known as sensors of non-methylated CpG. In contrast, the prokaryotic wH domains in DpnI and phi.HhiV4I act as sensor...
The intracellular endosymbiotic proteobacteria Wolbachia have evolved across the phyla nematoda and arthropoda. In Wolbachia phylogeny, supergroup F is the only clade known so far with members from both arthropod and filarial nematode hosts and therefore can provide unique insights into their evolution and biology. In this study, 4 new supergroup F...
The filarial parasites Mansonella ozzardi and Mansonella perstans , causative agents of mansonellosis, infect hundreds of millions of people worldwide, yet remain among the most understudied of the human filarial pathogens. M. ozzardi is highly prevalent in Latin American countries and Caribbean Islands, while M. perstans is predominantly found in...
Avoiding damage-induced sequencing errors is a critical step for the accurate identification of medium to rare frequency mutations in DNA samples. In the case of FFPE samples, deamination of cytosine moieties represents a major damage resulting in the loss of DNA material and sequencing errors. In this study, we demonstrated that, while damage from...
The filarial parasites Mansonella ozzardi and Mansonella perstans, causative agents of mansonellosis, infect hundreds of millions of people worldwide, yet remain among the most understudied of the human filarial pathogens. M. ozzardi is highly prevalent in Latin American countries and Caribbean Islands, while M. perstans is predominantly found in s...
Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransforme...
Covalent modifications of genomic DNA are crucial for most organisms to survive. Amplicon-based high throughput sequencing technologies erase all DNA modifications to retain only sequence information for the four canonical nucleobases, necessitating specialized technologies for ascertaining epigenetic information. To also capture base modification...
The luciferin sulfokinase (coelenterazine sulfotransferase) of Renilla was previously reported to activate the storage form, luciferyl sulfate (coelenterazine sulfate) to luciferin (coelenterazine), the substrate for the luciferase bioluminescence reaction. The gene coding for the coelenterazine sulfotransferase has not been identified. Here we use...
Phosphorothioation (PT), in which a non-bridging oxygen is replaced by a sulfur, is one of the rare modifications discovered in bacteria and archaea that occurs on the sugar-phosphate backbone as opposed to the nucleobase moiety of DNA. While PT modification is widespread in the prokaryotic kingdom, how PT modifications are distributed in the genom...
Nucleic acids in living organisms are more complex than the simple combinations of the four canonical nucleotides. Recent advances in biomedical research have led to the discovery of numerous naturally occurring nucleotide modifications and enzymes responsible for the synthesis of such modifications. In turn, these enzymes can be leveraged towards...
Covalent modifications of genomic DNA are crucial for most organisms to survive. Amplicon-based high throughput sequencing technologies erase all DNA modifications to retain only sequence information for the four canonical nucleobases, necessitating specialized technologies for ascertaining epigenetic information. To also capture base modification...
As the goal of a bacterium is to become bacteria, evolution has imposed continued selections for gene expression. The intracellular pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis, has adopted a fine-tuned response to survive its host’s methods to aggressively eradicate invaders. The development of microarrays and later RNA...
Phosphorothioation (PT), in which a non-bridging oxygen is replaced by a sulfur, is one of the rare modifications discovered in bacteria and archaea that occurs on the sugar-phosphate backbone as opposed to the nucleobase moiety of DNA. While PT modification is widespread in the prokaryotic kingdom, how PT modifications are distributed in the genom...
The intracellular endosymbiotic proteobacteria Wolbachia have evolved across the phyla nematoda and arthropoda. In Wolbachia phylogeny, supergroup F is the only clade known so far with members from both arthropod and filarial nematode hosts and therefore can provide unique insights into their evolution and biology. In this study, 4 new supergroup F...
Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering...
The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do...
Transposable elements (TE) are mobile genetic elements, present in all domains of life. They commonly encode a single transposase enzyme, that performs the excision and reintegration reactions, and these enzymes have been used in mutagenesis and creation of next-generation sequencing libraries. All transposases have some bias in the DNA sequence th...
The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested sub genomic RNAs used to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques...
Determination of eukaryotic Transcription Start Sites (TSS) has been based on methods that require the cap structure at the 5-prime end of transcripts derived from Pol-II RNA polymerase. Consequently, these methods do not reveal TSS derived from the other RNA polymerases which also play critical roles in various cell functions. To address this limi...
Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering...
Shotgun metagenomic sequencing is a powerful approach to study microbiomes in an unbiased manner and of increasing relevance for identifying novel enzymatic functions. However, the potential of metagenomics to relate from microbiome composition to function has thus far been underutilized. Here, we introduce the Metagenomics Genome-Phenome Associati...
Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional methods the 5′ and 3′ ends of poly(A) RNA cannot be identified unambiguously. This is due in part to the architecture of the nanopore/enzyme-motor complex, and in part to RNA de...
DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylas...
Alternative transcription units (ATUs) are dynamically encoded under different conditions and display overlapping patterns (sharing one or more genes) under a specific condition in bacterial genomes. Genome-scale identification of ATUs is essential for studying the emergence of human diseases caused by bacterial organisms. However, it is unrealisti...
Shotgun metagenomic sequencing is a powerful approach to study microbiomes in an unbiased manner and of increasing relevance for identifying novel enzymatic functions. However, the potential of metagenomics to relate from microbiome composition to function has thus far been underutilized. Here, we introduce the Metagenomics Genome-Phenome Associati...
DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylas...
The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA, leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisu...
Alternative transcription units (ATUs) are dynamically encoded under different conditions or environmental stimuli in bacterial genomes, and genome-scale identification of ATUs is essential for studying the emergence of human diseases caused by bacterial organisms. However, it is unrealistic to identify all ATUs using experimental techniques, due t...
Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional methods the 5′ and 3′ ends of poly(A) RNA cannot be identified unambiguously. This is due in part to the architecture of the nanopore/enzyme-motor complex, and in part to RNA de...
DNA methylation is important for gene regulation. The ability to accurately identify 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) gives us greater insight into potential gene regulatory mechanisms. Bisulfite sequencing (BS) is traditionally used to detect methylated Cs, however, BS does have its drawbacks. DNA is commonly damaged and d...
The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisul...
Mansonelliasis is a widespread yet neglected tropical infection of humans in Africa and South America caused by the filarial nematodes, Mansonella perstans , M. ozzardi, M. rodhaini and M. streptocerca. Clinical symptoms are non-distinct and diagnosis mainly relies on the detection of microfilariae in skin or blood. Species-specific DNA repeat sequ...
Methodologies for determining eukaryotic Transcription Start Sites (TSS) rely on the selection of the 5 prime canonical cap structure of Pol-II transcripts and are consequently ignoring entire classes of TSS derived from other RNA polymerases which play critical roles in various cell functions. To overcome this limitation, we developed ReCappable-s...
High-throughput sequencing is key for clinical diagnostic applications such as disease mutation profiling and identifying pathogens with high taxonomic specificity. Preparation of high quality next-generation sequencing (NGS) libraries is critical for obtaining accurate patient data, but clinical scientists are often limited by input DNA quality. I...
High-throughput sequencing is key for clinical diagnostic applications such as disease mutation profiling and identifying pathogens with high taxonomic specificity. Preparation of high quality next-generation sequencing (NGS) libraries is critical for obtaining accurate patient data, but clinical scientists are often limited by input DNA quality. I...
DNA isolated from blood draws (cell-free DNA (cfDNA)) or from archival material like formalin fixed paraffin embedded (FFPE) tissues have advanced the field of cancer genetics. DNA methylation (5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC)) is a key epigenetic factor that plays an important role in cellular processes and it’s misregul...
RAre DAmage and Repair sequencing (RADAR-seq) is a highly adaptable sequencing method that enables the identification and detection of rare DNA damage events for a wide variety of DNA lesions at single-molecule resolution on a genome-wide scale. In RADAR-seq, DNA lesions are replaced with a patch of modified bases that can be directly detected by P...
Dmrt1 is a highly conserved transcription factor, which is critically involved in regulation of gonad development of vertebrates. In medaka, a duplicate of dmrt1—acting as master sex-determining gene—has a tightly timely and spatially controlled gonadal expression pattern. In addition to transcriptional regulation, a sequence motif in the 3′ UTR (D...
Phylogeny and synteny analysis of Ol-bsf and Ol-cug-bp ohnologs.
(A) Circular cladogram representation of the phylogenetic tree of lrpprc proteins in jawed vertebrates (gnatosthomes). This phylogeny shows that lrpprc genes were retained as single copies in most jawed vertebrates even following whole genome duplications (red stars), e.g., the teleos...
Locomotor activity of Ol-bsf mutant fish versus wild type.
Locomotor activity (Ol-bsf mutants versus wild type) was determined at hatching stage (stage 39, 9 dpf) either under dark conditions (A) or with 10% light (approximately equal to 100 lux [panel B]) by measuring the total distance swum during a period of 5 minutes. (C) The total distance swu...
Presence of the D3U-box in different UTRs and coding sequence of gonadal genes.
Presence of the D3U-box (matrix-scan) was evaluated within the transcriptome of the medaka fish. D3U-box, dmrt1 3′ UTR box.
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Lrrprc and celf1, but not celf2, are expressed in mouse embryonic gonads.
(A to H) ISHs on sagittal sections of 14.5 dpc mouse embryos showed expression of lrrprc (A to D) and celf1 (E to H) most likely in germ cells within testis cords (B and F) and germ cells within the ovary (D and H). In contrast, no celf2 expression was detected in developing...
Ovarian phenotypes of the Ol-bsf mutant fish.
Morphological inspection of heterozygote mutant ovaries discloses a significant accumulation of small sized-oocytes compared to wild type (A) and (A1 to A5 for details and statistical analyses). (A) Overall size distribution of the oocytes in 9 wild-type and 9 Ol-BSF(+/−) adult ovaries. Each gonad (test...
Generation Ol-bsf knockout medaka fish after genome editing by CRISPR/Cas9 method.
(A) Several guide RNA were designed in order to target different locations on the Ol-bsf gene (targets 1, 2, and 6). (B) After injection of different combinations of guide RNAs together with the Cas9 mRNA, putative edited fish were subjected to RT-PCR using primer se...
Testicular phenotypes of the Ol-bsf mutant fish.
(A–J compared to K–T) Heterozygote mutant testes (A–J) exhibit a decreased number of spermatogonia with accumulation of type 2 spermatocytes, spermatids, and sperm within the most external layers of the seminiferous epithelium (arrowheads) compared to wild-type testes (K–T). Either 10 different wild-...
Analysis of morpholino efficiency and level of Ol-bsf down-regulation.
For in vivo transient down-regulation of Ol-bsf, a splice morpholino was designed to encompass the splice junction between exon 2 and intron 2 of the Ol-bsf gene in order to induce aberrant splicing and frame shit of the ORF. To show to what extend the splicing/activity of Ol-bs...
Real-time PCR quantification of Ol-cug-bp1, Ol-cug-bp2, and Ol-bsf during embryogenesis and in adult tissues.
(A and C) During embryonic development, both Ol-cug-bp ohnologs are expressed in a complementary manner. Being likely maternally deposited the expression of Ol-cug-bp1 rapidly decreases after mid-blastula transition (stage 10) to remain vir...
Ovarian phenotypes of the Ol-BSF mutant fish.
(A to R) Mid-sagittal sections of the ovaries utilized for counting the oocytes in S8 Fig. Each gonad (testes or ovaries) was sectioned through the mid-sagittal plan (see also Materials and methods). Underlying data for (A) can be found in S1 Data. Ol-BSF, Oryzias latipes Bicoid Stability Factor.
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Fertility test.
Egg numbers and fertilization rates were recorded over a period of 9 days for the following crosses: (A) male Ol-bsf (−/+) × female Ol-bsf (−/+); (B) male Ol-bsf (−/+) × female wild type; (C) male wild type × female Ol-bsf (−/+). Underlying data for (A to C) can be found in S1 Data. BSF, bicoid stability factor.
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Microarray data and mitochondrial gene quantification.
(A) Microarray. Adult testes of either bsf+/− or wild-type animals were subjected to microarray (see Materials and methods). GO term analysis reveals that in mutant testes partially depleted for the bsf gene, rRNA processing is particularly affected. Ol-bsf and Ol-cug-bp2 are down- and up-regul...
Gene evolution of cugbp elav-like family member 2 genes in some teleosts. celf2, cugbp elav-like family member 2.
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Abstract The use of next-generation sequencing (NGS) has been instrumental in advancing biological research and clinical diagnostics. To fully utilize the power of NGS, complete, uniform coverage of the entire genome is required. In this study, we identified the primary sources of bias observed in sequence coverage across AT-rich regions of the hum...
Following the Comment of Stewart et al, we repeated our analysis on sequencing runs from The Cancer Genome Atlas (TCGA) using their suggested parameters. We found signs of oxidative damage in all sequence contexts and irrespective of the sequencing date, reaffirming that DNA damage affects mutation-calling pipelines in their ability to accurately i...
Current methods for genome-wide analysis of gene expression require fragmentation of original transcripts into small fragments for short-read sequencing. In bacteria, the resulting fragmented information hides operon complexity. Additionally, in vivo processing of transcripts confounds the accurate identification of the 5' and 3' ends of operons. H...
Next-generation sequencing (NGS) methods are used extensively to profile mutations present in diseased human tissues. These genomic approaches hold great promise for personalized medicine but sequencing accuracy is essential for proper patient diagnosis and determining a treatment plan. A common source of DNA for genomic profiling is formalin-fixed...
Gene expression in bacteria is organized into operons, functional units of genomic DNA containing clusters of genes under the control of single promoters. The definition of an operon relies on the precise identification of the start of transcription (TSS), the end of transcription and (TTS) and the relation between the TSS and the TTS for each tran...
Current methods for genome-wide analysis of gene expression requires shredding original transcripts into small fragments for short-read sequencing. In bacteria, the resulting fragmented information hides operon complexity. Additionally, in-vivo processing of transcripts confounds the accurate identification of the 5’ and 3’ ends of operons. Here we...
Targeted cancer therapy based on genomic alterations can be remarkably effective. Currently, cancer genome profiling using next generation sequencing (NGS) is routinely applied in cancer care to guide personalized treatment. The accuracy of this profiling directly impacts therapeutic choices and the outcomes of patient care. We previously showed th...
When is a mutation a true genetic variant?
Large-scale sequencing studies have set out to determine the low-frequency pathogenic genetic variants in individuals and populations. However, Chen et al. demonstrate that many so-called low-frequency genetic variants in large public databases may be due to DNA damage. They scored libraries sequenced with...
Bacteria respond to their environment by regulating mRNA synthesis, often by altering the genomic sites at which RNA polymerase initiates transcription. Here, we investigate genome-wide changes in transcription start site (TSS) usage by Clostridium phytofermentans, a model bacterium for fermentation of lignocellulosic biomass. We quantify expressio...
The gene regulatory network (GRN) that supports neural stem cell (NS cell) self-renewal has so far been poorly characterised. Knowledge of the central transcription factors (TFs), the non-coding gene regulatory regions that they bind to and the genes whose expression they modulate will be crucial in unlocking the full therapeutic potential of these...
Pervasive mutations in somatic cells generate a heterogeneous genomic population within an organism and may result in serious medical conditions. While cancer is the most studied disease associated with somatic variations, recent advances in single cell and ultra deep sequencing indicate that a number of phenotypes and pathologies are impacted by c...
Targeted cancer therapy based on genomic alterations can be remarkably effective, and has made significant strides with the recent advances in next-generation sequencing (NGS) technology. Although samples of blood and other bodily fluids are being actively explored for early disease diagnosis and treatment monitoring, DNA isolated from FFPE samples...