Publications

@article{vergara_multi-ancestry_2020b,

title = {Multi-ancestry fine mapping of interferon lambda and the outcome of acute hepatitis C virus infection},

author = {Candelaria Vergara and Priya Duggal and Chloe L Thio and Ana Valencia and Thomas R O’ Brien and Rachel Latanich and Winston Timp and Eric O Johnson and Alex H Kral and Alessandra Mangia and James J Goedert and Valeria Piazzola and Shruti H Mehta and Gregory D Kirk and Marion G Peters and Sharyne M Donfield and Brian R Edlin and Michael P Busch and Graeme Alexander and Edward L Murphy and Arthur Y Kim and Georg M Lauer and Raymond T Chung and Matthew E Cramp and Andrea L Cox and Salim I Khakoo and Hugo R Rosen and Laurent Alric and Sarah J Wheelan and Genevieve L Wojcik and David L Thomas and Margaret A Taub},

url = {https://www.nature.com/articles/s41435-020-00115-3},

doi = {10.1038/s41435-020-00115-3},

issn = {1476-5470},

year  = {2020},

date = {2020-10-28},

urldate = {2020-10-28},

journal = {Genes & Immunity},

volume = {21},

issue = {6-8},

pages = {1--12},

abstract = {Clearance of acute infection with hepatitis C virus (HCV) is associated with the chr19q13.13 region containing the rs368234815 (TT/ΔG) polymorphism. We fine-mapped this region to detect possible causal variants that may contribute to HCV clearance. First, we performed sequencing of IFNL1-IFNL4 region in 64 individuals sampled according to rs368234815 genotype: TT/clearance (N = 16) and ΔG/persistent (N = 15) (genotype-outcome concordant) or TT/persistent (N = 19) and ΔG/clearance (N = 14) (discordant). 25 SNPs had a difference in counts of alternative allele textgreater5 between clearance and persistence individuals. Then, we evaluated those markers in an association analysis of HCV clearance conditioning on rs368234815 in two groups of European (692 clearance/1 025 persistence) and African ancestry (320 clearance/1 515 persistence) individuals. 10/25 variants were associated (P textless 0.05) in the conditioned analysis leaded by rs4803221 (P value = 4.9 × 10−04) and rs8099917 (P value = 5.5 × 10−04). In the European ancestry group, individuals with the haplotype rs368234815ΔG/rs4803221C were 1.7× more likely to clear than those with the rs368234815ΔG/rs4803221G haplotype (P value = 3.6 × 10−05). For another nearby SNP, the haplotype of rs368234815ΔG/rs8099917T was associated with HCV clearance compared to rs368234815ΔG/rs8099917G (OR: 1.6, P value = 1.8 × 10−04). We identified four possible causal variants: rs368234815, rs12982533, rs10612351 and rs4803221. Our results suggest a main signal of association represented by rs368234815, with contributions from rs4803221, and/or nearby SNPs including rs8099917.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{scott_reference_2020,

title = {A Reference Genome Sequence for Giant Sequoia},

author = {Alison D Scott and Aleksey V Zimin and Daniela Puiu and Rachael Workman and Monica Britton and Sumaira Zaman and Madison Caballero and Andrew C Read and Adam J Bogdanove and Emily Burns and Jill Wegrzyn and Winston Timp and Steven L Salzberg and David B Neale},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642918/},

doi = {10.1534/g3.120.401612},

issn = {2160-1836},

year  = {2020},

date = {2020-09-18},

urldate = {2020-09-18},

journal = {G3: Genes|Genomes|Genetics},

volume = {10},

number = {11},

pages = {3907--3919},

abstract = {The giant sequoia (Sequoiadendron giganteum) of California are massive, long-lived trees that grow along the U.S. Sierra Nevada mountains. Genomic data are limited in giant sequoia and producing a reference genome sequence has been an important goal to allow marker development for restoration and management. Using deep-coverage Illumina and Oxford Nanopore sequencing, combined with Dovetail chromosome conformation capture libraries, the genome was assembled into eleven chromosome-scale scaffolds containing 8.125 Gbp of sequence. Iso-Seq transcripts, assembled from three distinct tissues, was used as evidence to annotate a total of 41,632 protein-coding genes. The genome was found to contain, distributed unevenly across all 11 chromosomes and in 63 orthogroups, over 900 complete or partial predicted NLR genes, of which 375 are supported by annotation derived from protein evidence and gene modeling. This giant sequoia reference genome sequence represents the first genome sequenced in the Cupressaceae family, and lays a foundation for using genomic tools to aid in giant sequoia conservation and management.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{gale_identification_2020,

title = {Identification of Essential Genes and Fluconazole Susceptibility Genes in Candida glabrata by Profiling Hermes Transposon Insertions},

author = {Andrew N Gale and Rima M Sakhawala and Anton Levitan and Roded Sharan and Judith Berman and Winston Timp and Kyle W Cunningham},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534453/},

doi = {10.1534/g3.120.401595},

issn = {2160-1836},

year  = {2020},

date = {2020-08-20},

urldate = {2020-08-20},

journal = {G3: Genes|Genomes|Genetics},

volume = {10},

number = {10},

pages = {3859--3870},

abstract = {Within the budding yeasts, the opportunistic pathogen Candida glabrata and other members of the Nakaseomyces clade have developed virulence traits independently from C. albicans and C. auris. To begin exploring the genetic basis of C. glabrata virulence and its innate resistance to antifungals, we launched the Hermes transposon from a plasmid and sequenced more than 500,000 different semi-random insertions throughout the genome. With machine learning, we identified 1278 protein-encoding genes (25% of total) that could not tolerate transposon insertions and are likely essential for C. glabrata fitness in vitro. Interestingly, genes involved in mRNA splicing were less likely to be essential in C. glabrata than their orthologs in S. cerevisiae, whereas the opposite is true for genes involved in kinetochore function and chromosome segregation. When a pool of insertion mutants was challenged with the first-line antifungal fluconazole, insertions in several known resistance genes (e.g., PDR1, CDR1, PDR16, PDR17, UPC2A, DAP1, STV1) and 15 additional genes (including KGD1, KGD2, YHR045W) became hypersensitive to fluconazole. Insertions in 200 other genes conferred significant resistance to fluconazole, two-thirds of which function in mitochondria and likely down-regulate Pdr1 expression or function. Knockout mutants of KGD2 and IDH2, which consume and generate alpha-ketoglutarate in mitochondria, exhibited increased and decreased resistance to fluconazole through a process that depended on Pdr1. These findings establish the utility of transposon insertion profiling in forward genetic investigations of this important pathogen of humans.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{miga_telomere--telomere_2020b,

title = {Telomere-to-telomere assembly of a complete human X chromosome},

author = {Karen H Miga and Sergey Koren and Arang Rhie and Mitchell R Vollger and Ariel Gershman and Andrey Bzikadze and Shelise Brooks and Edmund Howe and David Porubsky and Glennis A Logsdon and Valerie A Schneider and Tamara Potapova and Jonathan Wood and William Chow and Joel Armstrong and Jeanne Fredrickson and Evgenia Pak and Kristof Tigyi and Milinn Kremitzki and Christopher Markovic and Valerie Maduro and Amalia Dutra and Gerard G Bouffard and Alexander M Chang and Nancy F Hansen and Amy B Wilfert and Françoise Thibaud-Nissen and Anthony D Schmitt and Jon-Matthew Belton and Siddarth Selvaraj and Megan Y Dennis and Daniela C Soto and Ruta Sahasrabudhe and Gulhan Kaya and Josh Quick and Nicholas J Loman and Nadine Holmes and Matthew Loose and Urvashi Surti and Rosa ana Risques and Tina Graves A Lindsay and Robert Fulton and Ira Hall and Benedict Paten and Kerstin Howe and Winston Timp and Alice Young and James C Mullikin and Pavel A Pevzner and Jennifer L Gerton and Beth A Sullivan and Evan E Eichler and Adam M Phillippy},

url = {https://www.nature.com/articles/s41586-020-2547-7},

doi = {10.1038/s41586-020-2547-7},

issn = {1476-4687},

year  = {2020},

date = {2020-07-01},

urldate = {2020-07-01},

journal = {Nature},

volume = {585},

issue = {7823},

pages = {79-84},

abstract = {After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a de novo human genome assembly that surpasses the continuity of GRCh382, along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the textasciitilde3.1 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). These novel sequences will be integrated into future human reference genome releases. Additionally, a complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. Our results demonstrate that finishing the entire human genome is now within reach and the data presented here will enable ongoing efforts to complete the remaining human chromosomes.},

note = {Publisher: Nature Publishing Group},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{marrano_high-quality_2020,

title = {High-quality chromosome-scale assembly of the walnut (Juglans regia L.) reference genome},

author = {Annarita Marrano and Monica Britton and Paulo A Zaini and Aleksey V Zimin and Rachael E Workman and Daniela Puiu and Luca Bianco and Erica Adele Di Pierro and Brian J Allen and Sandeep Chakraborty and Michela Troggio and Charles A Leslie and Winston Timp and Abhaya Dandekar and Steven L Salzberg and David B Neale},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7238675/},

doi = {10.1093/gigascience/giaa050},

issn = {2047-217X},

year  = {2020},

date = {2020-05-20},

urldate = {2020-05-20},

journal = {GigaScience},

volume = {9},

issue = {5},

pages = {giaa050},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{mckelvey_characterization_2020b,

title = {Characterization of Allele-Specific Regulation of Telomerase Reverse Transcriptase in Promoter Mutant Thyroid Cancer Cell Lines},

author = {Brittany A McKelvey and Timothy Gilpatrick and Yongchun Wang and Winston Timp and Christopher B Umbricht and Martha A Zeiger},

url = {https://www.liebertpub.com/doi/abs/10.1089/thy.2020.0055},

doi = {10.1089/thy.2020.0055},

issn = {1050-7256},

year  = {2020},

date = {2020-03-01},

urldate = {2020-03-01},

journal = {Thyroid},

volume = {30},

issue = {10},

pages = {1470-1481},

abstract = {Background: Telomerase reverse transcriptase (TERT) promoter mutations play a role in carcinogenesis and are found in both tumors and cancer cell lines. TERT promoter methylation, transcription factor binding, chromatin remodeling, and alternative splicing are also known to play an integral role in TERT regulation.Methods: Using nanopore Cas9 targeted sequencing, we characterized allele-specific methylation in thyroid cancer cell lines heterozygous for the TERT promoter mutation. Furthermore, using chromatin immunoprecipitation followed by Sanger sequencing, we probed allele-specific binding of the transcription factors GABPA (GA binding protein transcription factor subunit alpha) and MYC, as well as the chromatin marks H3K4me3 and H3K27me3. Finally, using coding single nucleotide polymorphisms and the long-read sequencing, we examined complementary DNA for monoallelic expression (MAE).Results: We found the mutant TERT promoter allele to be significantly less methylated than wild type, while more methylated in the gene body in heterozygous TERT mutant cell lines. We demonstrated that the transcriptional activators GABPA and MYC bind only to the mutant TERT allele. In addition, the activating and repressive chromatin marks H3K4me3 and H3K27me3, respectively, bind mutant and wild-type alleles exclusively. Finally, in heterozygous mutant cell lines, TERT exhibits MAE from the mutant allele only.Conclusions: In summary, by employing new long-read sequencing methods, we were able to definitively demonstrate allele-specific DNA methylation, histone modifications, transcription factor binding, and the resulting monoallelic transcription in cell lines with heterozygous TERT mutations.},

note = {Publisher: Mary Ann Liebert, Inc., publishers},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{olson_framework_2020b,

title = {A framework for assessing 16S rRNA marker-gene survey data analysis methods using mixtures.},

author = {Nathan D Olson and Senthil M Kumar and Shan Li and Domenick J Braccia and Stephanie Hao and Winston Timp and Marc L Salit and Colin O Stine and Hector Corrada Bravo},

url = {https://doi.org/10.1186/s40168-020-00812-1},

doi = {10.1186/s40168-020-00812-1},

issn = {2049-2618},

year  = {2020},

date = {2020-03-01},

journal = {Microbiome},

volume = {8},

number = {1},

pages = {35},

abstract = {There are a variety of bioinformatic pipelines and downstream analysis methods for analyzing 16S rRNA marker-gene surveys. However, appropriate assessment datasets and metrics are needed as there is limited guidance to decide between available analysis methods. Mixtures of environmental samples are useful for assessing analysis methods as one can evaluate methods based on calculated expected values using unmixed sample measurements and the mixture design. Previous studies have used mixtures of environmental samples to assess other sequencing methods such as RNAseq. But no studies have used mixtures of environmental to assess 16S rRNA sequencing.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Gilpatrick604173b,

title = {Targeted nanopore sequencing with Cas9-guided adapter ligation},

author = {Timothy Gilpatrick and Isac Lee and James E Graham and Etienne Raimondeau and Rebecca Bowen and Andrew Heron and Bradley Downs and Saraswati Sukumar and Fritz J Sedlazeck and Winston Timp},

url = {https://doi.org/10.1038/s41587-020-0407-5},

doi = {10.1038/s41587-020-0407-5},

issn = {1546-1696},

year  = {2020},

date = {2020-02-01},

urldate = {2020-02-01},

journal = {Nature Biotechnology},

volume = {38},

issue = {4},

pages = {433-438},

abstract = {Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods1–4 are limited by the loss of native modifications, short read length, high input requirements, low yield or long protocols. In the present study, we describe nanopore Cas9-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adapters for nanopore sequencing. We show that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants, structural variations and CpG methylation. We apply nCATS to four cell lines, to a cell-line-derived xenograft, and to normal and paired tumor/normal primary human breast tissue. Median sequencing coverage was 675× using a MinION flow cell and 34× using the smaller Flongle flow cell. The nCATS sequencing requires only textasciitilde3 μg of genomic DNA and can target a large number of loci in a single reaction. The method will facilitate the use of long-read sequencing in research and in the clinic.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Aganezov04092020,

title = {Comprehensive analysis of structural variants in breast cancer genomes using single-molecule sequencing},

author = {Sergey Aganezov and Sara Goodwin and Rachel M Sherman and Fritz J Sedlazeck and Gayatri Arun and Sonam Bhatia and Isac Lee and Melanie Kirsche and Robert Wappel and Melissa Kramer and Karen Kostroff and David L Spector and Winston Timp and Richard W McCombie and Michael C Schatz},

url = {http://genome.cshlp.org/content/early/2020/09/02/gr.260497.119.abstract},

doi = {10.1101/gr.260497.119},

year  = {2020},

date = {2020-01-01},

urldate = {2020-01-01},

journal = {Genome Research},

volume = {30},

issue = {9},

pages = {1258-1273},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Roach05022020,

title = {The full-length transcriptome of C. elegans using direct RNA sequencing},

author = {Nathan P Roach and Norah Sadowski and Amelia F Alessi and Winston Timp and James Taylor and John K Kim},

url = {http://genome.cshlp.org/content/early/2020/02/05/gr.251314.119.abstract},

doi = {10.1101/gr.251314.119},

year  = {2020},

date = {2020-01-01},

urldate = {2020-01-01},

journal = {Genome Research},

volume = {30},

issue = {2},

pages = {299-312},

abstract = {Current transcriptome annotations have largely relied on short read lengths intrinsic to the most widely used high-throughput cDNA sequencing technologies. For example, in the annotation of the Caenorhabditis elegans transcriptome, more than half of the transcript isoforms lack full-length support and instead rely on inference from short reads that do not span the full length of the isoform. We applied nanopore-based direct RNA sequencing to characterize the developmental polyadenylated transcriptome of C. elegans. Taking advantage of long reads spanning the full length of mRNA transcripts, we provide support for 23,865 splice isoforms across 14,611 genes, without the need for computational reconstruction of gene models. Of the isoforms identified, 3452 are novel splice isoforms not present in the WormBase WS265 annotation. Furthermore, we identified 16,342 isoforms in the 3′ untranslated region (3′ UTR), 2640 of which are novel and do not fall within 10 bp of existing 3′-UTR data sets and annotations. Combining 3′ UTRs and splice isoforms, we identified 28,858 full-length transcript isoforms. We also determined that poly(A) tail lengths of transcripts vary across development, as do the strengths of previously reported correlations between poly(A) tail length and expression level, and poly(A) tail length and 3′-UTR length. Finally, we have formatted this data as a publicly accessible track hub, enabling researchers to explore this data set easily in a genome browser.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Timpeaax8978,

title = {Beyond mass spectrometry, the next step in proteomics},

author = {Winston Timp and Gregory Timp},

url = {https://advances.sciencemag.org/content/6/2/eaax8978},

doi = {10.1126/sciadv.aax8978},

year  = {2020},

date = {2020-01-01},

urldate = {2020-01-01},

journal = {Science Advances},

volume = {6},

number = {2},

pages = {eaax8978},

publisher = {American Association for the Advancement of Science},

abstract = {Proteins can be the root cause of a disease, and they can be used to cure it. The need to identify these critical actors was recognized early (1951) by Sanger; the first biopolymer sequenced was a peptide, insulin. With the advent of scalable, single-molecule DNA sequencing, genomics and transcriptomics have since propelled medicine through improved sensitivity and lower costs, but proteomics has lagged behind. Currently, proteomics relies mainly on mass spectrometry (MS), but instead of truly sequencing, it classifies a protein and typically requires about a billion copies of a protein to do it. Here, we offer a survey that illuminates a few alternatives with the brightest prospects for identifying whole proteins and displacing MS for sequencing them. These alternatives all boast sensitivity superior to MS and promise to be scalable and seem to be adaptable to bioinformatics tools for calling the sequence of amino acids that constitute a protein.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{workman_nanopore_2019b,

title = {Nanopore native RNA sequencing of a human poly(A) transcriptome},

author = {Rachael E Workman and Alison D Tang and Paul S Tang and Miten Jain and John R Tyson and Roham Razaghi and Philip C Zuzarte and Timothy Gilpatrick and Alexander Payne and Joshua Quick and Norah Sadowski and Nadine Holmes and Jaqueline Goes de Jesus and Karen L Jones and Cameron M Soulette and Terrance P Snutch and Nicholas Loman and Benedict Paten and Matthew Loose and Jared T Simpson and Hugh E Olsen and Angela N Brooks and Mark Akeson and Winston Timp},

url = {https://www.nature.com/articles/s41592-019-0617-2},

doi = {10.1038/s41592-019-0617-2},

issn = {1548-7105},

year  = {2019},

date = {2019-11-01},

urldate = {2019-11-01},

journal = {Nature Methods},

volume = {17},

issue = {1},

pages = {114},

abstract = {Directly sequencing RNA strands through a nanopore retains the full length of the transcript and allows for analysis of polyA tail length, transcript haplotypes and base modifications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Sakowskie00392-19b,

title = {Current State of and Future Opportunities for Prediction in Microbiome Research: Report from the Mid-Atlantic Microbiome Meet-up in Baltimore on 9 January 2019},

author = {Eric Sakowski and Gherman Uritskiy and Rachel Cooper and Maya Gomes and Michael R McLaren and Jacquelyn S Meisel and Rebecca L Mickol and David C Mintz and Emmanuel F Mongodin and Mihai Pop and Mohammad Arifur Rahman and Alvaro Sanchez and Winston Timp and Jeseth Delgado Vela and Carly Muletz Wolz and Joseph P Zackular and Jessica Chopyk and Seth Commichaux and Meghan Davis and Douglas Dluzen and Sukirth M Ganesan and Muyideen Haruna and Dan Nasko and Mary J Regan and Saul Sarria and Nidhi Shah and Brook Stacy and Dylan Taylor and Jocelyne DiRuggiero and Sarah P Preheim},

editor = {Jack A Gilbert},

url = {https://msystems.asm.org/content/4/5/e00392-19},

doi = {10.1128/mSystems.00392-19},

year  = {2019},

date = {2019-10-08},

urldate = {2019-10-01},

journal = {mSystems},

volume = {4},

number = {5},

pages = {e00392-19},

publisher = {American Society for Microbiology Journals},

abstract = {Accurate predictions across multiple fields of microbiome research have far-reaching benefits to society, but there are few widely accepted quantitative tools to make accurate predictions about microbial communities and their functions. More discussion is needed about the current state of microbiome analysis and the tools required to overcome the hurdles preventing development and implementation of predictive analyses. We summarize the ideas generated by participants of the Mid-Atlantic Microbiome Meet-up in January 2019. While it was clear from the presentations that most fields have advanced beyond simple associative and descriptive analyses, most fields lack essential elements needed for the development and application of accurate microbiome predictions. Participants stressed the need for standardization, reproducibility, and accessibility of quantitative tools as key to advancing predictions in microbiome analysis. We highlight hurdles that participants identified and propose directions for future efforts that will advance the use of prediction in microbiome research.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{allen_epigenetic_2019b,

title = {Epigenetic changes induced by Bacteroides fragilis toxin (BFT)},

author = {Jawara Allen and Stephanie Hao and Cynthia L Sears and Winston Timp},

url = {https://iai.asm.org/content/early/2019/03/14/IAI.00447-18},

doi = {10.1128/IAI.00447-18},

issn = {0019-9567, 1098-5522},

year  = {2019},

date = {2019-03-01},

urldate = {2019-03-01},

journal = {Infection and Immunity},

volume = {87},

issue = {6},

pages = {e00447-18},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{briem_mir-203a_2019b,

title = {MiR-203a is differentially expressed during branching morphogenesis and EMT in breast progenitor cells and is a repressor of peroxidasin},

author = {Eirikur Briem and Zuzana Budkova and Anna Karen Sigurdardottir and Bylgja Hilmarsdottir and Jennifer Kricker and Winston Timp and Magnus Karl Magnusson and Gunnhildur Asta Traustadottir and Thorarinn Gudjonsson},

url = {http://www.sciencedirect.com/science/article/pii/S0925477318300972},

doi = {10.1016/j.mod.2018.11.002},

issn = {0925-4773},

year  = {2019},

date = {2019-02-01},

journal = {Mechanisms of Development},

volume = {155},

pages = {34--47},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{10.1172/jci.insight.130848b,

title = {Transmission and clearance of potential procarcinogenic bacteria during fecal microbiota transplantation for recurrent Clostridioides difficile},

author = {Julia L Drewes and Alina Corona and Uriel Sanchez and Yunfan Fan and Suchitra K Hourigan and Melissa Weidner and Sarah D Sidhu and Patricia J Simner and Hao Wang and Winston Timp and Maria Oliva-Hemker and Cynthia L Sears},

url = {https://insight.jci.org/articles/view/130848},

doi = {10.1172/jci.insight.130848},

year  = {2019},

date = {2019-01-01},

journal = {JCI Insight},

volume = {4},

number = {19},

publisher = {The American Society for Clinical Investigation},

abstract = {BACKGROUND Fecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridioides difficile infection (rCDI) in adults and children, but donor stool samples are currently screened for only a limited number of potential pathogens. We sought to determine whether putative procarcinogenic bacteria (enterotoxigenic Bacteroides fragilis, Fusobacterium nucleatum, and Escherichia coli harboring the colibactin toxin) could be durably transmitted from donors to patients during FMT.METHODS Stool samples were collected from 11 pediatric rCDI patients and their respective FMT donors prior to FMT as well as from the patients at 2–10 weeks, 10–20 weeks, and 6 months after FMT. Bacterial virulence factors in stool DNA extracts and stool cultures were measured by quantitative PCR: Bacteroides fragilis toxin (bft), Fusobacterium adhesin A (fadA), and Escherichia coli colibactin (clbB).RESULTS Four of 11 patients demonstrated sustained acquisition of a procarcinogenic bacteria. Whole genome sequencing was performed on colony isolates from one of these donor/recipient pairs and demonstrated that clbB+ E. coli strains present in the recipient after FMT were identical to a strain present in the donor, confirming strain transmission. Conversely, 2 patients exhibited clearance of procarcinogenic bacteria following FMT from a negative donor.CONCLUSION Both durable transmission and clearance of procarcinogenic bacteria occurred following FMT, suggesting that additional studies on appropriate screening measures for FMT donors and the long-term consequences and/or benefits of FMT are warranted.FUNDING Crohn’s & Colitis Foundation, the Bloomberg~Kimmel Institute for Cancer Immunotherapy at Johns Hopkins University School of Medicine, the National Cancer Institute, and the Canadian Institutes of Health Research.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{avin_characterization_2019b,

title = {Characterization of human telomerase reverse transcriptase promoter methylation and transcription factor binding in differentiated thyroid cancer cell lines},

author = {Brittany A Avin and Yongchun Wang and Timothy Gilpatrick and Rachael E Workman and Isac Lee and Winston Timp and Christopher B Umbricht and Martha A Zeiger},

doi = {10.1002/gcc.22735},

issn = {1098-2264},

year  = {2019},

date = {2019-01-01},

urldate = {2019-01-01},

journal = {Genes, Chromosomes & Cancer},

volume = {58},

issue = {8},

pages = {530-540},

abstract = {Telomerase reverse transcriptase (TERT) activation plays an important role in cancer development by enabling the immortalization of cells. TERT regulation is multifaceted, and its promoter methylation has been implicated in controlling expression through alteration in transcription factor binding. We have characterized TERT promoter methylation, transcription factor binding, and TERT expression levels in five differentiated thyroid cancer (DTC) cell lines and six normal thyroid tissue samples by targeted bisulfite sequencing, ChIP-qPCR, and qRT-PCR. DTC cell lines express varying levels of TERT and exhibit TERT promoter methylation patterns similar to patterns seen in other telomerase positive cancer cell lines. The minimal promoter immediately surrounding the transcription start site is hypomethylated, while further upstream portions show dense methylation. In contrast, the TERT promoter in normal thyroid tissue is largely unmethylated throughout and expresses TERT minimally. Transcription factor binding is also affected by TERT mutation status. The E-twenty-six (ETS) factor GABPA exhibits TERT binding in the TERT mutant DTC cells only, and allele-specific methylation patterns at the minimal promoter were observed as well, which may indicate allele-specific factor recruitment at the minimal promoter. Furthermore, we identified binding sites for activators MYC and GSC in the hypermethylated upstream region, pointing to its possible importance in TERT regulation. Overall, TERT expression and telomerase activity depend on the interplay of multiple regulatory mechanisms including TERT promoter methylation, mutation status, and recruitment of transcription factors. This work explores of the interplay between these regulatory mechanisms and offers insight into cellular control of active telomerase in human cancer.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{TammaAAC.01923-18b,

title = {Applying Rapid Whole Genome Sequencing to Predict Phenotypic Antimicrobial Susceptibility Testing Results Among Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates},

author = {Pranita D Tamma and Yunfan Fan and Yehudit Bergman and Geo Pertea and Abida Kazmi and Shawna Lewis and Karen C Carroll and Michael C Schatz and Winston Timp and Patricia J Simner},

url = {https://aac.asm.org/content/early/2018/10/23/AAC.01923-18},

doi = {10.1128/AAC.01923-18},

issn = {0066-4804},

year  = {2019},

date = {2019-01-01},

journal = {Antimicrobial Agents and Chemotherapy},

volume = {63},

number = {1},

pages = {e01923--18},

publisher = {American Society for Microbiology Journals},

abstract = {Objective: Standard antimicrobial susceptibility testing (AST) approaches lead to delays in the selection of optimal antimicrobial therapy. We sought to determine the accuracy of antimicrobial resistance (AMR) determinants identified by Nanopore whole genome sequencing in predicting AST results.Methods: Using a cohort of 40 clinical isolates (21 carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae, 10 non-carbapenemase-producing carbapenem resistant K. pneumoniae, and 9 carbapenem-susceptible K. pneumoniae), three separate sequencing and analysis pipelines were performed: (1) a real-time Nanopore analysis approach identifying acquired AMR genes, (2) an assembly-based Nanopore approach identifying acquired AMR genes and chromosomal mutations, and (3) an approach using short read correction of Nanopore assemblies. The short read correction of Nanopore assemblies served as the reference standard to determine the accuracy of Nanopore sequencing results.Results: With the real-time analysis approach, full annotation of acquired AMR genes occurred within 8 hours of subcultured isolates. Assemblies sufficient for full resistance gene and single nucleotide polymorphism annotation were available within 14 hours from subcultured isolates. The overall agreement of genotypic results and anticipated AST results for the 40 K. pneumoniae isolates was 77% (range 30-100%) and 92% (range 80-100%) for the real-time approach and the assembly approach, respectively. Evaluating the patients contributing the 40 isolates, the real-time approach and assembly approach could shorten the median time to effective antibiotic therapy by 20 hours and 26 hours, respectively, compared to standard AST.Conclusions: Nanopore sequencing offers a rapid approach to both accurately identify resistance mechanisms as well as predict AST results for K. pneumoniae isolates. Bioinformatics improvements enabling real-time alignment coupled with rapid extraction and library preparation will further enhance the accuracy and workflow of the Nanopore real-time approach.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{vanaja2018lossb,

title = {A Loss of Epigenetic Control Can Promote Cell Death through Reversing the Balance of Pathways in a Signaling Network},

author = {Kiran G Vanaja and Winston Timp and Andrew P Feinberg and Andre Levchenko},

doi = {10.1016/j.molcel.2018.08.025},

issn = {1097-4164},

year  = {2018},

date = {2018-10-01},

journal = {Molecular Cell},

volume = {72},

number = {1},

pages = {60--70.e3},

abstract = {Epigenetic control of regulatory networks is only partially understood. Expression of Insulin-like growth factor-II (IGF2) is controlled by genomic imprinting, mediated by silencing of the maternal allele. Loss of imprinting of IGF2 (LOI) is linked to intestinal and colorectal cancers, causally in murine models and epidemiologically in humans. However, the molecular underpinnings of the LOI phenotype are not clear. Surprisingly, in LOI cells, we find a reversal of the relative activities of two canonical signaling pathways triggered by IGF2, causing further rebalancing between pro- and anti-apoptotic signaling. A predictive mathematical model shows that this network rebalancing quantitatively accounts for the effect of receptor tyrosine kinase inhibition in both WT and LOI cells. This mechanism also quantitatively explains both the stable LOI phenotype and the therapeutic window for selective killing of LOI cells, and thus prevention of epigenetically controlled cancers. These findings suggest a framework for understanding epigenetically modified cell signaling.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{simner_antibiotic_2018b,

title = {Antibiotic pressure on the acquisition and loss of antibiotic resistance genes in Klebsiella pneumoniae},

author = {Patricia J Simner and Annukka A R Antar and Stephanie Hao and James Gurtowski and Pranita D Tamma and Clare Rock and Belita N A Opene and Tsigereda Tekle and Karen C Carroll and Michael C Schatz and Winston Timp},

doi = {10.1093/jac/dky121},

issn = {1460-2091},

year  = {2018},

date = {2018-04-01},

journal = {The Journal of Antimicrobial Chemotherapy},

abstract = {Objectives: In this study, we characterize a concurrent disseminated infection with a virulent hypermucoviscous (HMV) Klebsiella pneumoniae and an OXA-181-producing XDR K. pneumoniae from a patient with recent hospitalization in India. During exposure to meropenem therapy, the highly susceptible HMV K. pneumoniae became resistant to carbapenems, consistent with the acquisition of blaOXA-181. 

Methods: Twelve K. pneumoniae isolates were recovered from the patient and the hospital room environment over a 3 month hospitalization. Phenotypic and molecular studies were completed to characterize the isolates. Oxford Nanopore and Illumina MiSeq WGS were performed to study phylogeny (MLST and SNPs), plasmids and virulence genes and demonstrate changes in the organism's resistome that occurred over time. 

Results: WGS revealed that the HMV K. pneumoniae belonged to ST23 and harboured an IncH1B virulence plasmid, while the XDR K. pneumoniae belonged to ST147 and possessed two MDR plasmids (IncR and IncFII), the blaOXA-181-bearing ColKP3 plasmid and chromosomal mutations conferring the XDR phenotype. Sequential isolates demonstrated plasmid diversification (fusion of the IncR and IncFII plasmids), mobilization of resistance elements (ompK35 inactivation by ISEcp1-blaCTX-M-15 mobilization, varying numbers of resistance genes on plasmid scaffolds) and chromosomal mutations (mutations in mgrB) leading to further antibiotic resistance that coincided with antibiotic pressure. Importantly, the HMV strain in this study was unable to preserve the carbapenem-resistant phenotype without the selective pressure of meropenem. 

Conclusions: To the best of our knowledge, we are the first to report a carbapenem-resistant HMV K. pneumoniae strain in the USA. Ultimately, this case demonstrates the role of antibiotic pressure in the acquisition and loss of important genetic elements.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{tamma_successful_2018b,

title = {Successful Treatment of Persistent Burkholderia cepacia Complex Bacteremia with Ceftazidime-Avibactam},

author = {Pranita D Tamma and Yunfan Fan and Yehudit Bergman and Anna C Sick-Samuels and Alice J Hsu and Winston Timp and Patricia J Simner},

doi = {10.1128/AAC.02213-17},

issn = {1098-6596},

year  = {2018},

date = {2018-04-01},

journal = {Antimicrobial Agents and Chemotherapy},

volume = {62},

number = {4},

abstract = {We report our clinical experience treating a 2-month-old infant with congenital diaphragmatic hernia who experienced prolonged bacteremia with Burkholderia cepacia complex (Bcc) despite conventional antibiotic therapy and appropriate source control measures. The infection resolved after initiation of ceftazidime-avibactam. Whole-genome sequencing revealed that the isolate most closely resembled B. contaminans and identified the mechanism of resistance that likely contributed to clinical cure with this agent. Ceftazidime-avibactam should be considered salvage therapy for Bcc infections if other treatment options have been exhausted.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{workman_high_2018b,

title = {High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing},

author = {Rachael Workman and Renee Fedak and Duncan Kilburn and Stephanie Hao and Kelvin Liu and Winston Timp},

url = {https://www.nature.com/protocolexchange/protocols/6785},

year  = {2018},

date = {2018-04-01},

journal = {Nature Protocols Exchange},

abstract = {Single molecule sequencing requires optimized sample and library preparation protocols to obtain long-read lengths and high sequencing yields. Numerous protocols exist for the extraction of DNA from plant species, but the genomic DNA from these extractions is either too low yield, of insufficient purity for sensitive sequencing platforms, e.g. nanopore sequencing, too fragmented to achieve long reads, or otherwise unattainable from recalcitrant adult tissue. This renders many plant sequencing projects cost prohibitive or methodologically intractable. Existing protocols are also labor intensive, taking days to complete. Our protocol described here yields micrograms of high molecular weight gDNA from a single gram of adult or seedling leaf tissue in only a few hours, and produces high quality sequencing libraries for the Oxford Nanopore system, with typical yields ranging from 3-10 Gb per R9.4.1 flowcell and producing reads averaging 5-8 kb, with read length N50s ranging from 6-30 kb depending on the style},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{workman_single-molecule_2018b,

title = {Single-molecule, full-length transcript sequencing provides insight into the extreme metabolism of the ruby-throated hummingbird Archilochus colubris},

author = {Rachael E Workman and Alexander M Myrka and William G Wong and Elizabeth Tseng and Kenneth C Welch and Winston Timp},

url = {https://academic.oup.com/gigascience/article/7/3/giy009/4860431},

doi = {10.1093/gigascience/giy009},

year  = {2018},

date = {2018-03-01},

urldate = {2018-03-01},

journal = {GigaScience},

volume = {7},

issue = {3},

pages = {giy009},

abstract = {BackgroundHummingbirds oxidize ingested nectar sugars directly to fuel foraging but cannot sustain this fuel use during fasting periods, such as during the night or during long-distance migratory flights. Instead, fasting hummingbirds switch to oxidizing stored lipids that are derived from ingested sugars. The hummingbird liver plays a key role in moderating energy homeostasis and this remarkable capacity for fuel switching. Additionally, liver is the principle location of de novo lipogenesis, which can occur at exceptionally high rates, such as during premigratory fattening. Yet understanding how this tissue and whole organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation.FindingsWe generated a de novo transcriptome of the hummingbird liver using PacBio full-length cDNA sequencing (Iso-Seq), yielding 8.6Gb of sequencing data, or 2.6M reads from 4 different size fractions. We analyzed data using the SMRTAnalysis v3.1 Iso-Seq pipeline, then clustered isoforms into gene families to generate de novo gene contigs using Cogent. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. Finally, we closely examined homology of critical lipid metabolism genes between our transcriptome data and avian and human genomes.ConclusionsWe confirmed high levels of sequence divergence within hummingbird lipogenic enzymes, suggesting a high probability of adaptive divergent function in the hepatic lipogenic pathways. Our results leverage cutting-edge technology and a novel bioinformatics pipeline to provide a first direct look at the transcriptome of this incredible organism.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{10.1371/journal.pone.0209408b,

title = {Protein engineering strategies for improving the selective methylation of target CpG sites by a dCas9-directed cytosine methyltransferase in bacteria},

author = {Tina Xiong and Dahlia Rohm and Rachael E Workman and Lauren Roundtree and Carl D Novina and Winston Timp and Marc Ostermeier},

url = {https://doi.org/10.1371/journal.pone.0209408},

doi = {10.1371/journal.pone.0209408},

year  = {2018},

date = {2018-01-01},

journal = {PLoS One},

volume = {13},

number = {12},

pages = {1-18},

publisher = {Public Library of Science},

abstract = {Mammalian gene expression is a complex process regulated in part by CpG methylation. The ability to target methylation for de novo gene regulation could have therapeutic and research applications. We have previously developed a dCas9-MC/MN protein for targeting CpG methylation. dCas9-MC/MN is composed of an artificially split M.SssI methyltransferase (MC/MN), with the MC fragment fused to a nuclease-null CRISPR/Cas9 (dCas9). Guide RNAs directed dCas9-MC/MN to methylate target sites in E. coli and human cells but also caused some low-level off-target methylation. Here, in E. coli, we show that shortening the dCas9-MC linker increases methylation of CpG sites located at select distances from the dCas9 binding site. Although a shortened linker decreased methylation of other CpGs proximal to the target site, it did not reduce off-target methylation of more distant CpG sites. Instead, targeted mutagenesis of the methyltransferase’s DNA binding domain, designed to reduce DNA affinity, significantly and preferentially reduced methylation of such sites.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{lee_whole_2017b,

title = {Whole genome DNA methylation sequencing of the chicken retina, cornea and brain},

author = {Isac Lee and Bejan A Rasoul and Ashton S Holub and Alannah Lejeune and Raymond A Enke and Winston Timp},

url = {https://www.nature.com/articles/sdata2017148},

doi = {10.1038/sdata.2017.148},

issn = {2052-4463},

year  = {2017},

date = {2017-10-10},

urldate = {2017-10-10},

journal = {Scientific Data},

volume = {4},

issue = {1},

pages = {1-8},

abstract = {Data Descriptor},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{xiong_targeted_2017b,

title = {Targeted DNA methylation in human cells using engineered dCas9-methyltransferases},

author = {Tina Xiong and Glenna E Meister and Rachael E Workman and Nathaniel C Kato and Michael J Spellberg and Fulya Turker and Winston Timp and Marc Ostermeier and Carl D Novina},

url = {http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5532369/},

doi = {10.1038/s41598-017-06757-0},

issn = {2045-2322},

year  = {2017},

date = {2017-07-27},

urldate = {2017-07-27},

journal = {Scientific Reports},

volume = {7},

issue = {1},

pages = {1-14},

abstract = {Mammalian genomes exhibit complex patterns of gene expression regulated, in part, by DNA methylation. The advent of engineered DNA methyltransferases (MTases) to target DNA methylation to specific sites in the genome will accelerate many areas of biological research. However, targeted MTases require clear design rules to direct site-specific DNA methylation and minimize the unintended effects of off-target DNA methylation. Here we report a targeted MTase composed of an artificially split CpG MTase (sMTase) with one fragment fused to a catalytically-inactive Cas9 (dCas9) that directs the functional assembly of sMTase fragments at the targeted CpG site. We precisely map RNA-programmed DNA methylation to targeted CpG sites as a function of distance and orientation from the protospacer adjacent motif (PAM). Expression of the dCas9-sMTase in mammalian cells led to predictable and efficient (up to textasciitilde70%) DNA methylation at targeted sites. Multiplexing sgRNAs enabled targeting methylation to multiple sites in a single promoter and to multiple sites in multiple promoters. This programmable de novo MTase tool might be used for studying mechanisms of initiation, spreading and inheritance of DNA methylation, and for therapeutic gene silencing.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{kandathil_presence_2017b,

title = {Presence of Human Hepegivirus-1 in a Cohort of People Who Inject Drugs},

author = {Abraham J Kandathil and Florian P Breitwieser and Jaiprasath Sachithanandham and Matthew Robinson and Shruti H Mehta and Winston Timp and Steven L Salzberg and David L Thomas and Ashwin Balagopal},

doi = {10.7326/M17-0085},

issn = {1539-3704},

year  = {2017},

date = {2017-06-06},

journal = {Annals of Internal Medicine},

volume = {167},

number = {1},

pages = {1--7},

abstract = {Background: Next-generation metagenomic sequencing (NGMS) has opened new frontiers in microbial discovery but has been clinically characterized in only a few settings. 

Objective: To explore the plasma virome of persons who inject drugs and to characterize the sensitivity and accuracy of NGMS compared with quantitative clinical standards. 

Design: Longitudinal and cross-sectional studies. 

Setting: A clinical trial (ClinicalTrials.gov: NCT01285050) and a well-characterized cohort study of persons who have injected drugs. 

Participants: Persons co-infected with hepatitis C virus (HCV) and HIV. 

Measurements: Viral nucleic acid in plasma by NGMS and quantitative polymerase chain reaction (PCR). 

Results: Next-generation metagenomic sequencing generated a total of 600 million reads, which included the expected HIV and HCV RNA sequences. HIV and HCV reads were consistently identified only when samples contained more than 10000 copies/mL or IU/mL, respectively, as determined by quantitative PCR. A novel RNA virus, human hepegivirus-1 (HHpgV-1), was also detected by NGMS in 4 samples from 2 persons in the clinical trial. Through use of a quantitative PCR assay for HHpgV-1, infection was also detected in 17 (10.9%) of 156 members of a cohort of persons who injected drugs. In these persons, HHpgV-1 viremia persisted for a median of at least 4538 days and was associated with detection of other bloodborne viruses, such as HCV RNA and SEN virus D. 

Limitation: The medical importance of HHpgV-1 infection is unknown. 

Conclusion: Although NGMS is insensitive for detection of viruses with relatively low plasma nucleic acid concentrations, it may have broad potential for discovery of new viral infections of possible medical importance, such as HHpgV-1. 

Primary Funding Source: National Institutes of Health.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{simpson_detecting_2017b,

title = {Detecting DNA cytosine methylation using nanopore sequencing},

author = {Jared T Simpson and Rachael E Workman and P C Zuzarte and Matei David and L J Dursi and Winston Timp},

url = {http://www.nature.com/nmeth/journal/v14/n4/full/nmeth.4184.html},

doi = {10.1038/nmeth.4184},

issn = {1548-7091},

year  = {2017},

date = {2017-04-01},

journal = {Nature Methods},

volume = {14},

number = {4},

pages = {407--410},

abstract = {In nanopore sequencing devices, electrolytic current signals are sensitive to base modifications, such as 5-methylcytosine (5-mC). Here we quantified the strength of this effect for the Oxford Nanopore Technologies MinION sequencer. By using synthetically methylated DNA, we were able to train a hidden Markov model to distinguish 5-mC from unmethylated cytosine. We applied our method to sequence the methylome of human DNA, without requiring special steps for library preparation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{credle_multiplexed_2017b,

title = {Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization},

author = {Joel J Credle and Christopher Y Itoh and Tiezheng Yuan and Rajni Sharma and Erick R Scott and Rachael E Workman and Yunfan Fan and Franck Housseau and Nicolas J Llosa and Robert W Bell and Heather Miller and Sean X Zhang and Winston Timp and Benjamin H Larman},

doi = {10.1093/nar/gkx471},

year  = {2017},

date = {2017-01-01},

journal = {Nucleic Acids Research},

volume = {45},

number = {14},

pages = {e128--e128},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Luog3.300107.2017b,

title = {First Draft Genome Sequence of the Pathogenic Fungus Lomentospora prolificans (formerly Scedosporium prolificans)},

author = {Ruibang Luo and Aleksey Zimin and Rachael Workman and Yunfan Fan and Geo Pertea and Nina Grossman and Maggie P Wear and Bei Jia and Heather Miller and Arturo Casadevall and Winston Timp and Sean X Zhang and Steven L Salzberg},

doi = {10.1534/g3.117.300107},

year  = {2017},

date = {2017-01-01},

journal = {G3: Genes, Genomes, Genetics},

volume = {7},

number = {11},

pages = {3831--3836},

publisher = {G3: Genes, Genomes, Genetics},

abstract = {Here we describe the sequencing and assembly of the pathogenic fungus Lomentospora prolificans using a combination of short, highly accurate Illumina reads and additional coverage in very long Oxford Nanopore reads. The resulting assembly is highly contiguous, containing a total of 37,627,092 bp with over 98% of the sequence in just 26 scaffolds. Annotation identified 8,896 protein-coding genes. Pulsed-field gel analysis suggests that this organism contains at least 7 and possibly 11 chromosomes, the two longest of which have sizes corresponding closely to the sizes of the longest scaffolds, at 6.6 and 5.7 Mb.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{norris_nanopore_2016b,

title = {Nanopore sequencing detects structural variants in cancer},

author = {Alexis L Norris and Rachael E Workman and Yunfan Fan and James R Eshleman and Winston Timp},

doi = {10.1080/15384047.2016.1139236},

issn = {1555-8576},

year  = {2016},

date = {2016-03-01},

journal = {Cancer Biology & Therapy},

volume = {17},

number = {3},

pages = {246--253},

abstract = {Despite advances in sequencing, structural variants (SVs) remain difficult to reliably detect due to the short read length (textless300 bp) of 2nd generation sequencing. Not only do the reads (or paired-end reads) need to straddle a breakpoint, but repetitive elements often lead to ambiguities in the alignment of short reads. We propose to use the long-reads (up to 20 kb) possible with 3rd generation sequencing, specifically nanopore sequencing on the MinION. Nanopore sequencing relies on a similar concept to a Coulter counter, reading the DNA sequence from the change in electrical current resulting from a DNA strand being forced through a nanometer-sized pore embedded in a membrane. Though nanopore sequencing currently has a relatively high mismatch rate that precludes base substitution and small frameshift mutation detection, its accuracy is sufficient for SV detection because of its long reads. In fact, long reads in some cases may improve SV detection efficiency. We have tested nanopore sequencing to detect a series of well-characterized SVs, including large deletions, inversions, and translocations that inactivate the CDKN2A/p16 and SMAD4/DPC4 tumor suppressor genes in pancreatic cancer. Using PCR amplicon mixes, we have demonstrated that nanopore sequencing can detect large deletions, translocations and inversions at dilutions as low as 1:100, with as few as 500 reads per sample. Given the speed, small footprint, and low capital cost, nanopore sequencing could become the ideal tool for the low-level detection of cancer-associated SVs needed for molecular relapse, early detection, or therapeutic monitoring.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_think_2014b,

title = {Think Small: Nanopores for Sensing and Synthesis},

author = {Winston Timp and Allison M Nice and E M Nelson and V Kurz and K McKelvey and Gregory Timp},

doi = {10.1109/ACCESS.2014.2369506},

issn = {2169-3536},

year  = {2014},

date = {2014-11-01},

journal = {IEEE Access},

volume = {2},

pages = {1396--1408},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_large_2014b,

title = {Large hypomethylated blocks as a universal defining epigenetic alteration in human solid tumors},

author = {Winston Timp and Hector Corrada Bravo and Oliver G McDonald and Michael Goggins and Chris Umbricht and Martha Zeiger and Andrew P Feinberg and Rafael A Irizarry},

doi = {10.1186/s13073-014-0061-y},

issn = {1756-994X},

year  = {2014},

date = {2014-08-01},

journal = {Genome Medicine},

volume = {6},

number = {8},

pages = {61},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{kurz_epigenetic_2013b,

title = {Epigenetic Memory Emerging from Integrated Transcription Bursts},

author = {Volker Kurz and Edward M Nelson and Nicolas Perry and Winston Timp and Gregory Timp},

url = {http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3785891/},

doi = {10.1016/j.bpj.2013.08.010},

issn = {0006-3495},

year  = {2013},

date = {2013-09-01},

journal = {Biophysical Journal},

volume = {105},

number = {6},

pages = {1526--1532},

abstract = {Some autonomous bacteria coordinate their actions using quorum-sensing (QS) signals to affect gene expression. However, noise in the gene environment can compromise the cellular response. By exercising precise control over a cell’s genes and its microenvironment, we have studied the key positive autoregulation element by which the lux QS system integrates noisy signals into an epigenetic memory. We observed transcriptional bursting of the lux receptor in cells stimulated by near-threshold levels of QS ligand. The bursts are integrated over time into an epigenetic memory that confers enhanced sensitivity to the ligand. An emergent property of the system is manifested in pattern formation among phenotypes within a chemical gradient.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_cancer_2013b,

title = {Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host},

author = {Winston Timp and Andrew P Feinberg},

doi = {10.1038/nrc3486},

issn = {1474-1768},

year  = {2013},

date = {2013-07-01},

journal = {Nature Reviews Cancer},

volume = {13},

number = {7},

pages = {497--510},

abstract = {Although at the genetic level cancer is caused by diverse mutations, epigenetic modifications are characteristic of all cancers, from apparently normal precursor tissue to advanced metastatic disease, and these epigenetic modifications drive tumour cell heterogeneity. We propose a unifying model of cancer in which epigenetic dysregulation allows rapid selection for tumour cell survival at the expense of the host. Mechanisms involve both genetic mutations and epigenetic modifications that disrupt the function of genes that regulate the epigenome itself. Several exciting recent discoveries also point to a genome-scale disruption of the epigenome that involves large blocks of DNA hypomethylation, mutations of epigenetic modifier genes and alterations of heterochromatin in cancer (including large organized chromatin lysine modifications (LOCKs) and lamin-associated domains (LADs)), all of which increase epigenetic and gene expression plasticity. Our model suggests a new approach to cancer diagnosis and therapy that focuses on epigenetic dysregulation and has great potential for risk detection and chemoprevention.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nelson_using_2012b,

title = {Using a Nanopore for Single Molecule Detection and Single Cell Transfection},

author = {Edward M Nelson and Volker Kurz and Jiwook Shim and Winston Timp and Gregory Timp},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3384492/},

doi = {10.1039/c2an35571j},

issn = {0003-2654},

year  = {2012},

date = {2012-07-01},

journal = {The Analyst},

volume = {137},

number = {13},

pages = {3020--3027},

abstract = {We assert that it is possible to trap and identify proteins, and even (conceivably) manipulate proteins secreted from a single cell (i.e. the secretome) through transfection via electroporation by exploiting the exquisite control over the electrostatic potential available in a nanopore. These capabilities may be leveraged for single cell analysis and transfection with single molecule resolution, ultimately enabling a careful scrutiny of tissue heterogeneity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_dna_2012b,

title = {DNA base-calling from a nanopore using a Viterbi algorithm},

author = {Winston Timp and Jeffrey Comer and Aleksei Aksimentiev},

doi = {10.1016/j.bpj.2012.04.009},

issn = {1542-0086},

year  = {2012},

date = {2012-05-01},

journal = {Biophysical Journal},

volume = {102},

number = {10},

pages = {L37--39},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{mcdonald_genome-scale_2011b,

title = {Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition},

author = {Oliver G McDonald and Hao Wu and Winston Timp and Akiko Doi and Andrew P Feinberg},

doi = {10.1038/nsmb.2084},

issn = {1545-9985},

year  = {2011},

date = {2011-08-01},

journal = {Nature Structural & Molecular Biology},

volume = {18},

number = {8},

pages = {867--874},

abstract = {Epithelial-to-mesenchymal transition (EMT) is an extreme example of cell plasticity that is important for normal development, injury repair and malignant progression. Widespread epigenetic reprogramming occurs during stem cell differentiation and malignant transformation, but EMT-related epigenetic reprogramming is poorly understood. Here we investigated epigenetic modifications during EMT mediated by transforming growth factor beta. Although DNA methylation was unchanged during EMT, we found a global reduction in the heterochromatin mark H3 Lys9 dimethylation (H3K9Me2), an increase in the euchromatin mark H3 Lys4 trimethylation (H3K4Me3) and an increase in the transcriptional mark H3 Lys36 trimethylation (H3K36Me3). These changes depended largely on lysine-specific demethylase-1 (Lsd1), and loss of Lsd1 function had marked effects on EMT-driven cell migration and chemoresistance. Genome-scale mapping showed that chromatin changes were mainly specific to large organized heterochromatin K9 modifications (LOCKs), which suggests that EMT is characterized by reprogramming of specific chromatin domains across the genome.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{hansen_increased_2011b,

title = {Increased methylation variation in epigenetic domains across cancer types},

author = {Kasper Daniel Hansen and Winston Timp and Héctor Corrada Bravo and Sarven Sabunciyan and Benjamin Langmead and Oliver G McDonald and Bo Wen and Hao Wu and Yun Liu and Dinh Diep and Eirikur Briem and Kun Zhang and Rafael A Irizarry and Andrew P Feinberg},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145050/},

doi = {10.1038/ng.865},

issn = {1061-4036},

year  = {2011},

date = {2011-06-01},

urldate = {2019-04-27},

journal = {Nature Genetics},

volume = {43},

number = {8},

pages = {768--775},

abstract = {Tumor heterogeneity is a major barrier to effective cancer diagnosis and treatment. We recently identified cancer-specific differentially DNA-methylated regions (cDMRs) in colon cancer, which also distinguish normal tissue types from each other, suggesting that these cDMRs might be generalized across cancer types. Here we show stochastic methylation variation of the same cDMRs, distinguishing cancer from normal, in colon, lung, breast, thyroid, and Wilms tumors, with intermediate variation in adenomas. Whole genome bisulfite sequencing shows these variable cDMRs are related to loss of sharply delimited methylation boundaries at CpG islands. Furthermore, we find hypomethylation of discrete blocks encompassing half the genome, with extreme gene expression variability. Genes associated with the cDMRs and large blocks are involved in mitosis and matrix remodeling, respectively. These data suggest a model for cancer involving loss of epigenetic stability of well-defined genomic domains that underlies increased methylation variability in cancer and could contribute to tumor heterogeneity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_nanopore_2010b,

title = {Nanopore Sequencing: Electrical Measurements of the Code of Life},

author = {Winston Timp and Utkur M Mirsaidov and Deqiang Wang and Jeff Comer and Aleksei Aksimentiev and Gregory Timp},

doi = {10.1109/TNANO.2010.2044418},

issn = {1536-125X},

year  = {2010},

date = {2010-05-01},

journal = {IEEE Transactions on Nanotechnology},

volume = {9},

number = {3},

pages = {281--294},

abstract = {Sequencing a single molecule of deoxyribonucleic acid (DNA) using a nanopore is a revolutionary concept because it combines the potential for long read lengths (textgreater5 kbp) with high speed (1 bp/10 ns), while obviating the need for costly amplification procedures due to the exquisite single molecule sensitivity. The prospects for implementing this concept seem bright. The cost savings from the removal of required reagents, coupled with the speed of nanopore sequencing places the $1000 genome within grasp. However, challenges remain: high fidelity reads demand stringent control over both the molecular configuration in the pore and the translocation kinetics. The molecular configuration determines how the ions passing through the pore come into contact with the nucleotides, while the translocation kinetics affect the time interval in which the same nucleotides are held in the constriction as the data is acquired. Proteins like α-hemolysin and its mutants offer exquisitely precise self-assembled nanopores and have demonstrated the facility for discriminating individual nucleotides, but it is currently difficult to design protein structure ab initio, which frustrates tailoring a pore for sequencing genomic DNA. Nanopores in solid-state membranes have been proposed as an alternative because of the flexibility in fabrication and ease of integration into a sequencing platform. Preliminary results have shown that with careful control of the dimensions of the pore and the shape of the electric field, control of DNA translocation through the pore is possible. Furthermore, discrimination between different base pairs of DNA may be feasible. Thus, a nanopore promises inexpensive, reliable, high-throughput sequencing, which could thrust genomic science into personal medicine.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_jamming_2009b,

title = {Jamming prokaryotic cell-to-cell communications in a model biofilm},

author = {Winston Timp and Utkur Mirsaidov and Paul Matsudaira and Gregory Timp},

doi = {10.1039/b810157d},

issn = {1473-0197},

year  = {2009},

date = {2009-04-01},

journal = {Lab on a Chip},

volume = {9},

number = {7},

pages = {925--934},

abstract = {We report on the physical parameters governing prokaryotic cell-to-cell signaling in a model biofilm. The model biofilm is comprised of bacteria that are genetically engineered to transmit and receive quorum-sensing (QS) signals. The model is formed using arrays of time-shared, holographic optical traps in conjunction with microfluidics to precisely position bacteria, and then encapsulated within a hydrogel that mimics the extracellular matrix. Using fluorescent protein reporters functionally linked to QS genes, we assay the intercellular signaling. We find that there isn't a single cell density for which QS-regulated genes are induced or repressed. On the contrary, cell-to-cell signaling is largely governed by diffusion, and is acutely sensitive to mass-transfer to the surroundings and the cell location. These observations are consistent with the view that QS-signals act simply as a probe measuring mixing, flow, or diffusion in the microenvironment of the cell.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{mirsaidov_nanoelectromechanics_2009b,

title = {Nanoelectromechanics of methylated DNA in a synthetic nanopore},

author = {U Mirsaidov and W Timp and X Zou and V Dimitrov and K Schulten and A P Feinberg and G Timp},

doi = {10.1016/j.bpj.2008.12.3760},

issn = {1542-0086},

year  = {2009},

date = {2009-02-01},

journal = {Biophysical Journal},

volume = {96},

number = {4},

pages = {L32--34},

abstract = {Methylation of cytosine is a covalent modification of DNA that can be used to silence genes, orchestrating a myriad of biological processes including cancer. We have discovered that a synthetic nanopore in a membrane comparable in thickness to a protein binding site can be used to detect methylation. We observe a voltage threshold for permeation of methylated DNA through a textless2 nm diameter pore, which we attribute to the stretching transition; this can differ by textgreater1 V/20 nm depending on the methylation level, but not the DNA sequence.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_new_2009b,

title = {A new link between epigenetic progenitor lesions in cancer and the dynamics of signal transduction},

author = {Winston Timp and Andre Levchenko and Andrew P Feinberg},

doi = {10.4161/cc.8.3.7542},

issn = {1551-4005},

year  = {2009},

date = {2009-02-01},

journal = {Cell Cycle (Georgetown, Tex.)},

volume = {8},

number = {3},

pages = {383--390},

abstract = {Our recent study of the mechanism by which an epigenetic alteration, loss of imprinting (LOI) of Igf2, increases tumor risk, revealed a strong relationship between IGF2 dosage, the dynamics of signaling along the IGF2 axis, cell proliferation and tumor risk.(1) Colon epithelia in a mouse model with LOI of Igf2 showed increased sensitivity to IGF1R blockade and abrogation of premalignant lesion development in LOI(+) mice. These results are consistent with the epigenetic progenitor model of cancer,(2) in which epigenetic changes precede and heighten risk of cancer in response to oncogenic mutations. Thus, one can envision a highly targeted and focused chemoprevention strategy targeted to signaling pathways in nonmalignant cells that have undergone an epigenetic lesion, rather than a broad approach toward reversing epigenetic lesions that may have unintended consequences affecting the whole epigenome.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{mirsaidov_live_2008b,

title = {Live cell lithography: using optical tweezers to create synthetic tissue},

author = {Utkur Mirsaidov and Jan Scrimgeour and Winston Timp and Kaethe Beck and Mustafa Mir and Paul Matsudaira and Gregory Timp},

doi = {10.1039/b807987k},

issn = {1473-0197},

year  = {2008},

date = {2008-12-01},

journal = {Lab on a Chip},

volume = {8},

number = {12},

pages = {2174--2181},

abstract = {We demonstrate a new method for creating synthetic tissue that has the potential to capture the three-dimensional (3D) complexity of a multi-cellular organism with submicron precision. Using multiple laminar fluid flows in a microfluidic network, we convey cells to an assembly area where multiple, time-shared optical tweezers are used to organize them into a complex array. The cells are then encapsulated in a 30 microm x 30 microm x 45 microm volume of photopolymerizable hydrogel that mimicks an extra-cellular matrix. To extend the size, shape and constituency of the array without loss of viability, we then step to an adjacent location while maintaining registration with the reference array, and repeat the process. Using this step-and-repeat method, we formed a heterogeneous array of E. coli genetically engineered with a lac switch that is functionally linked to fluorescence reporters. We then induced the array using ligands through a microfluidic network and followed the space-time development of the fluorescence to evaluate viability and metabolic activity.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{mirsaidov_optimal_2008b,

title = {Optimal optical trap for bacterial viability},

author = {Utkur Mirsaidov and Winston Timp and Kaethe Timp and Mustafa Mir and Paul Matsudaira and Gregory Timp},

doi = {10.1103/PhysRevE.78.021910},

issn = {1539-3755},

year  = {2008},

date = {2008-08-01},

journal = {Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics},

volume = {78},

number = {2 Pt 1},

pages = {021910},

abstract = {Optical trapping is a powerful tool for the micromanipulation of living cells--especially bacteria--but photodamage induced by the laser beam can adversely affect viability. We have explored optical trapping conditions in the near infrared (840-930 nm) that preserve the viability of E. coli, as measured by gene expression of green fluorescent protein. We have found that time-sharing the optical traps, i.e., dwelling only 10 micros-1 ms on the cell, improves viability relative to continuous wave (CW) exposure for the same exposure time. We have also observed that similar to CW traps the photodamage in a time-shared trap depends weakly on wavelength, but linearly on peak power, implying an effect induced by single photon absorption. Taken altogether, integrating the exposure time and peak power, the data indicate that there is a lethal energy dose of about 5 J for E. coli. Thus a single parameter--the energy--can be used to describe the limitation on viability.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_chapter_2008b,

title = {Chapter 14: Electron microscopy of hydrated samples},

author = {Winston Timp and Paul Matsudaira},

doi = {10.1016/S0091-679X(08)00614-6},

issn = {0091-679X},

year  = {2008},

date = {2008-01-01},

journal = {Methods in Cell Biology},

volume = {89},

pages = {391--407},

abstract = {Conventional electron microscopy offers a substantial resolution advantage over light microscopy, but requires difficult and often destructive preparation techniques. Recent advances in electron microscopy allow for imaging of hydrated samples, retaining the resolution advantage while removing the difficulty in preparation. Two new techniques, environmental scanning electron microscopy and wet electron microscopy offer this advantage, allowing for new possibilities in biological imaging.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{kaneda_enhanced_2007b,

title = {Enhanced sensitivity to IGF-II signaling links loss of imprinting of IGF2 to increased cell proliferation and tumor risk},

author = {Atsushi Kaneda and Chiaochun J Wang and Raymond Cheong and Winston Timp and Patrick Onyango and Bo Wen and Christine A Iacobuzio-Donahue and Rolf Ohlsson and Rita Andraos and Mark A Pearson and Alexei A Sharov and Dan L Longo and Minoru S H Ko and Andre Levchenko and Andrew P Feinberg},

doi = {10.1073/pnas.0710359105},

issn = {1091-6490},

year  = {2007},

date = {2007-12-01},

journal = {Proceedings of the National Academy of Sciences of the United States of America},

volume = {104},

number = {52},

pages = {20926--20931},

abstract = {Loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2), leading to abnormal activation of the normally silent maternal allele, is a common human epigenetic population variant associated with a 5-fold increased frequency of colorectal neoplasia. Here, we show first that LOI leads specifically to increased expression of proliferation-related genes in mouse intestinal crypts. Surprisingly, LOI(+) mice also have enhanced sensitivity to IGF-II signaling, not simply increased IGF-II levels, because in vivo blockade with NVP-AEW541, a specific inhibitor of the IGF-II signaling receptor, showed reduction of proliferation-related gene expression to levels half that seen in LOI(-) mice. Signal transduction assays in microfluidic chips confirmed this enhanced sensitivity with marked augmentation of Akt/PKB signaling in LOI(+) cells at low doses of IGF-II, which was reduced in the presence of the inhibitor to levels below those found in LOI(-) cells, and was associated with increased expression of the IGF1 and insulin receptor genes. We exploited this increased IGF-II sensitivity to develop an in vivo chemopreventive strategy using the azoxymethane (AOM) mutagenesis model. LOI(+) mice treated with AOM showed a 60% increase in premalignant aberrant crypt foci (ACF) formation over LOI(-) mice. In vivo IGF-II blockade with NVP-AEW541 abrogated this effect, reducing ACF to a level 30% lower even than found in exposed LOI(-) mice. Thus, LOI increases cancer risk in a counterintuitive way, by increasing the sensitivity of the IGF-II signaling pathway itself, providing a previously undescribed epigenetic chemoprevention strategy in which cells with LOI are "IGF-II addicted" and undergo reduced tumorigenesis in the colon upon IGF-II pathway blockade.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{zeskind_nucleic_2007b,

title = {Nucleic acid and protein mass mapping by live-cell deep-ultraviolet microscopy},

author = {Benjamin J Zeskind and Caroline D Jordan and Winston Timp and Linda Trapani and Guichy Waller and Victor Horodincu and Daniel J Ehrlich and Paul Matsudaira},

doi = {10.1038/nmeth1053},

issn = {1548-7091},

year  = {2007},

date = {2007-07-01},

journal = {Nature Methods},

volume = {4},

number = {7},

pages = {567--569},

abstract = {We developed a deep-ultraviolet (UV) microscope capable of imaging cell mitosis and motility at 280 nm for 45 min with minimal UV-induced toxicity, and for 6 h before the onset of visible cell death in cultured human and mouse cells. Combined with computational methods that convert the intensity of each pixel into an estimate of mass, deep-UV microscopy images generate maps of nucleic acid mass, protein mass and fluorescence yield in unlabeled cells.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{akselrod_laser-guided_2006b,

title = {Laser-guided assembly of heterotypic three-dimensional living cell microarrays},

author = {G M Akselrod and W Timp and U Mirsaidov and Q Zhao and C Li and R Timp and K Timp and P Matsudaira and G Timp},

doi = {10.1529/biophysj.106.084079},

issn = {0006-3495},

year  = {2006},

date = {2006-11-01},

journal = {Biophysical Journal},

volume = {91},

number = {9},

pages = {3465--3473},

abstract = {We have assembled three-dimensional heterotypic networks of living cells in hydrogel without loss of viability using arrays of time-multiplexed, holographic optical traps. The hierarchical control of the cell positions is achieved with, to our knowledge, unprecedented submicron precision, resulting in arrays with an intercell separation textless400 nm. In particular, we have assembled networks of Swiss 3T3 fibroblasts surrounded by a ring of bacteria. We have also demonstrated the ability to manipulate hundreds of Pseudomonas aeruginosa simultaneously into two- and three-dimensional arrays with a time-averaged power textless2 mW per trap. This is the first time to our knowledge that living cell arrays of such complexity have been synthesized, and it represents a milestone in synthetic biology and tissue engineering.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{timp_wet_2006b,

title = {Wet electron microscopy with quantum dots},

author = {Winston Timp and Nicki Watson and Alon Sabban and Ory Zik and Paul Matsudaira},

doi = {10.2144/000112239},

issn = {0736-6205},

year  = {2006},

date = {2006-09-01},

journal = {BioTechniques},

volume = {41},

number = {3},

pages = {295--298},

abstract = {Wet electron microscopy (EM) is a new imaging method with the potential to allow higher spatial resolution of samples. In contrast to most EM methods, it requires little time to perform and does not require complicated equipment or difficult steps. We used this method on a common murine macrophage cell line, IC-21, in combination with various stains and preparations, to collect high resolution images of the actin cytoskeleton. Most importantly, we demonstrated the use of quantum dots in conjunction with this technique to perform light/electron correlation microscopy. We found that wet EM is a useful tool that fits into a niche between the simplicity of light microscopy and the high spatial resolution of EM.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}