Publications

@article{pmid36827376,

title = {Microscale combinatorial stimulation of human myeloid cells reveals inflammatory priming by viral ligands},

author = {Miguel Reyes and Samantha M Leff and Matteo Gentili and Nir Hacohen and Paul C Blainey},

doi = {10.1126/sciadv.ade5090},

issn = {2375-2548},

year  = {2023},

date = {2023-02-01},

journal = {Sci Adv},

volume = {9},

number = {8},

pages = {eade5090},

abstract = {Cells sense a wide variety of signals and respond by adopting complex transcriptional states. Most single-cell profiling is carried out today at cellular baseline, blind to cells' potential spectrum of functional responses. Exploring the space of cellular responses experimentally requires access to a large combinatorial perturbation space. Single-cell genomics coupled with multiplexing techniques provide a useful tool for characterizing cell states across several experimental conditions. However, current multiplexing strategies require programmatic handling of many samples in macroscale arrayed formats, precluding their application in large-scale combinatorial analysis. Here, we introduce StimDrop, a method that combines antibody-based cell barcoding with parallel droplet processing to automatically formulate cell population × stimulus combinations in a microfluidic device. We applied StimDrop to profile the effects of 512 sequential stimulation conditions on human dendritic cells. Our results demonstrate that priming with viral ligands potentiates hyperinflammatory responses to a second stimulus, and show transcriptional signatures consistent with this phenomenon in myeloid cells of patients with severe COVID-19.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid36347254,

title = {The phenotypic landscape of essential human genes},

author = {Luke Funk and Kuan-Chung Su and Jimmy Ly and David Feldman and Avtar Singh and Brittania Moodie and Paul C Blainey and Iain M Cheeseman},

url = {https://vesuvius.wi.mit.edu/},

doi = {10.1016/j.cell.2022.10.017},

issn = {1097-4172},

year  = {2022},

date = {2022-11-01},

urldate = {2022-11-01},

journal = {Cell},

volume = {185},

number = {24},

pages = {4634--4653.e22},

abstract = {Understanding the basis for cellular growth, proliferation, and function requires determining the roles of essential genes in diverse cellular processes, including visualizing their contributions to cellular organization and morphology. Here, we combined pooled CRISPR-Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules. Analysis of >31 million individual cells identified measurable phenotypes for >90% of gene knockouts, implicating gene targets in specific cellular processes. Clustering of phenotypic similarities based on hundreds of quantitative parameters further revealed co-functional genes across diverse cellular activities, providing predictions for gene functions and associations. By conducting pooled live-cell screening of ∼450,000 cell division events for 239 genes, we additionally identified diverse genes with functional contributions to chromosome segregation. Our work establishes a resource detailing the consequences of disrupting core cellular processes that represents the functional landscape of essential human genes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid36366905,

title = {Pooled genetic screens with image-based profiling},

author = {Russell T Walton and Avtar Singh and Paul C Blainey},

doi = {10.15252/msb.202110768},

issn = {1744-4292},

year  = {2022},

date = {2022-11-01},

journal = {Mol Syst Biol},

volume = {18},

number = {11},

pages = {e10768},

abstract = {Spatial structure in biology, spanning molecular, organellular, cellular, tissue, and organismal scales, is encoded through a combination of genetic and epigenetic factors in individual cells. Microscopy remains the most direct approach to exploring the intricate spatial complexity defining biological systems and the structured dynamic responses of these systems to perturbations. Genetic screens with deep single-cell profiling via image features or gene expression programs have the capacity to show how biological systems work in detail by cataloging many cellular phenotypes with one experimental assay. Microscopy-based cellular profiling provides information complementary to next-generation sequencing (NGS) profiling and has only recently become compatible with large-scale genetic screens. Optical screening now offers the scale needed for systematic characterization and is poised for further scale-up. We discuss how these methodologies, together with emerging technologies for genetic perturbation and microscopy-based multiplexed molecular phenotyping, are powering new approaches to reveal genotype-phenotype relationships.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35241837,

title = {Designing sensitive viral diagnostics with machine learning},

author = {Hayden C Metsky and Nicole L Welch and Priya P Pillai and Nicholas J Haradhvala and Laurie Rumker and Sreekar Mantena and Yibin B Zhang and David K Yang and Cheri M Ackerman and Juliane Weller and Paul C Blainey and Cameron Myhrvold and Michael Mitzenmacher and Pardis C Sabeti},

doi = {10.1038/s41587-022-01213-5},

issn = {1546-1696},

year  = {2022},

date = {2022-07-01},

journal = {Nat Biotechnol},

volume = {40},

number = {7},

pages = {1123--1131},

abstract = {Design of nucleic acid-based viral diagnostics typically follows heuristic rules and, to contend with viral variation, focuses on a genome's conserved regions. A design process could, instead, directly optimize diagnostic effectiveness using a learned model of sensitivity for targets and their variants. Toward that goal, we screen 19,209 diagnostic-target pairs, concentrated on CRISPR-based diagnostics, and train a deep neural network to accurately predict diagnostic readout. We join this model with combinatorial optimization to maximize sensitivity over the full spectrum of a virus's genomic variation. We introduce Activity-informed Design with All-inclusive Patrolling of Targets (ADAPT), a system for automated design, and use it to design diagnostics for 1,933 vertebrate-infecting viral species within 2 hours for most species and within 24 hours for all but three. We experimentally show that ADAPT's designs are sensitive and specific to the lineage level and permit lower limits of detection, across a virus's variation, than the outputs of standard design techniques. Our strategy could facilitate a proactive resource of assays for detecting pathogens.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35706396,

title = {Epidemiology and genomics of a slow outbreak of methicillin-resistant  (MRSA) in a neonatal intensive care unit: Successful chronic decolonization of MRSA-positive healthcare personnel},

author = {Kathleen A Quan and Mohamad R A Sater and Cherry Uy and Robin Clifton-Koeppel and Linda L Dickey and William Wilson and Pat Patton and Wayne Chang and Pamela Samuelson and Georgia K Lagoudas and Teri Allen and Lenny Merchant and Rick Gannotta and Cassiana E Bittencourt and J C Soto and Kaye D Evans and Paul C Blainey and John Murray and Dawn Shelton and Helen S Lee and Matthew Zahn and Julia Wolfe and Keith Madey and Jennifer Yim and Shruti K Gohil and Yonatan H Grad and Susan S Huang},

doi = {10.1017/ice.2022.133},

issn = {1559-6834},

year  = {2022},

date = {2022-06-01},

journal = {Infect Control Hosp Epidemiol},

pages = {1--8},

abstract = {OBJECTIVE: To describe the genomic analysis and epidemiologic response related to a slow and prolonged methicillin-resistant  (MRSA) outbreak.



DESIGN: Prospective observational study.



SETTING: Neonatal intensive care unit (NICU).



METHODS: We conducted an epidemiologic investigation of a NICU MRSA outbreak involving serial baby and staff screening to identify opportunities for decolonization. Whole-genome sequencing was performed on MRSA isolates.



RESULTS: A NICU with excellent hand hygiene compliance and longstanding minimal healthcare-associated infections experienced an MRSA outbreak involving 15 babies and 6 healthcare personnel (HCP). In total, 12 cases occurred slowly over a 1-year period (mean, 30.7 days apart) followed by 3 additional cases 7 months later. Multiple progressive infection prevention interventions were implemented, including contact precautions and cohorting of MRSA-positive babies, hand hygiene observers, enhanced environmental cleaning, screening of babies and staff, and decolonization of carriers. Only decolonization of HCP found to be persistent carriers of MRSA was successful in stopping transmission and ending the outbreak. Genomic analyses identified bidirectional transmission between babies and HCP during the outbreak.



CONCLUSIONS: In comparison to fast outbreaks, outbreaks that are "slow and sustained" may be more common to units with strong existing infection prevention practices such that a series of breaches have to align to result in a case. We identified a slow outbreak that persisted among staff and babies and was only stopped by identifying and decolonizing persistent MRSA carriage among staff. A repeated decolonization regimen was successful in allowing previously persistent carriers to safely continue work duties.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35130561,

title = {Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants},

author = {Nicole L Welch and Meilin Zhu and Catherine Hua and Juliane Weller and Marzieh Ezzaty Mirhashemi and Tien G Nguyen and Sreekar Mantena and Matthew R Bauer and Bennett M Shaw and Cheri M Ackerman and Sri Gowtham Thakku and Megan W Tse and Jared Kehe and Marie-Martine Uwera and Jacqueline S Eversley and Derek A Bielwaski and Graham McGrath and Joseph Braidt and Jeremy Johnson and Felecia Cerrato and Gage K Moreno and Lydia A Krasilnikova and Brittany A Petros and Gabrielle L Gionet and Ewa King and Richard C Huard and Samantha K Jalbert and Michael L Cleary and Nicholas A Fitzgerald and Stacey B Gabriel and Glen R Gallagher and Sandra C Smole and Lawrence C Madoff and Catherine M Brown and Matthew W Keller and Malania M Wilson and Marie K Kirby and John R Barnes and Daniel J Park and Katherine J Siddle and Christian T Happi and Deborah T Hung and Michael Springer and Bronwyn L MacInnis and Jacob E Lemieux and Eric Rosenberg and John A Branda and Paul C Blainey and Pardis C Sabeti and Cameron Myhrvold},

doi = {10.1038/s41591-022-01734-1},

issn = {1546-170X},

year  = {2022},

date = {2022-05-01},

journal = {Nat Med},

volume = {28},

number = {5},

pages = {1083--1094},

abstract = {The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting. We further developed an mCARMEN panel to enable the identification of 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 2,088 patient specimens with near-perfect concordance to sequencing-based variant classification. Lastly, we implemented a combined Cas13 and Cas12 approach that enables quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples. The mCARMEN platform enables high-throughput surveillance of multiple viruses and variants simultaneously, enabling rapid detection of SARS-CoV-2 variants.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35450424,

title = {Multiplexed detection of bacterial nucleic acids using Cas13 in droplet microarrays},

author = {Sri Gowtham Thakku and Cheri M Ackerman and Cameron Myhrvold and Roby P Bhattacharyya and Jonathan Livny and Peijun Ma and Giselle Isabella Gomez and Pardis C Sabeti and Paul C Blainey and Deborah T Hung},

doi = {10.1093/pnasnexus/pgac021},

issn = {2752-6542},

year  = {2022},

date = {2022-03-01},

journal = {PNAS Nexus},

volume = {1},

number = {1},

pages = {pgac021},

abstract = {Rapid and accurate diagnosis of infections is fundamental to individual patient care and public health management. Nucleic acid detection methods are critical to this effort, but are limited either in the breadth of pathogens targeted or by the expertise and infrastructure required. We present here a high-throughput system that enables rapid identification of bacterial pathogens, bCARMEN, which utilizes: (1) modular CRISPR-Cas13-based nucleic acid detection with enhanced sensitivity and specificity; and (2) a droplet microfluidic system that enables thousands of simultaneous, spatially multiplexed detection reactions at nanoliter volumes; and (3) a novel preamplification strategy that further enhances sensitivity and specificity. We demonstrate bCARMEN is capable of detecting and discriminating 52 clinically relevant bacterial species and several key antibiotic resistance genes. We further develop a simple proof of principle workflow using stabilized reagents and cell phone camera optical readout, opening up the possibility of a rapid point-of-care multiplexed bacterial pathogen identification and antibiotic susceptibility testing.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid35022620,

title = {Pooled genetic perturbation screens with image-based phenotypes},

author = {David Feldman and Luke Funk and Anna Le and Rebecca J Carlson and Michael D Leiken and FuNien Tsai and Brian Soong and Avtar Singh and Paul C Blainey},

doi = {10.1038/s41596-021-00653-8},

issn = {1750-2799},

year  = {2022},

date = {2022-02-01},

journal = {Nat Protoc},

volume = {17},

number = {2},

pages = {476--512},

abstract = {Discovery of the genetic components underpinning fundamental and disease-related processes is being rapidly accelerated by combining efficient, programmable genetic engineering with phenotypic readouts of high spatial, temporal and/or molecular resolution. Microscopy is a fundamental tool for studying cell biology, but its lack of high-throughput sequence readouts hinders integration in large-scale genetic screens. Optical pooled screens using in situ sequencing provide massively scalable integration of barcoded lentiviral libraries (e.g., CRISPR perturbation libraries) with high-content imaging assays, including dynamic processes in live cells. The protocol uses standard lentiviral vectors and molecular biology, providing single-cell resolution of phenotype and engineered genotype, scalability to millions of cells and accurate sequence reads sufficient to distinguish >10 perturbations. In situ amplification takes ~2 d, while sequencing can be performed in ~1.5 h per cycle. The image analysis pipeline provided enables fully parallel automated sequencing analysis using a cloud or cluster computing environment.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34739314,

title = {Positive interactions are common among culturable bacteria},

author = {Jared Kehe and Anthony Ortiz and Anthony Kulesa and Jeff Gore and Paul C Blainey and Jonathan Friedman},

doi = {10.1126/sciadv.abi7159},

issn = {2375-2548},

year  = {2021},

date = {2021-11-01},

journal = {Sci Adv},

volume = {7},

number = {45},

pages = {eabi7159},

abstract = {Interspecies interactions shape the structure and function of microbial communities. In particular, positive, growth-promoting interactions can substantially affect the diversity and productivity of natural and engineered communities. However, the prevalence of positive interactions and the conditions in which they occur are not well understood. To address this knowledge gap, we used kChip, an ultrahigh-throughput coculture platform, to measure 180,408 interactions among 20 soil bacteria across 40 carbon environments. We find that positive interactions, often described to be rare, occur commonly and primarily as parasitisms between strains that differ in their carbon consumption profiles. Notably, nongrowing strains are almost always promoted by strongly growing strains (85%), suggesting a simple positive interaction–mediated approach for cultivation, microbiome engineering, and microbial consortium design.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34112975,

title = {Random access DNA memory using Boolean search in an archival file storage system},

author = {James L Banal and Tyson R Shepherd and Joseph Berleant and Hellen Huang and Miguel Reyes and Cheri M Ackerman and Paul C Blainey and Mark Bathe},

doi = {10.1038/s41563-021-01021-3},

issn = {1476-4660},

year  = {2021},

date = {2021-09-01},

journal = {Nat Mater},

volume = {20},

number = {9},

pages = {1272--1280},

abstract = {DNA is an ultrahigh-density storage medium that could meet exponentially growing worldwide demand for archival data storage if DNA synthesis costs declined sufficiently and if random access of files within exabyte-to-yottabyte-scale DNA data pools were feasible. Here, we demonstrate a path to overcome the second barrier by encapsulating data-encoding DNA file sequences within impervious silica capsules that are surface labelled with single-stranded DNA barcodes. Barcodes are chosen to represent file metadata, enabling selection of sets of files with Boolean logic directly, without use of amplification. We demonstrate random access of image files from a prototypical 2-kilobyte image database using fluorescence sorting with selection sensitivity of one in 10 files, which thereby enables one in 10 selection capability using N optical channels. Our strategy thereby offers a scalable concept for random access of archival files in large-scale molecular datasets.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34212403,

title = {The future of antibiotics begins with discovering new combinations},

author = {Meilin Zhu and Megan W Tse and Juliane Weller and Julie Chen and Paul C Blainey},

doi = {10.1111/nyas.14649},

issn = {1749-6632},

year  = {2021},

date = {2021-07-01},

journal = {Ann N Y Acad Sci},

volume = {1496},

number = {1},

pages = {82--96},

abstract = {Antibiotic resistance is a worldwide and growing clinical problem. With limited drug development in the antibacterial space, combination therapy has emerged as a promising strategy to combat multidrug-resistant bacteria. Antibacterial combinations can improve antibiotic efficacy and suppress antibacterial resistance through independent, synergistic, or even antagonistic activities. Combination therapies are famously used to treat viral and mycobacterial infections and cancer. However, antibacterial combinations are only now emerging as a common treatment strategy for other bacterial infections owing to challenges in their discovery, development, regulatory approval, and commercial/clinical deployment. Here, we focus on discovery-where the sheer scale of combinatorial chemical spaces represents a significant challenge-and discuss how combination therapy can impact the treatment of bacterial infections. Despite these challenges, recent advancements, including new in silico methods, theoretical frameworks, and microfluidic platforms, are poised to identify the new and efficacious antibacterial combinations needed to revitalize the antibacterial drug pipeline.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid34103408,

title = {Plasma from patients with bacterial sepsis or severe COVID-19 induces suppressive myeloid cell production from hematopoietic progenitors in vitro},

author = {Miguel Reyes and Michael R Filbin and Roby P Bhattacharyya and Abraham Sonny and Arnav Mehta and Kianna Billman and Kyle R Kays and Mayra Pinilla-Vera and Maura E Benson and Lisa A Cosimi and Deborah T Hung and Bruce D Levy and Alexandra-Chloe Villani and Moshe Sade-Feldman and Rebecca M Baron and Marcia B Goldberg and Paul C Blainey and Nir Hacohen},

doi = {10.1126/scitranslmed.abe9599},

issn = {1946-6242},

year  = {2021},

date = {2021-06-01},

journal = {Sci Transl Med},

volume = {13},

number = {598},

abstract = {Bacterial sepsis and severe COVID-19 share similar clinical manifestations and are both associated with dysregulation of the myeloid cell compartment. We previously reported an expanded CD14 monocyte state, MS1, in patients with bacterial sepsis and validated expansion of this cell subpopulation in publicly available transcriptomics data. Here, using published datasets, we show that the gene expression program associated with MS1 correlated with sepsis severity and was up-regulated in monocytes from patients with severe COVID-19. To examine the ontogeny and function of MS1 cells, we developed a cellular model for inducing CD14 MS1 monocytes from healthy bone marrow hematopoietic stem and progenitor cells (HSPCs). We found that plasma from patients with bacterial sepsis or COVID-19 induced myelopoiesis in HSPCs in vitro and expression of the MS1 gene program in monocytes and neutrophils that differentiated from these HSPCs. Furthermore, we found that plasma concentrations of IL-6, and to a lesser extent IL-10, correlated with increased myeloid cell output from HSPCs in vitro and enhanced expression of the MS1 gene program. We validated the requirement for these two cytokines to induce the MS1 gene program through CRISPR-Cas9 editing of their receptors in HSPCs. Using this cellular model system, we demonstrated that induced MS1 cells were broadly immunosuppressive and showed decreased responsiveness to stimulation with a synthetic RNA analog. Our in vitro study suggests a potential role for systemic cytokines in inducing myelopoiesis during severe bacterial or SARS-CoV-2 infection.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33355294,

title = {DENT-seq for genome-wide strand-specific identification of DNA single-strand break sites with single-nucleotide resolution},

author = {Joshua J Elacqua and Navpreet Ranu and Sarah E DiIorio and Paul C Blainey},

doi = {10.1101/gr.265223.120},

issn = {1549-5469},

year  = {2021},

date = {2021-01-01},

journal = {Genome Res},

volume = {31},

number = {1},

pages = {75--87},

abstract = {DNA single-strand breaks (SSBs), or "nicks," are the most common form of DNA damage. Oxidative stress, endogenous enzyme activities, and other processes cause tens of thousands of nicks per cell per day. Accumulation of nicks, caused by high rates of occurrence or defects in repair enzymes, has been implicated in multiple diseases. However, improved methods for nick analysis are needed to characterize the mechanisms of these processes and learn how the location and number of nicks affect cells, disease progression, and health outcomes. In addition to natural processes, including DNA repair, leading genome editing technologies rely on nuclease activity, including nick generation, at specific target sites. There is currently a pressing need for methods to study off-target nicking activity genome-wide to evaluate the side effects of emerging genome editing tools on cells and organisms. Here, we developed a new method, DENT-seq, for efficient strand-specific profiling of nicks in complex DNA samples with single-nucleotide resolution and low false-positive rates. DENT-seq produces a single deep sequence data set enriched for reads near nick sites and establishes a readily detectable mutational signal that allows for determination of the nick site and strand with single-base resolution at penetrance as low as one strand per thousand. We apply DENT-seq to profile the off-target activity of the Nb.BsmI nicking endonuclease and an engineered spCas9 nickase. DENT-seq will be useful in exploring the activity of engineered nucleases in genome editing and other biotechnological applications as well as spontaneous and therapeutic-associated strand breaks.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33293579,

title = {A validated lineage-derived somatic truth data set enables benchmarking in cancer genome analysis},

author = {Megan Shand and Jose Soto and Lee Lichtenstein and David Benjamin and Yossi Farjoun and Yehuda Brody and Yosef Maruvka and Paul C Blainey and Eric Banks},

doi = {10.1038/s42003-020-01460-9},

issn = {2399-3642},

year  = {2020},

date = {2020-12-01},

journal = {Commun Biol},

volume = {3},

number = {1},

pages = {744},

abstract = {Existing cancer benchmark data sets for human sequencing data use germline variants, synthetic methods, or expensive validations, none of which are satisfactory for providing a large collection of true somatic variation across a whole genome. Here we propose a data set, Lineage derived Somatic Truth (LinST), of short somatic mutations in the HT115 colon cancer cell-line, that are validated using a known cell lineage that includes thousands of mutations and a high confidence region covering 2.7 gigabases per sample.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid33234154,

title = {CloneSifter: enrichment of rare clones from heterogeneous cell populations},

author = {David Feldman and FuNien Tsai and Anthony J Garrity and Ryan O'Rourke and Lisa Brenan and Patricia Ho and Elizabeth Gonzalez and Silvana Konermann and Cory M Johannessen and Rameen Beroukhim and Pratiti Bandopadhayay and Paul C Blainey},

doi = {10.1186/s12915-020-00911-3},

issn = {1741-7007},

year  = {2020},

date = {2020-11-01},

journal = {BMC Biol},

volume = {18},

number = {1},

pages = {177},

abstract = {BACKGROUND: Many biological processes, such as cancer metastasis, organismal development, and acquisition of resistance to cytotoxic therapy, rely on the emergence of rare sub-clones from a larger population. Understanding how the genetic and epigenetic features of diverse clones affect clonal fitness provides insight into molecular mechanisms underlying selective processes. While large-scale barcoding with NGS readout has facilitated cellular fitness assessment at the population level, this approach does not support characterization of clones prior to selection. Single-cell genomics methods provide high biological resolution, but are challenging to scale across large populations to probe rare clones and are destructive, limiting further functional analysis of important clones.



RESULTS: Here, we develop CloneSifter, a methodology for tracking and enriching rare clones throughout their response to selection. CloneSifter utilizes a CRISPR sgRNA-barcode library that facilitates the isolation of viable cells from specific clones within the barcoded population using a sequence-specific retrieval reporter. We demonstrate that CloneSifter can measure clonal fitness of cancer cell models in vitro and retrieve targeted clones at abundance as low as 1 in 1883 in a heterogeneous cell population.



CONCLUSIONS: CloneSifter provides a means to track and access specific and rare clones of interest across dynamic changes in population structure to comprehensively explore the basis of these changes.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32349121,

title = {Massively multiplexed nucleic acid detection with Cas13},

author = {Cheri M Ackerman and Cameron Myhrvold and Sri Gowtham Thakku and Catherine A Freije and Hayden C Metsky and David K Yang and Simon H Ye and Chloe K Boehm and Tinna-Sólveig F Kosoko-Thoroddsen and Jared Kehe and Tien G Nguyen and Amber Carter and Anthony Kulesa and John R Barnes and Vivien G Dugan and Deborah T Hung and Paul C Blainey and Pardis C Sabeti},

doi = {10.1038/s41586-020-2279-8},

issn = {1476-4687},

year  = {2020},

date = {2020-06-01},

journal = {Nature},

volume = {582},

number = {7811},

pages = {277--282},

abstract = {The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples while simultaneously testing for many pathogens. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents self-organize in a microwell array to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32066974,

title = {An immune-cell signature of bacterial sepsis},

author = {Miguel Reyes and Michael R Filbin and Roby P Bhattacharyya and Kianna Billman and Thomas Eisenhaure and Deborah T Hung and Bruce D Levy and Rebecca M Baron and Paul C Blainey and Marcia B Goldberg and Nir Hacohen},

doi = {10.1038/s41591-020-0752-4},

issn = {1546-170X},

year  = {2020},

date = {2020-03-01},

journal = {Nat Med},

volume = {26},

number = {3},

pages = {333--340},

abstract = {Dysregulation of the immune response to bacterial infection can lead to sepsis, a condition with high mortality. Multiple whole-blood gene-expression studies have defined sepsis-associated molecular signatures, but have not resolved changes in transcriptional states of specific cell types. Here, we used single-cell RNA-sequencing to profile the blood of people with sepsis (n = 29) across three clinical cohorts with corresponding controls (n = 36). We profiled total peripheral blood mononuclear cells (PBMCs, 106,545 cells) and dendritic cells (19,806 cells) across all subjects and, on the basis of clustering of their gene-expression profiles, defined 16 immune-cell states. We identified a unique CD14 monocyte state that is expanded in people with sepsis and validated its power in distinguishing these individuals from controls using public transcriptomic data from subjects with different disease etiologies and from multiple geographic locations (18 cohorts, n = 1,467 subjects). We identified a panel of surface markers for isolation and quantification of the monocyte state and characterized its epigenomic and functional phenotypes, and propose a model for its induction from human bone marrow. This study demonstrates the utility of single-cell genomics in discovering disease-associated cytologic signatures and provides insight into the cellular basis of immune dysregulation in bacterial sepsis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32373614,

title = {Single-Cell Transcriptional Profiling of Cells Derived From Regenerating Alveolar Ducts},

author = {Alexandra B Ysasi and Robert D Bennett and Willi Wagner and Cristian D Valenzuela and Andrew B Servais and Akira Tsuda and Saumyadipta Pyne and Shuqiang Li and Jonna Grimsby and Prapti Pokharel and Kenneth J Livak and Maximilian Ackermann and Paul C Blainey and Steven J Mentzer},

doi = {10.3389/fmed.2020.00112},

issn = {2296-858X},

year  = {2020},

date = {2020-01-01},

journal = {Front Med (Lausanne)},

volume = {7},

pages = {112},

abstract = {Lung regeneration occurs in a variety of adult mammals after surgical removal of one lung (pneumonectomy). Previous studies of murine post-pneumonectomy lung growth have identified regenerative "hotspots" in subpleural alveolar ducts; however, the cell-types participating in this process remain unclear. To identify the single cells participating in post-pneumonectomy lung growth, we used laser microdissection, enzymatic digestion and microfluidic isolation. Single-cell transcriptional analysis of the murine alveolar duct cells was performed using the C1 integrated fluidic circuit (Fluidigm) and a custom PCR panel designed for lung growth and repair genes. The multi-dimensional data set was analyzed using visualization software based on the tSNE algorithm. The analysis identified 6 cell clusters; 1 cell cluster was present only after pneumonectomy. This post-pneumonectomy cluster was significantly less transcriptionally active than 3 other clusters and may represent a transitional cell population. A provisional cluster identity for 4 of the 6 cell clusters was obtained by embedding bulk transcriptional data into the tSNE analysis. The transcriptional pattern of the 6 clusters was further analyzed for genes associated with lung repair, matrix production, and angiogenesis. The data demonstrated that multiple cell-types (clusters) transcribed genes linked to these basic functions. We conclude that the coordinated gene expression across multiple cell clusters is likely a response to a shared regenerative microenvironment within the subpleural alveolar ducts.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31659048,

title = {Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium},

author = {Stephanie Tzouanas Schmidt and Feiqiao Brian Yu and Paul C Blainey and Andrew P May and Stephen R Quake},

doi = {10.1073/pnas.1904273116},

issn = {1091-6490},

year  = {2019},

date = {2019-11-01},

journal = {Proc Natl Acad Sci U S A},

volume = {116},

number = {46},

pages = {23100--23105},

abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) systems have been effectively harnessed to engineer the genomes of organisms from across the tree of life. Nearly all currently characterized Cas9 proteins are derived from mesophilic bacteria, and canonical Cas9 systems are challenged by applications requiring enhanced stability or elevated temperatures. We discovered IgnaviCas9, a Cas9 protein from a hyperthermophilic Ignavibacterium identified through mini-metagenomic sequencing of samples from a hot spring. IgnaviCas9 is active at temperatures up to 100 °C in vitro, which enables DNA cleavage beyond the 44 °C limit of  Cas9 (SpyCas9) and the 70 °C limit of both  Cas9 (GeoCas9) and  T12 Cas9 (ThermoCas9). As a potential application of this enzyme, we demonstrate that IgnaviCas9 can be used in bacterial RNA-seq library preparation to remove unwanted cDNA from 16s ribosomal rRNA without increasing the number of steps, thus underscoring the benefits provided by its exceptional thermostability in improving molecular biology and genomic workflows. IgnaviCas9 is an exciting addition to the CRISPR-Cas9 toolbox and expands its temperature range.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31853478,

title = {Simultaneous profiling of gene expression and chromatin accessibility in single cells},

author = {Miguel Reyes and Kianna Billman and Nir Hacohen and Paul C Blainey},

doi = {10.1002/adbi.201900065},

issn = {2366-7478},

year  = {2019},

date = {2019-11-01},

journal = {Adv Biosyst},

volume = {3},

number = {11},

abstract = {Profiling multiple omic layers in a single cell enables the discovery and analysis of biological phenomena that are not apparent from analysis of mono-omic data. While methods for multi-omic profiling have been reported, their adoption has been limited due to high cost and complex workflows. Here, we present a simple method for joint profiling of gene expression and chromatin accessibility in tens to hundreds of single cells. We assess the quality of resulting single cell ATAC- and RNA-seq data across three cell types, examine the link between accessibility and expression at the  and  loci in human primary T cells and monocytes, and compare the accuracy of clustering solutions for mono-omic and combined data. The new method allows biological laboratories to perform simultaneous profiling of gene expression and chromatin accessibility using standard reagents and instrumentation. This technique, in conjunction with other advances in multi-omic profiling, will enable highly-resolved cell state classification and more specific mechanistic hypothesis generation than is possible with mono-omic analysis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31626775,

title = {Optical Pooled Screens in Human Cells},

author = {David Feldman and Avtar Singh and Jonathan L Schmid-Burgk and Rebecca J Carlson and Anja Mezger and Anthony J Garrity and Feng Zhang and Paul C Blainey},

doi = {10.1016/j.cell.2019.09.016},

issn = {1097-4172},

year  = {2019},

date = {2019-10-01},

journal = {Cell},

volume = {179},

number = {3},

pages = {787--799.e17},

abstract = {Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31558769,

title = {Multiplexed and high-throughput neuronal fluorescence imaging with diffusible probes},

author = {Syuan-Ming Guo and Remi Veneziano and Simon Gordonov and Li Li and Eric Danielson and Karen Perez de Arce and Demian Park and Anthony B Kulesa and Eike-Christian Wamhoff and Paul C Blainey and Edward S Boyden and Jeffrey R Cottrell and Mark Bathe},

doi = {10.1038/s41467-019-12372-6},

issn = {2041-1723},

year  = {2019},

date = {2019-09-01},

journal = {Nat Commun},

volume = {10},

number = {1},

pages = {4377},

abstract = {Synapses contain hundreds of distinct proteins whose heterogeneous expression levels are determinants of synaptic plasticity and signal transmission relevant to a range of diseases. Here, we use diffusible nucleic acid imaging probes to profile neuronal synapses using multiplexed confocal and super-resolution microscopy. Confocal imaging is performed using high-affinity locked nucleic acid imaging probes that stably yet reversibly bind to oligonucleotides conjugated to antibodies and peptides. Super-resolution PAINT imaging of the same targets is performed using low-affinity DNA imaging probes to resolve nanometer-scale synaptic protein organization across nine distinct protein targets. Our approach enables the quantitative analysis of thousands of synapses in neuronal culture to identify putative synaptic sub-types and co-localization patterns from one dozen proteins. Application to characterize synaptic reorganization following neuronal activity blockade reveals coordinated upregulation of the post-synaptic proteins PSD-95, SHANK3 and Homer-1b/c, as well as increased correlation between synaptic markers in the active and synaptic vesicle zones.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31186361,

title = {Massively parallel screening of synthetic microbial communities},

author = {Jared Kehe and Anthony Kulesa and Anthony Ortiz and Cheri M Ackerman and Sri Gowtham Thakku and Daniel Sellers and Seppe Kuehn and Jeff Gore and Jonathan Friedman and Paul C Blainey},

doi = {10.1073/pnas.1900102116},

issn = {1091-6490},

year  = {2019},

date = {2019-06-01},

journal = {Proc Natl Acad Sci U S A},

volume = {116},

number = {26},

pages = {12804--12809},

abstract = {Microbial communities have numerous potential applications in biotechnology, agriculture, and medicine. Nevertheless, the limited accuracy with which we can predict interspecies interactions and environmental dependencies hinders efforts to rationally engineer beneficial consortia. Empirical screening is a complementary approach wherein synthetic communities are combinatorially constructed and assayed in high throughput. However, assembling many combinations of microbes is logistically complex and difficult to achieve on a timescale commensurate with microbial growth. Here, we introduce the kChip, a droplets-based platform that performs rapid, massively parallel, bottom-up construction and screening of synthetic microbial communities. We first show that the kChip enables phenotypic characterization of microbes across environmental conditions. Next, in a screen of ∼100,000 multispecies communities comprising up to 19 soil isolates, we identified sets that promote the growth of the model plant symbiont  in a manner robust to carbon source variation and the presence of additional species. Broadly, kChip screening can identify multispecies consortia possessing any optically assayable function, including facilitation of biocontrol agents, suppression of pathogens, degradation of recalcitrant substrates, and robustness of these functions to perturbation, with many applications across basic and applied microbial ecology.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31009452,

title = {A Bayesian model of acquisition and clearance of bacterial colonization incorporating within-host variation},

author = {Marko Järvenpää and Mohamad R Abdul Sater and Georgia K Lagoudas and Paul C Blainey and Loren G Miller and James A McKinnell and Susan S Huang and Yonatan H Grad and Pekka Marttinen},

doi = {10.1371/journal.pcbi.1006534},

issn = {1553-7358},

year  = {2019},

date = {2019-04-01},

journal = {PLoS Comput Biol},

volume = {15},

number = {4},

pages = {e1006534},

abstract = {Bacterial populations that colonize a host can play important roles in host health, including serving as a reservoir that transmits to other hosts and from which invasive strains emerge, thus emphasizing the importance of understanding rates of acquisition and clearance of colonizing populations. Studies of colonization dynamics have been based on assessment of whether serial samples represent a single population or distinct colonization events. With the use of whole genome sequencing to determine genetic distance between isolates, a common solution to estimate acquisition and clearance rates has been to assume a fixed genetic distance threshold below which isolates are considered to represent the same strain. However, this approach is often inadequate to account for the diversity of the underlying within-host evolving population, the time intervals between consecutive measurements, and the uncertainty in the estimated acquisition and clearance rates. Here, we present a fully Bayesian model that provides probabilities of whether two strains should be considered the same, allowing us to determine bacterial clearance and acquisition from genomes sampled over time. Our method explicitly models the within-host variation using population genetic simulation, and the inference is done using a combination of Approximate Bayesian Computation (ABC) and Markov Chain Monte Carlo (MCMC). We validate the method with multiple carefully conducted simulations and demonstrate its use in practice by analyzing a collection of methicillin resistant Staphylococcus aureus (MRSA) isolates from a large recently completed longitudinal clinical study. An R-code implementation of the method is freely available at: https://github.com/mjarvenpaa/bacterial-colonization-model.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30814220,

title = {Hydrogenotrophic methanogenesis in archaeal phylum Verstraetearchaeota reveals the shared ancestry of all methanogens},

author = {Bojk A Berghuis and Feiqiao Brian Yu and Frederik Schulz and Paul C Blainey and Tanja Woyke and Stephen R Quake},

doi = {10.1073/pnas.1815631116},

issn = {1091-6490},

year  = {2019},

date = {2019-03-01},

journal = {Proc Natl Acad Sci U S A},

volume = {116},

number = {11},

pages = {5037--5044},

abstract = {Methanogenic archaea are major contributors to the global carbon cycle and were long thought to belong exclusively to the euryarchaeal phylum. Discovery of the methanogenesis gene cluster methyl-coenzyme M reductase (Mcr) in the Bathyarchaeota, and thereafter the Verstraetearchaeota, led to a paradigm shift, pushing back the evolutionary origin of methanogenesis to predate that of the Euryarchaeota. The methylotrophic methanogenesis found in the non-Euryarchaota distinguished itself from the predominantly hydrogenotrophic methanogens found in euryarchaeal orders as the former do not couple methanogenesis to carbon fixation through the reductive acetyl-CoA [Wood-Ljungdahl pathway (WLP)], which was interpreted as evidence for independent evolution of the two methanogenesis pathways. Here, we report the discovery of a complete and divergent hydrogenotrophic methanogenesis pathway in a thermophilic order of the Verstraetearchaeota, which we have named Candidatus Methanohydrogenales, as well as the presence of the WLP in the crenarchaeal order Desulfurococcales. Our findings support the ancient origin of hydrogenotrophic methanogenesis, suggest that methylotrophic methanogenesis might be a later adaptation of specific orders, and provide insight into how the transition from hydrogenotrophic to methylotrophic methanogenesis might have occurred.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30712876,

title = {Commensal Microbiota Promote Lung Cancer Development via γδ T Cells},

author = {Chengcheng Jin and Georgia K Lagoudas and Chen Zhao and Susan Bullman and Arjun Bhutkar and Bo Hu and Samuel Ameh and Demi Sandel and Xu Sue Liang and Sarah Mazzilli and Mark T Whary and Matthew Meyerson and Ronald Germain and Paul C Blainey and James G Fox and Tyler Jacks},

doi = {10.1016/j.cell.2018.12.040},

issn = {1097-4172},

year  = {2019},

date = {2019-02-01},

journal = {Cell},

volume = {176},

number = {5},

pages = {998--1013.e16},

abstract = {Lung cancer is closely associated with chronic inflammation, but the causes of inflammation and the specific immune mediators have not been fully elucidated. The lung is a mucosal tissue colonized by a diverse bacterial community, and pulmonary infections commonly present in lung cancer patients are linked to clinical outcomes. Here, we provide evidence that local microbiota provoke inflammation associated with lung adenocarcinoma by activating lung-resident γδ T cells. Germ-free or antibiotic-treated mice were significantly protected from lung cancer development induced by Kras mutation and p53 loss. Mechanistically, commensal bacteria stimulated Myd88-dependent IL-1β and IL-23 production from myeloid cells, inducing proliferation and activation of Vγ6Vδ1 γδ T cells that produced IL-17 and other effector molecules to promote inflammation and tumor cell proliferation. Our findings clearly link local microbiota-immune crosstalk to lung tumor development and thereby define key cellular and molecular mediators that may serve as effective targets in lung cancer intervention.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30746468,

title = {Multiplexed enrichment and genomic profiling of peripheral blood cells reveal subset-specific immune signatures},

author = {Miguel Reyes and Dwayne Vickers and Kianna Billman and Thomas Eisenhaure and Paul Hoover and Edward P Browne and Deepak A Rao and Nir Hacohen and Paul C Blainey},

doi = {10.1126/sciadv.aau9223},

issn = {2375-2548},

year  = {2019},

date = {2019-01-01},

journal = {Sci Adv},

volume = {5},

number = {1},

pages = {eaau9223},

abstract = {Specialized immune cell subsets are involved in autoimmune disease, cancer immunity, and infectious disease through a diverse range of functions mediated by overlapping pathways and signals. However, subset-specific responses may not be detectable in analyses of whole blood samples, and no efficient approach for profiling cell subsets at high throughput from small samples is available. We present a low-input microfluidic system for sorting immune cells into subsets and profiling their gene expression. We validate the system's technical performance against standard subset isolation and library construction protocols and demonstrate the importance of subset-specific profiling through in vitro stimulation experiments. We show the ability of this integrated platform to identify subset-specific disease signatures by profiling four immune cell subsets in blood from patients with systemic lupus erythematosus (SLE) and matched control subjects. The platform has the potential to make multiplexed subset-specific analysis routine in many research laboratories and clinical settings.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30256981,

title = {Targeting individual cells by barcode in pooled sequence libraries},

author = {Navpreet Ranu and Alexandra-Chloé Villani and Nir Hacohen and Paul C Blainey},

doi = {10.1093/nar/gky856},

issn = {1362-4962},

year  = {2019},

date = {2019-01-01},

journal = {Nucleic Acids Res},

volume = {47},

number = {1},

pages = {e4},

abstract = {Transcriptional profiling of thousands of single cells in parallel by RNA-seq is now routine. However, due to reliance on pooled library preparation, targeting analysis to particular cells of interest is difficult. Here, we present a multiplexed PCR method for targeted sequencing of select cells from pooled single-cell sequence libraries. We demonstrated this molecular enrichment method on multiple cell types within pooled single-cell RNA-seq libraries produced from primary human blood cells. We show how molecular enrichment can be combined with FACS to efficiently target ultra-rare cell types, such as the recently identified AXL+SIGLEC6+ dendritic cell (AS DC) subset, in order to reduce the required sequencing effort to profile single cells by 100-fold. Our results demonstrate that DNA barcodes identifying cells within pooled sequencing libraries can be used as targets to enrich for specific molecules of interest, for example reads from a set of target cells.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30459213,

title = {Quantification of somatic mutation flow across individual cell division events by lineage sequencing},

author = {Yehuda Brody and Robert J Kimmerling and Yosef E Maruvka and David Benjamin and Joshua J Elacqua and Nicholas J Haradhvala and Jaegil Kim and Kent W Mouw and Kristjana Frangaj and Amnon Koren and Gad Getz and Scott R Manalis and Paul C Blainey},

doi = {10.1101/gr.238543.118},

issn = {1549-5469},

year  = {2018},

date = {2018-12-01},

journal = {Genome Res},

volume = {28},

number = {12},

pages = {1901--1918},

abstract = {Mutation data reveal the dynamic equilibrium between DNA damage and repair processes in cells and are indispensable to the understanding of age-related diseases, tumor evolution, and the acquisition of drug resistance. However, available genome-wide methods have a limited ability to resolve rare somatic variants and the relationships between these variants. Here, we present lineage sequencing, a new genome sequencing approach that enables somatic event reconstruction by providing quality somatic mutation call sets with resolution as high as the single-cell level in subject lineages. Lineage sequencing entails sampling single cells from a population and sequencing subclonal sample sets derived from these cells such that knowledge of relationships among the cells can be used to jointly call variants across the sample set. This approach integrates data from multiple sequence libraries to support each variant and precisely assigns mutations to lineage segments. We applied lineage sequencing to a human colon cancer cell line with a DNA polymerase epsilon () proofreading deficiency (HT115) and a human retinal epithelial cell line immortalized by constitutive telomerase expression (RPE1). Cells were cultured under continuous observation to link observed single-cell phenotypes with single-cell mutation data. The high sensitivity, specificity, and resolution of the data provide a unique opportunity for quantitative analysis of variation in mutation rate, spectrum, and correlations among variants. Our data show that mutations arrive with nonuniform probability across sublineages and that DNA lesion dynamics may cause strong correlations between certain mutations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29899149,

title = {Combinatorial drug discovery in nanoliter droplets},

author = {Anthony Kulesa and Jared Kehe and Juan E Hurtado and Prianca Tawde and Paul C Blainey},

doi = {10.1073/pnas.1802233115},

issn = {1091-6490},

year  = {2018},

date = {2018-06-01},

journal = {Proc Natl Acad Sci U S A},

volume = {115},

number = {26},

pages = {6685--6690},

abstract = {Combinatorial drug treatment strategies perturb biological networks synergistically to achieve therapeutic effects and represent major opportunities to develop advanced treatments across a variety of human disease areas. However, the discovery of new combinatorial treatments is challenged by the sheer scale of combinatorial chemical space. Here, we report a high-throughput system for nanoliter-scale phenotypic screening that formulates a chemical library in nanoliter droplet emulsions and automates the construction of chemical combinations en masse using parallel droplet processing. We applied this system to predict synergy between more than 4,000 investigational and approved drugs and a panel of 10 antibiotics against , a model gram-negative pathogen. We found a range of drugs not previously indicated for infectious disease that synergize with antibiotics. Our validated hits include drugs that synergize with the antibiotics vancomycin, erythromycin, and novobiocin, which are used against gram-positive bacteria but are not effective by themselves to resolve gram-negative infections.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29361033,

title = {Single-particle trajectories reveal two-state diffusion-kinetics of hOGG1 proteins on DNA},

author = {Christian L Vestergaard and Paul C Blainey and Henrik Flyvbjerg},

doi = {10.1093/nar/gky004},

issn = {1362-4962},

year  = {2018},

date = {2018-03-01},

journal = {Nucleic Acids Res},

volume = {46},

number = {5},

pages = {2446--2458},

abstract = {We reanalyze trajectories of hOGG1 repair proteins diffusing on DNA. A previous analysis of these trajectories with the popular mean-squared-displacement approach revealed only simple diffusion. Here, a new optimal estimator of diffusion coefficients reveals two-state kinetics of the protein. A simple, solvable model, in which the protein randomly switches between a loosely bound, highly mobile state and a tightly bound, less mobile state is the simplest possible dynamic model consistent with the data. It yields accurate estimates of hOGG1's (i) diffusivity in each state, uncorrupted by experimental errors arising from shot noise, motion blur and thermal fluctuations of the DNA; (ii) rates of switching between states and (iii) rate of detachment from the DNA. The protein spends roughly equal time in each state. It detaches only from the loosely bound state, with a rate that depends on pH and the salt concentration in solution, while its rates for switching between states are insensitive to both. The diffusivity in the loosely bound state depends primarily on pH and is three to ten times higher than in the tightly bound state. We propose and discuss some new experiments that take full advantage of the new tools of analysis presented here.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29093105,

title = {Trends in Antibiotic Susceptibility in Staphylococcus aureus in Boston, Massachusetts, from 2000 to 2014},

author = {Sanjat Kanjilal and Mohamad R Abdul Sater and Maile Thayer and Georgia K Lagoudas and Soohong Kim and Paul C Blainey and Yonatan H Grad},

doi = {10.1128/JCM.01160-17},

issn = {1098-660X},

year  = {2018},

date = {2018-01-01},

journal = {J Clin Microbiol},

volume = {56},

number = {1},

abstract = {The rate of infection by methicillin-resistant  (MRSA) has declined over the past decade, but it is unclear whether this represents a decline in  infections overall. To evaluate the trends in the annual rates of infection by  subtypes and mean antibiotic resistance, we conducted a 15-year retrospective observational study at two tertiary care institutions in Boston, MA, of 31,753 adult inpatients with  isolated from clinical specimens. We inferred the gain and loss of methicillin resistance through genome sequencing of 180 isolates from 2016. The annual rates of infection by  declined from 2003 to 2014 by 4.2% (2.7% to 5.6%), attributable to an annual decline in MRSA of 10.9% (9.3% to 12.6%). Penicillin-susceptible  (PSSA) increased by 6.1% (4.2% to 8.1%) annually, and rates of methicillin-susceptible penicillin-resistant  (MSSA) did not change. Resistance in  decreased from 2000 to 2014 by 0.8 antibiotics (0.7 to 0.8). Within common MRSA clonal complexes, 3/14 MSSA and 2/21 PSSA isolates arose from the loss of resistance-conferring genes. Overall, in two tertiary care institutions in Boston, MA, a decline in  infections has been accompanied by a shift toward increased antibiotic susceptibility. The rise in PSSA makes penicillin an increasingly viable treatment option.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28994817,

title = {A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA},

author = {Kan Xiong and Paul C Blainey},

doi = {10.3791/55923},

issn = {1940-087X},

year  = {2017},

date = {2017-10-01},

journal = {J Vis Exp},

number = {128},

abstract = {We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-λ-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to >80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28678007,

title = {Microfluidic-based mini-metagenomics enables discovery of novel microbial lineages from complex environmental samples},

author = {Feiqiao Brian Yu and Paul C Blainey and Frederik Schulz and Tanja Woyke and Mark A Horowitz and Stephen R Quake},

doi = {10.7554/eLife.26580},

issn = {2050-084X},

year  = {2017},

date = {2017-07-01},

journal = {Elife},

volume = {6},

abstract = {Metagenomics and single-cell genomics have enabled genome discovery from unknown branches of life. However, extracting novel genomes from complex mixtures of metagenomic data can still be challenging and represents an ill-posed problem which is generally approached with ad hoc methods. Here we present a microfluidic-based mini-metagenomic method which offers a statistically rigorous approach to extract novel microbial genomes while preserving single-cell resolution. We used this approach to analyze two hot spring samples from Yellowstone National Park and extracted 29 new genomes, including three deeply branching lineages. The single-cell resolution enabled accurate quantification of genome function and abundance, down to 1% in relative abundance. Our analyses of genome level SNP distributions also revealed low to moderate environmental selection. The scale, resolution, and statistical power of microfluidic-based mini-metagenomics make it a powerful tool to dissect the genomic structure of microbial communities while effectively preserving the fundamental unit of biology, the single cell.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28329759,

title = {Corrigendum: Mobile genes in the human microbiome are structured from global to individual scales},

author = {I L Brito and S Yilmaz and K Huang and L Xu and S D Jupiter and A P Jenkins and W Naisilisili and M Tamminen and C S Smillie and J R Wortman and B W Birren and R J Xavier and P C Blainey and A K Singh and D Gevers and E J Alm},

doi = {10.1038/nature20774},

issn = {1476-4687},

year  = {2017},

date = {2017-04-01},

journal = {Nature},

volume = {544},

number = {7648},

pages = {124},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid28128213,

title = {High-throughput automated microfluidic sample preparation for accurate microbial genomics},

author = {Soohong Kim and Joachim De Jonghe and Anthony B Kulesa and David Feldman and Tommi Vatanen and Roby P Bhattacharyya and Brittany Berdy and James Gomez and Jill Nolan and Slava Epstein and Paul C Blainey},

doi = {10.1038/ncomms13919},

issn = {2041-1723},

year  = {2017},

date = {2017-01-01},

journal = {Nat Commun},

volume = {8},

pages = {13919},

abstract = {Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (∼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence ∼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27813331,

title = {Sliding on DNA: From Peptides to Small Molecules},

author = {Kan Xiong and Graham S Erwin and Aseem Z Ansari and Paul C Blainey},

doi = {10.1002/anie.201606768},

issn = {1521-3773},

year  = {2016},

date = {2016-11-01},

journal = {Angew Chem Int Ed Engl},

volume = {55},

number = {48},

pages = {15110--15114},

abstract = {Many DNA binding proteins utilize one-dimensional (1D) diffusion along DNA to accelerate their DNA target recognition. Although 1D diffusion of proteins along DNA has been studied for decades, a quantitative understanding is only beginning to emerge and few chemical tools are available to apply 1D diffusion as a design principle. Recently, we discovered that peptides can bind and slide along DNA-even transporting cargo along DNA. Such molecules are known as molecular sleds. Here, to advance our understanding of structure-function relationships governing sequence nonspecific DNA interaction of natural molecular sleds and to explore the potential for controlling sliding activity, we test the DNA binding and sliding activities of chemically modified peptides and analogs, and show that synthetic small molecules can slide on DNA. We found new ways to control molecular sled activity, novel small-molecule synthetic sleds, and molecular sled activity in N-methylpyrrole/N-methylimidazole polyamides that helps explain how these molecules locate rare target sites.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27479330,

title = {Virtual microfluidics for digital quantification and single-cell sequencing},

author = {Liyi Xu and Ilana L Brito and Eric J Alm and Paul C Blainey},

doi = {10.1038/nmeth.3955},

issn = {1548-7105},

year  = {2016},

date = {2016-09-01},

journal = {Nat Methods},

volume = {13},

number = {9},

pages = {759--762},

abstract = {We have developed hydrogel-based virtual microfluidics as a simple and robust alternative to complex engineered microfluidic systems for the compartmentalization of nucleic acid amplification reactions. We applied in-gel digital multiple displacement amplification (dMDA) to purified DNA templates, cultured bacterial cells and human microbiome samples in the virtual microfluidics system, and demonstrated whole-genome sequencing of single-cell MDA products with excellent coverage uniformity and markedly reduced chimerism compared with products of liquid MDA reactions.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27573117,

title = {Funding: Interdisciplinary challenges},

author = {Paul C Blainey},

doi = {10.1038/nmicrobiol.2016.118},

issn = {2058-5276},

year  = {2016},

date = {2016-07-01},

journal = {Nat Microbiol},

volume = {1},

number = {8},

pages = {16118},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid27409808,

title = {Mobile genes in the human microbiome are structured from global to individual scales},

author = {I L Brito and S Yilmaz and K Huang and L Xu and S D Jupiter and A P Jenkins and W Naisilisili and M Tamminen and C S Smillie and J R Wortman and B W Birren and R J Xavier and P C Blainey and A K Singh and D Gevers and E J Alm},

doi = {10.1038/nature18927},

issn = {1476-4687},

year  = {2016},

date = {2016-07-01},

journal = {Nature},

volume = {535},

number = {7612},

pages = {435--439},

abstract = {Recent work has underscored the importance of the microbiome in human health, and has largely attributed differences in phenotype to differences in the species present among individuals. However, mobile genes can confer profoundly different phenotypes on different strains of the same species. Little is known about the function and distribution of mobile genes in the human microbiome, and in particular whether the gene pool is globally homogenous or constrained by human population structure. Here, we investigate this question by comparing the mobile genes found in the microbiomes of 81 metropolitan North Americans with those of 172 agrarian Fiji islanders using a combination of single-cell genomics and metagenomics. We find large differences in mobile gene content between the Fijian and North American microbiomes, with functional variation that mirrors known dietary differences such as the excess of plant-based starch degradation genes found in Fijian individuals. Notably, we also observed differences between the mobile gene pools of neighbouring Fijian villages, even though microbiome composition across villages is similar. Finally, we observe high rates of recombination leading to individual-specific mobile elements, suggesting that the abundance of some genes may reflect environmental selection rather than dispersal limitation. Together, these data support the hypothesis that human activities and behaviours provide selective pressures that shape mobile gene pools, and that acquisition of mobile genes is important for colonizing specific human populations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26857546,

title = {Molecular sled sequences are common in mammalian proteins},

author = {Kan Xiong and Paul C Blainey},

doi = {10.1093/nar/gkw035},

issn = {1362-4962},

year  = {2016},

date = {2016-03-01},

journal = {Nucleic Acids Res},

volume = {44},

number = {5},

pages = {2266--2273},

abstract = {Recent work revealed a new class of molecular machines called molecular sleds, which are small basic molecules that bind and slide along DNA with the ability to carry cargo along DNA. Here, we performed biochemical and single-molecule flow stretching assays to investigate the basis of sliding activity in molecular sleds. In particular, we identified the functional core of pVIc, the first molecular sled characterized; peptide functional groups that control sliding activity; and propose a model for the sliding activity of molecular sleds. We also observed widespread DNA binding and sliding activity among basic polypeptide sequences that implicate mammalian nuclear localization sequences and many cell penetrating peptides as molecular sleds. These basic protein motifs exhibit weak but physiologically relevant sequence-nonspecific DNA affinity. Our findings indicate that many mammalian proteins contain molecular sled sequences and suggest the possibility that substantial undiscovered sliding activity exists among nuclear mammalian proteins.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26831565,

title = {Molecular sled is an eleven-amino acid vehicle facilitating biochemical interactions via sliding components along DNA},

author = {Walter F Mangel and William J McGrath and Kan Xiong and Vito Graziano and Paul C Blainey},

doi = {10.1038/ncomms10202},

issn = {2041-1723},

year  = {2016},

date = {2016-02-01},

journal = {Nat Commun},

volume = {7},

pages = {10202},

abstract = {Recently, we showed the adenovirus proteinase interacts productively with its protein substrates in vitro and in vivo in nascent virus particles via one-dimensional diffusion along the viral DNA. The mechanism by which this occurs has heretofore been unknown. We show sliding of these proteins along DNA occurs on a new vehicle in molecular biology, a 'molecular sled' named pVIc. This 11-amino acid viral peptide binds to DNA independent of sequence. pVIc slides on DNA, exhibiting the fastest one-dimensional diffusion constant, 26±1.8 × 10(6) (bp)(2) s(-1). pVIc is a 'molecular sled,' because it can slide heterologous cargos along DNA, for example, a streptavidin tetramer. Similar peptides, for example, from the C terminus of β-actin or NLSIII of the p53 protein, slide along DNA. Characteristics of the 'molecular sled' in its milieu (virion, nucleus) have implications for how proteins in the nucleus of cells interact and imply a new form of biochemistry, one-dimensional biochemistry.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26695070,

title = {Tools for the Microbiome: Nano and Beyond},

author = {Julie S Biteen and Paul C Blainey and Zoe G Cardon and Miyoung Chun and George M Church and Pieter C Dorrestein and Scott E Fraser and Jack A Gilbert and Janet K Jansson and Rob Knight and Jeff F Miller and Aydogan Ozcan and Kimberly A Prather and Stephen R Quake and Edward G Ruby and Pamela A Silver and Sharif Taha and Ger van den Engh and Paul S Weiss and Gerard C L Wong and Aaron T Wright and Thomas D Young},

doi = {10.1021/acsnano.5b07826},

issn = {1936-086X},

year  = {2016},

date = {2016-01-01},

journal = {ACS Nano},

volume = {10},

number = {1},

pages = {6--37},

abstract = {The microbiome presents great opportunities for understanding and improving the world around us and elucidating the interactions that compose it. The microbiome also poses tremendous challenges for mapping and manipulating the entangled networks of interactions among myriad diverse organisms. Here, we describe the opportunities, technical needs, and potential approaches to address these challenges, based on recent and upcoming advances in measurement and control at the nanoscale and beyond. These technical needs will provide the basis for advancing the largely descriptive studies of the microbiome to the theoretical and mechanistic understandings that will underpin the discipline of microbiome engineering. We anticipate that the new tools and methods developed will also be more broadly useful in environmental monitoring, medicine, forensics, and other areas.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid26732280,

title = {A microfluidic platform enabling single-cell RNA-seq of multigenerational lineages},

author = {Robert J Kimmerling and Gregory Lee Szeto and Jennifer W Li and Alex S Genshaft and Samuel W Kazer and Kristofor R Payer and Jacob de Riba Borrajo and Paul C Blainey and Darrell J Irvine and Alex K Shalek and Scott R Manalis},

doi = {10.1038/ncomms10220},

issn = {2041-1723},

year  = {2016},

date = {2016-01-01},

journal = {Nat Commun},

volume = {7},

pages = {10220},

abstract = {We introduce a microfluidic platform that enables off-chip single-cell RNA-seq after multi-generational lineage tracking under controlled culture conditions. We use this platform to generate whole-transcriptome profiles of primary, activated murine CD8+ T-cell and lymphocytic leukemia cell line lineages. Here we report that both cell types have greater intra- than inter-lineage transcriptional similarity. For CD8+ T-cells, genes with functional annotation relating to lymphocyte differentiation and function--including Granzyme B--are enriched among the genes that demonstrate greater intra-lineage expression level similarity. Analysis of gene expression covariance with matched measurements of time since division reveals cell type-specific transcriptional signatures that correspond with cell cycle progression. We believe that the ability to directly measure the effects of lineage and cell cycle-dependent transcriptional profiles of single cells will be broadly useful to fields where heterogeneous populations of cells display distinct clonal trajectories, including immunology, cancer, and developmental biology.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid25353527,

title = {Optimal estimation of diffusion coefficients from single-particle trajectories},

author = {Christian L Vestergaard and Paul C Blainey and Henrik Flyvbjerg},

doi = {10.1103/PhysRevE.89.022726},

issn = {1550-2376},

year  = {2014},

date = {2014-02-01},

journal = {Phys Rev E Stat Nonlin Soft Matter Phys},

volume = {89},

number = {2},

pages = {022726},

abstract = {How does one optimally determine the diffusion coefficient of a diffusing particle from a single-time-lapse recorded trajectory of the particle? We answer this question with an explicit, unbiased, and practically optimal covariance-based estimator (CVE). This estimator is regression-free and is far superior to commonly used methods based on measured mean squared displacements. In experimentally relevant parameter ranges, it also outperforms the analytically intractable and computationally more demanding maximum likelihood estimator (MLE). For the case of diffusion on a flexible and fluctuating substrate, the CVE is biased by substrate motion. However, given some long time series and a substrate under some tension, an extended MLE can separate particle diffusion on the substrate from substrate motion in the laboratory frame. This provides benchmarks that allow removal of bias caused by substrate fluctuations in CVE. The resulting unbiased CVE is optimal also for short time series on a fluctuating substrate. We have applied our estimators to human 8-oxoguanine DNA glycolase proteins diffusing on flow-stretched DNA, a fluctuating substrate, and found that diffusion coefficients are severely overestimated if substrate fluctuations are not accounted for.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid25309876,

title = {Laser microdissection of the alveolar duct enables single-cell genomic analysis},

author = {Robert D Bennett and Alexandra B Ysasi and Janeil M Belle and Willi L Wagner and Moritz A Konerding and Paul C Blainey and Saumyadipta Pyne and Steven J Mentzer},

doi = {10.3389/fonc.2014.00260},

issn = {2234-943X},

year  = {2014},

date = {2014-01-01},

journal = {Front Oncol},

volume = {4},

pages = {260},

abstract = {Complex tissues such as the lung are composed of structural hierarchies such as alveoli, alveolar ducts, and lobules. Some structural units, such as the alveolar duct, appear to participate in tissue repair as well as the development of bronchioalveolar carcinoma. Here, we demonstrate an approach to conduct laser microdissection of the lung alveolar duct for single-cell PCR analysis. Our approach involved three steps. (1) The initial preparation used mechanical sectioning of the lung tissue with sufficient thickness to encompass the structure of interest. In the case of the alveolar duct, the precision-cut lung slices were 200 μm thick; the slices were processed using near-physiologic conditions to preserve the state of viable cells. (2) The lung slices were examined by transmission light microscopy to target the alveolar duct. The air-filled lung was sufficiently accessible by light microscopy that counterstains or fluorescent labels were unnecessary to identify the alveolar duct. (3) The enzymatic and microfluidic isolation of single cells allowed for the harvest of as few as several thousand cells for PCR analysis. Microfluidics based arrays were used to measure the expression of selected marker genes in individual cells to characterize different cell populations. Preliminary work suggests the unique value of this approach to understand the intra- and intercellular interactions within the regenerating alveolar duct.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid24524132,

title = {Dissecting genomic diversity, one cell at a time},

author = {Paul C Blainey and Stephen R Quake},

doi = {10.1038/nmeth.2783},

issn = {1548-7105},

year  = {2014},

date = {2014-01-01},

journal = {Nat Methods},

volume = {11},

number = {1},

pages = {19--21},

abstract = {Emerging technologies are bringing single-cell genome sequencing into the mainstream; this field has already yielded insights into the genetic architecture and variability between cells that highlight the dynamic nature of the genome.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid23298390,

title = {The future is now: single-cell genomics of bacteria and archaea},

author = {Paul C Blainey},

doi = {10.1111/1574-6976.12015},

issn = {1574-6976},

year  = {2013},

date = {2013-05-01},

journal = {FEMS Microbiol Rev},

volume = {37},

number = {3},

pages = {407--427},

abstract = {Interest in the expanding catalog of uncultivated microorganisms, increasing recognition of heterogeneity among seemingly similar cells, and technological advances in whole-genome amplification and single-cell manipulation are driving considerable progress in single-cell genomics. Here, the spectrum of applications for single-cell genomics, key advances in the development of the field, and emerging methodology for single-cell genome sequencing are reviewed by example with attention to the diversity of approaches and their unique characteristics. Experimental strategies transcending specific methodologies are identified and organized as a road map for future studies in single-cell genomics of environmental microorganisms. Over the next decade, increasingly powerful tools for single-cell genome sequencing and analysis will play key roles in accessing the genomes of uncultivated organisms, determining the basis of microbial community functions, and fundamental aspects of microbial population biology.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}