Publications

@article{Yeh2022,

title = {Treatment of Sindbis Virus-Infected Neurons with Antibody to E2 Alters Synthesis of Complete and nsP1-Expressing Defective Viral RNAs},

author = {Jane X Yeh and Yunfan Fan and Maggie L Bartlett and Xiaoyan Zhang and Norah Sadowski and Debra A Hauer and Winston Timp and Diane E Griffin},

editor = {Anne Moscona, Columbia University Medical College},

url = {https://journals.asm.org/doi/pdf/10.1128/mbio.02221-22},

doi = {http://dx.doi.org/10.1128/mbio.02221-22},

year  = {2022},

date = {2022-09-07},

urldate = {2022-09-07},

journal = {Mbio},

volume = {13},

issue = {5},

pages = {e02221-22},

abstract = {Alphaviruses are positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV), the prototype alphavirus, preferentially infects neurons in mice and is a model system for studying mechanisms of viral clearance from the nervous system. Antibody specific to the SINV E2 glycoprotein plays an important role in SINV clearance, and this effect is reproduced in cultures of infected mature neurons. To determine how anti-E2 antibody affects SINV RNA synthesis, Oxford Nanopore Technologies direct long-read RNA sequencing was used to sequence viral RNAs following antibody treatment of infected neurons. Differentiated AP-7 rat olfactory neuronal cells, an in vitro model for mature neurons, were infected with SINV and treated with anti-E2 antibody. Whole-cell RNA lysates were collected for sequencing of poly(A)-selected RNA 24, 48, and 72 h after infection. Three primary species …},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Long read mitochondrial genome sequencing using Cas9-guided adaptor ligation},

author = {Amy R Vandiver and Brittany Pielstick and Timothy Gilpatrick and Austin N Hoang and Hillary J Vernon and Jonathan Wanagat and Winston Timp},

url = {https://www.sciencedirect.com/science/article/pii/S1567724922000514},

doi = {https://doi.org/10.1016/j.mito.2022.06.003},

issn = {1567-7249},

year  = {2022},

date = {2022-07-01},

urldate = {2022-07-01},

journal = {Mitochondrion},

volume = {65},

pages = {176-183},

abstract = {The mitochondrial genome (mtDNA) is an important source of disease-causing genetic variability, but existing sequencing methods limit understanding, precluding phased measurement of mutations and clear detection of large sporadic deletions. We adapted a method for amplification-free sequence enrichment using Cas9 cleavage to obtain full length nanopore reads of mtDNA. We then utilized the long reads to phase mutations in a patient with an mtDNA-linked syndrome and demonstrated that this method can map age-induced mtDNA deletions. We believe this method will offer deeper insight into our understanding of mtDNA variation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Tiek2022,

title = {Temozolomide-induced guanine mutations create exploitable vulnerabilities of guanine-rich DNA and RNA regions in drug-resistant gliomas},

author = {Deanna M Tiek and Beril Erdogdu and Roham Razaghi and Lu Jin and Norah Sadowski and Carla Alamillo-Ferrer and J Robert Hogg and Bassem R Haddad and David H Drewry and Carrow I Wells and Julie E Pickett and Xiao Song and Anshika Goenka and Bo Hu and Samuel A Goldlust and William J Zuercher and Mihaela Pertea and Winston Timp and Shi-Yuan Cheng and Rebecca B Riggins},

url = {https://www.science.org/doi/full/10.1126/sciadv.abn3471},

doi = {10.1126/sciadv.abn3471},

issn = {2375-2548},

year  = {2022},

date = {2022-06-22},

urldate = {2022-06-22},

journal = {Science Advances},

volume = {8},

issue = {25},

pages = {eabn3471},

abstract = {Temozolomide (TMZ) is a chemotherapeutic agent that has been the first-line standard of care for the aggressive brain cancer glioblastoma (GBM) since 2005. Although initially beneficial, TMZ resistance is universal and second-line interventions are an unmet clinical need. Here, we took advantage of the known mechanism of action of TMZ to target guanines (G) and investigated G-rich G-quadruplex (G4) and splice site changes that occur upon TMZ resistance. We report that TMZ-resistant GBM has guanine mutations that disrupt the G-rich DNA G4s and splice sites that lead to deregulated alternative splicing. These alterations create vulnerabilities, which are selectively targeted by either the G4-stabilizing drug TMPyP4 or a novel splicing kinase inhibitor of cdc2-like kinase. Last, we show that the G4 and RNA binding protein EWSR1 aggregates in the cytoplasm in TMZ-resistant GBM cells and patient samples …},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chang2022,

title = {Epigenetic comparison of CHO hosts and clones reveals divergent methylation and transcription patterns across lineages},

author = {Meiping Chang and Amit Kumar and Swetha Kumar and Steven Huhn and Winston Timp and Michael Betenbaugh and Zhimei Du},

doi = {https://doi.org/10.1002/bit.28036},

year  = {2022},

date = {2022-04-01},

journal = {Biotechnology and Bioengineering},

volume = {119},

issue = {4},

pages = {1062-0176},

abstract = {In this study, we examined DNA methylation and transcription profiles of recombinant clones derived from two different Chinese hamster ovary hosts. We found striking epigenetic differences between the clones, with global hypomethylation in the host 1 clones that produce bispecific antibody with higher productivity and complex assembly efficiency. Whereas the methylation patterns were found mostly inherited from the host, the host 1 clones exhibited continued demethylation reflected by the hypomethylation of newly emerged differential methylation regions (DMRs) even at the clone development stage. Several interconnected biological functions and pathways including cell adhesion, regulation of ion transport, and cholesterol biosynthesis were significantly altered between the clones at the RNA expression level and contained DMR in the promoter and/or gene‐body of the transcripts, suggesting epigenetic …},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Altemose2022,

title = {Complete genomic and epigenetic maps of human centromeres},

author = {Nicolas Altemose and Glennis A Logsdon and Andrey V Bzikadze and Pragya Sidhwani and Sasha A Langley and Gina V Caldas and Savannah J Hoyt and Lev Uralsky and Fedor D Ryabov and Colin J Shew and Michael EG Sauria and Matthew Borchers and Ariel Gershman and Alla Mikheenko and Valery A Shepelev and Tatiana Dvorkina and Olga Kunyavskaya and Mitchell R Vollger and Arang Rhie and Ann M McCartney and Mobin Asri and Ryan Lorig-Roach and Kishwar Shafin and Julian K Lucas and Sergey Aganezov and Daniel Olson and Leonardo Gomes de Lima and Tamara Potapova and Gabrielle A Hartley and Marina Haukness and Peter Kerpedjiev and Fedor Gusev and Kristof Tigyi and Shelise Brooks and Alice Young and Sergey Nurk and Sergey Koren and Sofie R Salama and Benedict Paten and Evgeny I Rogaev and Aaron Streets and Gary H Karpen and Abby F Dernburg and Beth A Sullivan and Aaron F Straight and Travis J Wheeler and Jennifer L Gerton and Evan E Eichler and Adam M Phillippy and Winston Timp and Megan Y Dennis and Rachel J O’Neill and Justin M Zook and Michael C Schatz and Pavel A Pevzner and Mark Diekhans and Charles H Langley and Ivan A Alexandrov and Karen H Miga},

url = {https://www.science.org/doi/full/10.1126/science.abl4178},

doi = {10.1126/science.abl4178},

year  = {2022},

date = {2022-04-01},

urldate = {2022-04-01},

journal = {Science},

volume = {376},

issue = {6588},

pages = {eabl4178},

publisher = {Cold Spring Harbor Laboratory},

abstract = {Existing human genome assemblies have almost entirely excluded highly repetitive sequences within and near centromeres, limiting our understanding of their sequence, evolution, and essential role in chromosome segregation. Here, we present an extensive study of newly assembled peri/centromeric sequences representing 6.2% (189.9 Mb) of the first complete, telomere-to-telomere human genome assembly (T2T-CHM13). We discovered novel patterns of peri/centromeric repeat organization, variation, and evolution at both large and small length scales. We also found that inner kinetochore proteins tend to overlap the most recently duplicated subregions within centromeres. Finally, we compared chromosome X centromeres across a diverse panel of individuals and uncovered structural, epigenetic, and sequence variation at single-base resolution across these regions. In total, this work provides an unprecedented atlas of human centromeres to guide future studies of their complex and critical functions as well as their unique evolutionary dynamics.One-sentence summary Deep characterization of fully assembled human centromeres reveals their architecture and fine-scale organization, variation, and evolution.Competing Interest StatementSK and KHM have received travel funds to speak at symposia organized by Oxford Nanopore. W.T. has two patents (8,748,091 and 8,394,584) licensed to Oxford Nanopore Technologies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Sholes2022,

title = {Chromosome specific telomere lengths and the minimal functional telomere revealed by nanopore sequencing},

author = {Samantha L Sholes and Kayarash Karimian and Ariel Gershman and Thomas J Kelly and Winston Timp and Carol W Greider},

url = {https://genome.cshlp.org/content/32/4/616.full.pdf},

doi = {10.1101/gr.275868.121},

year  = {2022},

date = {2022-04-01},

urldate = {2021-01-01},

journal = {Genome Research},

volume = {32},

issue = {4},

pages = {616-628},

publisher = {Cold Spring Harbor Laboratory},

abstract = {We developed a method to tag telomeres and measure telomere length by nanopore sequencing in the yeast S. cerevisiae. Nanopore allows long read sequencing through the telomere, subtelomere and into unique chromosomal sequence, enabling assignment of telomere length to a specific chromosome end. We observed chromosome end specific telomere lengths that were stable over 120 cell divisions. These stable chromosome specific telomere lengths may be explained by stochastic clonal variation or may represent a new biological mechanism that maintains equilibrium unique to each chromosomes end. We examined the role of RIF1 and TEL1 in telomere length regulation and found that TEL1 is epistatic to RIF1 at most telomeres, consistent with the literature. However, at telomeres that lack subtelomeric Ytextquoteright sequences, tel1Δ rif1Δ double mutants had a very small, but significant, increase in telomere length compared to the tel1Δ single mutant, suggesting an influence of Ytextquoteright elements on telomere length regulation. We sequenced telomeres in a telomerase-null mutant (est2Δ) and found the minimal telomere length to be around 75bp. In these est2Δ mutants there were many apparent telomere recombination events at individual telomeres before the generation of survivors, and these events were significantly reduced in est2Δ rad52Δ double mutants. The rate of telomere shortening in the absence of telomerase was similar across all chromosome ends at about 5 bp per generation. This new method gives quantitative, high resolution telomere length measurement at each individual chromosome end, suggests possible new biological mechanisms regulating telomere length, and provides capability to test new hypotheses.Competing Interest StatementWinston Timp has two patents (8,748,091 and 8,394,584) licensed to Oxford Nanopore Technologies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Hoyt2021.07.12.451456,

title = {From telomere to telomere: the transcriptional and epigenetic state of human repeat elements},

author = {Savannah J Hoyt and Jessica M Storer and Gabrielle A Hartley and Patrick GS Grady and Ariel Gershman and Leonardo G de Lima and Charles Limouse and Reza Halabian and Luke Wojenski and Matias Rodriguez and Nicolas Altemose and Arang Rhie and Leighton J Core and Jennifer L Gerton and Wojciech Makalowski and Daniel Olson and Jeb Rosen and Arian FA Smit and Aaron F Straight and Mitchell R Vollger and Travis J Wheeler and Michael C Schatz and Evan E Eichler and Adam M Phillippy and Winston Timp and Karen H Miga and Rachel J O’Neill},

url = {https://www.science.org/doi/full/10.1126/science.abk3112},

doi = {10.1126/science.abk3112},

year  = {2022},

date = {2022-04-01},

urldate = {2021-01-01},

journal = {Science},

volume = {376},

issue = {6588},

pages = {eabk3112},

publisher = {Cold Spring Harbor Laboratory},

abstract = {Mobile elements and highly repetitive genomic regions are potent sources of lineage-specific genomic innovation and fingerprint individual genomes. Comprehensive analyses of large, composite or arrayed repeat elements and those found in more complex regions of the genome require a complete, linear genome assembly. Here we present the first de novo repeat discovery and annotation of a complete human reference genome, T2T-CHM13v1.0. We identified novel satellite arrays, expanded the catalog of variants and families for known repeats and mobile elements, characterized new classes of complex, composite repeats, and provided comprehensive annotations of retroelement transduction events. Utilizing PRO-seq to detect nascent transcription and nanopore sequencing to delineate CpG methylation profiles, we defined the structure of transcriptionally active retroelements in humans, including for the first time those found in centromeres. Together, these data provide expanded insight into the diversity, distribution and evolution of repetitive regions that have shaped the human genome.Competing Interest StatementKHM has received travel funds to speak at symposia organized by Oxford Nanopore. WT has two patents (8,748,091 and 8,394,584) licensed to Oxford Nanopore Technologies. All other authors declare that they have no competing interests.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Gershman2022,

title = {Epigenetic Patterns in a Complete Human Genome},

author = {Ariel Gershman and Michael EG Sauria and Xavi Guitart and Mitchell R Vollger and Paul W Hook and Savannah J Hoyt and Miten Jain and Alaina Shumate and Roham Razaghi and Sergey Koren and Nicolas Altemose and Gina V Caldas and Glennis A Logsdon and Arang Rhie and Evan E Eichler and Michael C Schatz and Rachel J O’Neill and Adam M Phillippy and Karen H Miga and Winston Timp},

url = {https://www.science.org/doi/full/10.1126/science.abj5089},

doi = {10.1126/science.abj5089},

issn = {0036-8075},

year  = {2022},

date = {2022-04-01},

urldate = {2021-01-01},

journal = {Science},

volume = {376},

issue = {6588},

pages = {eabj5089},

publisher = {Cold Spring Harbor Laboratory},

abstract = {The completion of the first telomere-to-telomere human genome, T2T-CHM13, enables exploration of the full epigenome, removing limitations previously imposed by the missing reference sequence. Existing epigenetic studies omit unassembled and unmappable genomic regions (e.g. centromeres, pericentromeres, acrocentric chromosome arms, subtelomeres, segmental duplications, tandem repeats). Leveraging the new assembly, we were able to measure enrichment of epigenetic marks with short reads using k-mer assisted mapping methods. This granted array-level enrichment information to characterize the epigenetic regulation of these satellite repeats. Using nanopore sequencing data, we generated base level maps of the most complete human methylome ever produced. We examined methylation patterns in satellite DNA and revealed organized patterns of methylation along individual molecules. When exploring the centromeric epigenome, we discovered a distinctive dip in centromere methylation consistent with active sites of kinetochore assembly. Through long-read chromatin accessibility measurements (nanoNOMe) paired to CUT&RUN data, we found the hypomethylated region was extremely inaccessible and paired to CENP-A/B binding. With long-reads we interrogated allele-specific, longrange epigenetic patterns in complex macro-satellite arrays such as those involved in X chromosome inactivation. Using the single molecule measurements we can clustered reads based on methylation status alone distinguishing epigenetically heterogeneous and homogeneous areas. The analysis provides a framework to investigate the most elusive regions of the human genome, applying both long and short-read technology to grant new insights into epigenetic regulation.Competing Interest StatementW.T. has two patents (8,748,091 and 8,394,584) licensed to Oxford Nanopore Technologies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Vollger2022,

title = {Segmental duplications and their variation in a complete human genome},

author = {Mitchell R Vollger and Xavi Guitart and Philip C Dishuck and Ludovica Mercuri and William T Harvey and Ariel Gershman and Mark Diekhans and Arvis Sulovari and Katherine M Munson and Alexandra P Lewis and Kendra Hoekzema and David Porubsky and Ruiyang Li and Sergey Nurk and Sergey Koren and Karen H Miga and Adam M Phillippy and Winston Timp and Mario Ventura and Evan E Eichler},

url = {https://www.science.org/doi/full/10.1126/science.abj6965},

doi = {10.1126/science.abj6965},

isbn = {0036-8075},

year  = {2022},

date = {2022-04-01},

urldate = {2021-01-01},

journal = {Science},

volume = {376},

issue = {6588},

pages = {eabj6965},

publisher = {Cold Spring Harbor Laboratory},

abstract = {Despite their importance in disease and evolution, highly identical segmental duplications (SDs) are among the last regions of the human reference genome (GRCh38) to be fully sequenced. Using a complete telomere-to-telomere human genome (T2T-CHM13), we present a comprehensive view of human SD organization. SDs account for nearly one-third of the additional sequence, increasing the genome-wide estimate from 5.4 to 7.0% [218 million base pairs (Mbp)]. An analysis of 268 human genomes shows that 91% of the previously unresolved T2T-CHM13 SD sequence (68.3 Mbp) better represents human copy number variation. Comparing long-read assemblies from human (n = 12) and nonhuman primate (n = 5) genomes, we systematically reconstruct the evolution and structural haplotype diversity of biomedically relevant and duplicated genes. This analysis reveals patterns of structural heterozygosity …},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Nurk2022,

title = {The complete sequence of a human genome},

author = {Sergey Nurk and Sergey Koren and Arang Rhie and Mikko Rautiainen and Andrey V Bzikadze and Alla Mikheenko and Mitchell R Vollger and Nicolas Altemose and Lev Uralsky and Ariel Gershman and Sergey Aganezov and Savannah J Hoyt and Mark Diekhans and Glennis A Logsdon and Michael Alonge and Stylianos E Antonarakis and Matthew Borchers and Gerard G Bouffard and Shelise Y Brooks and Gina V Caldas and Nae-Chyun Chen and Haoyu Cheng and Chen-Shan Chin and William Chow and Leonardo G de Lima and Philip C Dishuck and Richard Durbin and Tatiana Dvorkina and Ian T Fiddes and Giulio Formenti and Robert S Fulton and Arkarachai Fungtammasan and Erik Garrison and Patrick GS Grady and Tina A Graves-Lindsay and Ira M Hall and Nancy F Hansen and Gabrielle A Hartley and Marina Haukness and Kerstin Howe and Michael W Hunkapiller and Chirag Jain and Miten Jain and Erich D Jarvis and Peter Kerpedjiev and Melanie Kirsche and Mikhail Kolmogorov and Jonas Korlach and Milinn Kremitzki and Heng Li and Valerie V Maduro and Tobias Marschall and Ann M McCartney and Jennifer McDaniel and Danny E Miller and James C Mullikin and Eugene W Myers and Nathan D Olson and Benedict Paten and Paul Peluso and Pavel A Pevzner and David Porubsky and Tamara Potapova and Evgeny I Rogaev and Jeffrey A Rosenfeld and Steven L Salzberg and Valerie A Schneider and Fritz J Sedlazeck and Kishwar Shafin and Colin J Shew and Alaina Shumate and Ying Sims and Arian FA Smit and Daniela C Soto and Ivan Sović and Jessica M Storer and Aaron Streets and Beth A Sullivan and Françoise Thibaud-Nissen and James Torrance and Justin Wagner and Brian P Walenz and Aaron Wenger and Jonathan MD Wood and Chunlin Xiao and Stephanie M Yan and Alice C Young and Samantha Zarate and Urvashi Surti and Rajiv C McCoy and Megan Y Dennis and Ivan A Alexandrov and Jennifer L Gerton and Rachel J O’Neill and Winston Timp and Justin M Zook and Michael C Schatz and Evan E Eichler and Karen H Miga and Adam M Phillippy},

url = {https://www.science.org/doi/10.1126/science.abj6987},

doi = {10.1126/science.abj6987},

issn = {0036-8075},

year  = {2022},

date = {2022-04-01},

urldate = {2021-01-01},

journal = {Science},

volume = {376},

issue = {6588},

pages = {44-53},

publisher = {Cold Spring Harbor Laboratory},

abstract = {Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion–base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding. The completed regions include all centromeric satellite arrays, recent segmental duplications, and the short arms of all five acrocentric chromosomes, unlocking these complex regions of the genome to variational and functional studies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Stephenson2022,

title = {Direct detection of RNA modifications and structure using single molecule nanopore sequencing},

author = {William Stephenson and Roham Razaghi and Steven Busan and Kevin M Weeks and Winston Timp and Peter Smibert},

url = {https://www.sciencedirect.com/science/article/pii/S2666979X22000143},

doi = {10.1016/j.xgen.2022.100097},

issn = {2666-979X},

year  = {2022},

date = {2022-02-09},

urldate = {2020-05-01},

journal = {Cell Genomics},

volume = {2},

issue = {2},

pages = {100097},

publisher = {Cold Spring Harbor Laboratory},

abstract = {Modifications are present on many classes of RNA, including tRNA, rRNA, and mRNA. These modifications modulate diverse biological processes such as genetic recoding and mRNA export and folding. In addition, modifications can be introduced to RNA molecules using chemical probing strategies that reveal RNA structure and dynamics. Many methods exist to detect RNA modifications by short-read sequencing; however, limitations on read length inherent to short-read-based methods dissociate modifications from their native context, preventing single-molecule modification analysis. Here, we demonstrate direct RNA nanopore sequencing to detect endogenous and exogenous RNA modifications on long RNAs at the single-molecule level. We detect endogenous 2′-O-methyl and base modifications across E. coli and S. cerevisiae ribosomal RNAs as shifts in current signal and dwell times distally through …},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Neale2022,

title = {Assembled and annotated 26.5 Gbp coast redwood genome: a resource for estimating evolutionary adaptive potential and investigating hexaploid origin},

author = {David B Neale and Aleksey V Zimin and Sumaira Zaman and Alison D Scott and Bikash Shrestha and Rachael E Workman and Daniela Puiu and Brian J Allen and Zane J Moore and Manoj K Sekhwal and Amanda R De La Torre and Patrick E McGuire and Emily Burns and Winston Timp and Jill L Wegrzyn and Steven L Salzberg},

url = {https://academic.oup.com/g3journal/article/12/1/jkab380/6460957},

doi = {10.1093/g3journal/jkab380},

year  = {2022},

date = {2022-01-01},

journal = {G3},

volume = {12},

issue = {1},

pages = {jkab380},

abstract = {Sequencing, assembly, and annotation of the 26.5 Gbp hexaploid genome of coast redwood (Sequoia sempervirens) was completed leading toward discovery of genes related to climate adaptation and investigation of the origin of the hexaploid genome. Deep-coverage short-read Illumina sequencing data from haploid tissue from a single seed were combined with long-read Oxford Nanopore Technologies sequencing data from diploid needle tissue to create an initial assembly, which was then scaffolded using proximity ligation data to produce a highly contiguous final assembly, SESE 2.1, with a scaffold N50 size of 44.9 Mbp. The assembly included several scaffolds that span entire chromosome arms, confirmed by the presence of telomere and centromere sequences on the ends of the scaffolds. The structural annotation produced 118,906 genes with 113 containing introns that exceed 500 Kbp in length …},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Genomic insights into metabolic flux in ruby-throated hummingbirds},

author = {Ariel Gershman and Quinn Hauck and Morag Dick and Jerrica M Jamison and Michael Tassia and Xabier Agirrezabala and Saad Muhammad and Raafay Ali and Rachael E Workman and Mikel Valle and G William Wong and Kenneth C Welch and Winston Timp},

url = {https://www.biorxiv.org/content/biorxiv/early/2022/03/21/2022.03.21.485221.full.pdf},

doi = {10.1101/2022.03.21.485221},

year  = {2022},

date = {2022-01-01},

journal = {bioRxiv},

abstract = {Hummingbirds are very well adapted to sustain efficient and rapid metabolic shifts. They oxidize ingested nectar to directly fuel flight when foraging but have to switch to oxidizing stored lipids derived from ingested sugars during the night or long-distance migratory flights. Understanding how this organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. To explore these questions, we generated a chromosome level de novo genome assembly of the ruby-throated hummingbird (A. colubris) using a combination of long and short read sequencing and scaffolding using other existing assemblies. We then used hybrid long and short-read RNA-sequencing for a comprehensive transcriptome assembly and annotation. Our genomic and transcriptomic data found positive selection of key metabolic genes in nectivorous avian species and a deletion of critical genes (GLUT4, GCK) involved in glucostasis in other vertebrates. We found expression of fructose-specific GLUT5 putatively in place of insulin-sensitive GLUT4, with predicted protein models suggesting affinity for both fructose and glucose. Alternative isoforms may even act to sequester fructose to preclude limitations from transport in metabolism. Finally, we identified differentially expressed genes from fasted and fed hummingbirds suggesting key pathways for the rapid metabolic switch hummingbirds undergo.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Razaghi2022,

title = {Modbamtools: Analysis of single-molecule epigenetic data for long-range profiling, heterogeneity, and clustering},

author = {Roham Razaghi and Paul W Hook and Shujun Ou and Michael Schatz and Kasper D Hansen and Miten Jain and Winston Timp},

url = {https://www.biorxiv.org/content/10.1101/2022.07.07.499188v1},

doi = {https://doi.org/10.1101/2022.07.07.499188},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {bioRxiv},

abstract = {The advent of long-read sequencing methods provides new opportunities for profiling the epigenome - especially as the methylation signature comes for "free" when native DNA is sequenced on either Oxford Nanopore or Pacific Biosciences instruments. However, we lack tools to visualize and analyze data generated from these new sources. Recent efforts from the GA4GH consortium have standardized methods to encode modification location and probabilities in the BAM format. Leveraging this standard format, we developed a technology-agnostic tool, modbamtools to visualize, manipulate and compare base modification/methylation data in a fast and robust way. modbamtools can produce high quality, interactive, and publication-ready visualizations as well as provide modules for downstream analysis of base modifications. Modbamtools comprehensive manual and tutorial can be found at https://rrazaghi.github.io/modbamtools/.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kandathil2021,

title = {Plasma virome and the risk of blood-borne infection in persons with substance use disorder},

author = {Abraham J Kandathil and Andrea L Cox and Kimberly Page and David Mohr and Roham Razaghi and Khalil G Ghanem and Susan A Tuddenham and Yu-Hsiang Hsieh and Jennifer L Evans and Kelly E Coller and Winston Timp and David D Celentano and Stuart C Ray and David L Thomas},

url = {https://www.nature.com/articles/s41467-021-26980-8},

doi = {10.1038/s41467-021-26980-8},

issn = {2041-1723},

year  = {2021},

date = {2021-11-25},

journal = {Nature Communications},

volume = {12},

issue = {1},

pages = {1-7},

abstract = {There is an urgent need for innovative methods to reduce transmission of bloodborne pathogens like HIV and HCV among people who inject drugs (PWID). We investigate if PWID who acquire non-pathogenic bloodborne viruses like anelloviruses and pegiviruses might be at greater risk of acquiring a bloodborne pathogen. PWID who later acquire HCV accumulate more non-pathogenic viruses in plasma than matched controls who do not acquire HCV infection. Additionally, phylogenetic analysis of those non-pathogenic virus sequences reveals drug use networks. Here we find first in Baltimore and confirm in San Francisco that the accumulation of non-pathogenic viruses in PWID is a harbinger for subsequent acquisition of pathogenic viruses, knowledge that may guide the prioritization of the public health resources to combat HIV and HCV.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Mahmoud2021-zq,

title = {PRINCESS: comprehensive detection of haplotype resolved SNVs, SVs, and methylation},

author = {Medhat Mahmoud and Harshavardhan Doddapaneni and Winston Timp and Fritz J Sedlazeck},

year  = {2021},

date = {2021-09-01},

journal = {Genome Biology},

volume = {22},

number = {1},

pages = {268},

abstract = {Long-read sequencing has been shown to have advantages in 

 structural variation (SV) detection and methylation calling. Many 

 studies focus either on SV, methylation, or phasing of SNV; 

 however, only the combination of variants provides a 

 comprehensive insight into the sample and thus enables novel 

 findings in biology or medicine. PRINCESS is a structured 

 workflow that takes raw sequence reads and generates a fully 

 phased SNV, SV, and methylation call set within a few hours. 

 PRINCESS achieves high accuracy and long phasing even on low 

 coverage datasets and can resolve repetitive, complex medical 

 relevant genes that often escape detection. PRINCESS is publicly 

 available at https://github.com/MeHelmy/princess under the MIT 

 license.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Lietard2021,

title = {Chemical and photochemical error rates in light-directed synthesis of complex DNA libraries},

author = {Jory Lietard and Adrien Leger and Yaniv Erlich and Norah Sadowski and Winston Timp and Mark M Somoza},

url = {https://doi.org/10.1093/nar/gkab505},

doi = {10.1093/nar/gkab505},

issn = {0305-1048},

year  = {2021},

date = {2021-07-09},

urldate = {2021-07-09},

journal = {Nucleic Acids Research},

volume = {49},

issue = {12},

pages = {6687-6701},

abstract = {Nucleic acid microarrays are the only tools that can supply very large oligonucleotide libraries, cornerstones of the nascent fields of de novo gene assembly and DNA data storage. Although the chemical synthesis of oligonucleotides is highly developed and robust, it is not error free, requiring the design of methods that can correct or compensate for errors, or select for high-fidelity oligomers. However, outside the realm of array manufacturers, little is known about the sources of errors and their extent. In this study, we look at the error rate of DNA libraries synthesized by photolithography and dissect the proportion of deletion, insertion and substitution errors. We find that the deletion rate is governed by the photolysis yield. We identify the most important substitution error and correlate it to phosphoramidite coupling. Besides synthetic failures originating from the coupling cycle, we uncover the role of imperfections …},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Fan2021,

title = {Genome and transcriptome of a pathogenic yeast, Candida nivariensis},

author = {Yunfan Fan and Andrew N Gale and Anna Bailey and Kali Barnes and Kiersten Colotti and Michal Mass and Luke B Morina and Bailey Robertson and Remy Schwab and Niki Tselepidakis and Winston Timp},

url = {https://academic.oup.com/g3journal/advance-article-pdf/doi/10.1093/g3journal/jkab137/38337897/jkab137.pdf},

doi = {10.1093/g3journal/jkab137},

year  = {2021},

date = {2021-04-01},

urldate = {2021-04-01},

journal = {G3},

volume = {11},

issue = {7},

pages = {jkab137},

publisher = {Cold Spring Harbor Laboratory},

abstract = {We present a highly contiguous genome and transcriptome of the pathogenic yeast, Candida nivariensis. We sequenced both the DNA and RNA of this species using both the Oxford Nanopore Technologies (ONT) and Illumina platforms. We assembled the genome into an 11.8 Mb draft composed of 16 contigs with an N50 of 886 Kb, including a circular mitochondrial sequence of 28 Kb. Using direct RNA nanopore sequencing and Illumina cDNA sequencing, we constructed an annotation of our new assembly, supplemented by lifting over genes from Saccharomyces cerevisiae and Candida glabrata.Competing Interest StatementWT holds two patents (US 8,748,091 and US 8,394,584) licensed to ONT. YF and WT have received travel funding from ONT.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Polinskieabe8290,

title = {The American lobster genome reveals insights on longevity, neural, and immune adaptations},

author = {Jennifer M. Polinski and Aleksey V. Zimin and K. Fraser Clark and Andrea B. Kohn and Norah Sadowski and Winston Timp and Andrey Ptitsyn and Prarthana Khanna and Daria Y. Romanova and Peter Williams and Spencer J. Greenwood and Leonid L. Moroz and David R. Walt and Andrea G. Bodnar},

url = {https://advances.sciencemag.org/content/7/26/eabe8290},

doi = {10.1126/sciadv.abe8290},

year  = {2021},

date = {2021-01-01},

urldate = {2021-01-01},

journal = {Science Advances},

volume = {7},

number = {26},

pages = {eabe8290},

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

abstract = {The American lobster, Homarus americanus, is integral to marine ecosystems and supports an important commercial fishery. This iconic species also serves as a valuable model for deciphering neural networks controlling rhythmic motor patterns and olfaction. Here, we report a high-quality draft assembly of the H. americanus genome with 25,284 predicted gene models. Analysis of the neural gene complement revealed extraordinary development of the chemosensory machinery, including a profound diversification of ligand-gated ion channels and secretory molecules. The discovery of a novel class of chimeric receptors coupling pattern recognition and neurotransmitter binding suggests a deep integration between the neural and immune systems. A robust repertoire of genes involved in innate immunity, genome stability, cell survival, chemical defense, and cuticle formation represents a diversity of defense mechanisms essential to thrive in the benthic marine environment. Together, these unique evolutionary adaptations contribute to the longevity and ecological success of this long-lived benthic predator.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{thielen_jci_insight,

title = {Genomic diversity of SARS-CoV-2 during early introduction into the Baltimore–Washington metropolitan area},

author = {Peter M Thielen and Shirlee Wohl and Thomas Mehoke and Srividya Ramakrishnan and Melanie Kirsche and Oluwaseun Falade-Nwulia and Nídia S Trovão and Amanda Ernlund and Craig Howser and Norah Sadowski and Paul C Morris and Mark Hopkins and Matthew Schwartz and Yunfan Fan and Victoria Gniazdowski and Justin Lessler and Lauren Sauer and Michael C Schatz and Jared D Evans and Stuart C Ray and Winston Timp and Heba H Mostafa},

url = {https://insight.jci.org/articles/view/144350},

doi = {10.1172/jci.insight.144350},

year  = {2021},

date = {2021-01-01},

journal = {JCI Insight},

volume = {6},

number = {6},

publisher = {The American Society for Clinical Investigation},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Weinguny_cho_2021b,

title = {Subcloning induces changes in the DNA-methylation pattern of outgrowing Chinese hamster ovary cell colonies},

author = {Marcus Weinguny and Gerald Klanert and Peter Eisenhut and Isac Lee and Winston Timp and Nicole Borth},

url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/biot.202000350},

doi = {https://doi.org/10.1002/biot.202000350},

year  = {2021},

date = {2021-01-01},

journal = {Biotechnology Journal},

pages = {2000350},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{yee_metagenomic_2020b,

title = {Metagenomic next-generation sequencing of rectal swabs for the surveillance of antimicrobial-resistant organisms on the Illumina Miseq and Oxford MinION platforms},

author = {Rebecca Yee and Florian P Breitwieser and Stephanie Hao and Belita N A Opene and Rachael E Workman and Pranita D Tamma and Jennifer Dien-Bard and Winston Timp and Patricia J Simner},

url = {https://doi.org/10.1007/s10096-020-03996-4},

doi = {10.1007/s10096-020-03996-4},

issn = {1435-4373},

year  = {2021},

date = {2021-01-01},

urldate = {2020-08-01},

journal = {European Journal of Clinical Microbiology & Infectious Diseases},

volume = {40},

issue = {1},

pages = {95-102},

abstract = {Antimicrobial resistance (AMR) is a public health threat where efficient surveillance is needed to prevent outbreaks. Existing methods for detection of gastrointestinal colonization of multidrug-resistant organisms (MDRO) are limited to specific organisms or resistance mechanisms. Metagenomic next-generation sequencing (mNGS) is a more rapid and agnostic diagnostic approach for microbiome and resistome investigations. We determined if mNGS can detect MDRO from rectal swabs in concordance with standard microbiology results. We performed and compared mNGS performance on short-read Illumina MiSeq (N = 10) and long-read Nanopore MinION (N = 5) platforms directly from rectal swabs to detect vancomycin-resistant enterococci (VRE) and carbapenem-resistant Gram-negative organisms (CRO). We detected Enterococcus faecium (N = 8) and Enterococcus faecalis (N = 2) with associated van genes (9/10) in concordance with VRE culture-based results. We studied the microbiome and identified CRO, Pseudomonas aeruginosa (N = 1), Enterobacter cloacae (N = 1), and KPC-producing Klebsiella pneumoniae (N = 1). Nanopore real-time analysis detected the blaKPC gene in 2.3 min and provided genetic context (blaKPC harbored on pKPC_Kp46 IncF plasmid). Illumina sequencing provided accurate allelic variant determination (i.e., blaKPC-2) and strain typing of the K. pneumoniae (ST-15). We demonstrated an agnostic approach for surveillance of MDRO, examining advantages of both short- and long-read mNGS methods for AMR detection.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{10.1371/journal.ppat.1009537,

title = {A plasmid locus associated with textitKlebsiella clinical infections encodes a microbiome-dependent gut fitness factor},

author = {Jay Vornhagen and Christine M. Bassis and Srividya Ramakrishnan and Robert Hein and Sophia Mason and Yehudit Bergman and Nicole Sunshine and Yunfan Fan and Caitlyn L. Holmes and Winston Timp and Michael C. Schatz and Vincent B. Young and Patricia J. Simner and Michael A. Bachman},

url = {https://doi.org/10.1371/journal.ppat.1009537},

doi = {10.1371/journal.ppat.1009537},

year  = {2021},

date = {2021-01-01},

urldate = {2021-01-01},

journal = {PLOS Pathogens},

volume = {17},

number = {4},

pages = {1-34},

publisher = {Public Library of Science},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{clark2021epigenetically,

title = {Epigenetically regulated digital signaling defines epithelial innate immunity at the tissue level},

author = {Helen R Clark and Connor McKenney and Nathan M Livingston and Ariel Gershman and Seema Sajjan and Isaac S Chan and Andrew J Ewald and Winston Timp and Bin Wu and Abhyudai Singh and Sergi Regot},

year  = {2021},

date = {2021-01-01},

journal = {Nature Communications},

volume = {12},

number = {1},

pages = {1--13},

publisher = {Nature Publishing Group},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{10.1093/g3journal/jkaa047b,

title = {De novo genome assembly of the tobacco hornworm moth (Manduca sexta)},

author = {Ariel Gershman and Tatiana G Romer and Yunfan Fan and Roham Razaghi and Wendy A Smith and Winston Timp},

url = {https://doi.org/10.1093/g3journal/jkaa047},

doi = {10.1093/g3journal/jkaa047},

issn = {2160-1836},

year  = {2021},

date = {2021-01-01},

urldate = {2021-01-01},

journal = {G3 Genes|Genomes|Genetics},

volume = {11},

number = {1},

pages = {jkaa047},

abstract = {The tobacco hornworm, Manduca sexta, is a lepidopteran insect that is used extensively as a model system for studying insect biology, development, neuroscience, and immunity. However, current studies rely on the highly fragmented reference genome Msex_1.0, which was created using now-outdated technologies and is hindered by a variety of deficiencies and inaccuracies. We present a new reference genome for M. sexta, JHU_Msex_v1.0, applying a combination of modern technologies in a de novo assembly to increase continuity, accuracy, and completeness. The assembly is 470 Mb and is ∼20× more continuous than the original assembly, with scaffold N50 > 14 Mb. We annotated the assembly by lifting over existing annotations and supplementing with additional supporting RNA-based data for a total of 25,256 genes. The new reference assembly is accessible in annotated form for public use. We demonstrate that improved continuity of the M. sexta genome improves resequencing studies and benefits future research on M. sexta as a model organism.},

note = {jkaa047},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{lee_simultaneous_2020,

title = {Simultaneous profiling of chromatin accessibility and methylation on human cell lines with nanopore sequencing},

author = {Isac Lee and Roham Razaghi and Timothy Gilpatrick and Michael Molnar and Ariel Gershman and Norah Sadowski and Fritz J Sedlazeck and Kasper D Hansen and Jared T Simpson and Winston Timp},

url = {https://www.nature.com/articles/s41592-020-01000-7},

doi = {10.1038/s41592-020-01000-7},

issn = {1548-7105},

year  = {2020},

date = {2020-12-01},

journal = {Nature Methods},

volume = {17},

number = {12},

pages = {1191--1199},

abstract = {Probing epigenetic features on DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we use nanopore sequencing to evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA by applying GpC methyltransferase to exogenously label open chromatin. We performed nanopore sequencing of nucleosome occupancy and methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7 and MDA-MB-231). The single-molecule resolution allows footprinting of protein and nucleosome binding, and determination of the combinatorial promoter epigenetic signature on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, allowing us to generate a fully phased human epigenome, consisting of chromosome-level allele-specific profiles of CpG methylation and chromatin accessibility. We further apply this to a breast cancer model to evaluate differential methylation and accessibility between cancerous and noncancerous cells.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{kovaka_targeted_2020,

title = {Targeted nanopore sequencing by real-time mapping of raw electrical signal with UNCALLED},

author = {Sam Kovaka and Yunfan Fan and Bohan Ni and Winston Timp and Michael C Schatz},

url = {https://www.nature.com/articles/s41587-020-0731-9},

doi = {10.1038/s41587-020-0731-9},

issn = {1546-1696},

year  = {2020},

date = {2020-11-30},

urldate = {2020-11-30},

journal = {Nature Biotechnology},

volume = {39},

issue = {4},

pages = {1--11},

abstract = {Conventional targeted sequencing methods eliminate many of the benefits of nanopore sequencing, such as the ability to accurately detect structural variants or epigenetic modifications. The ReadUntil method allows nanopore devices to selectively eject reads from pores in real time, which could enable purely computational targeted sequencing. However, this requires rapid identification of on-target reads while most mapping methods require computationally intensive basecalling. We present UNCALLED (https://github.com/skovaka/UNCALLED), an open source mapper that rapidly matches streaming of nanopore current signals to a reference sequence. UNCALLED probabilistically considers k-mers that could be represented by the signal and then prunes the candidates based on the reference encoded within a Ferragina–Manzini index. We used UNCALLED to deplete sequencing of known bacterial genomes within a metagenomics community, enriching the remaining species 4.46-fold. UNCALLED also enriched 148 human genes associated with hereditary cancers to 29.6× coverage using one MinION flowcell, enabling accurate detection of single-nucleotide polymorphisms, insertions and deletions, structural variants and methylation in these genes.},

note = {Publisher: Nature Publishing Group},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pokrass_cell-cycle-dependent_2020b,

title = {Cell-Cycle-Dependent ERK Signaling Dynamics Direct Fate Specification in the Mammalian Preimplantation Embryo},

author = {Michael J Pokrass and Kathleen A Ryan and Tianchi Xin and Brittany Pielstick and Winston Timp and Valentina Greco and Sergi Regot},

doi = {10.1016/j.devcel.2020.09.013},

issn = {1878-1551},

year  = {2020},

date = {2020-11-09},

journal = {Developmental Cell},

volume = {55},

number = {3},

pages = {328--340.e5},

abstract = {Despite the noisy nature of single cells, multicellular organisms robustly generate different cell types from one zygote. This process involves dynamic cross regulation between signaling and gene expression that is difficult to capture with fixed-cell approaches. To study signaling dynamics and fate specification during preimplantation development, we generated a transgenic mouse expressing the ERK kinase translocation reporter and measured ERK activity in single cells of live embryos. Our results show primarily active ERK in both the inner cell mass and trophectoderm cells due to fibroblast growth factor (FGF) signaling. Strikingly, a subset of mitotic events results in a short pulse of ERK inactivity in both daughter cells that correlates with elevated endpoint NANOG levels. Moreover, endogenous tagging of Nanog in embryonic stem cells reveals that ERK inhibition promotes enhanced stabilization of NANOG protein after mitosis. Our data show that cell cycle, signaling, and differentiation are coordinated during preimplantation development.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@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{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{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{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{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{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{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{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{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}

}