Publications

Bartsch, Michael B.; Krishnakumar, Raga K.; Sinha, Anupama S.; Bird, Sara W.; Schoeniger, Joseph S.; Patel, Kamlesh P.

Abstract not provided.

Nucleic Acids Research

Schoeniger, Joseph S.; Hudson, Corey H.; Bent, Zachary W.; Sinha, Anupama S.; Williams, Kelly P.

Virulence genes on mobile DNAs such as genomic islands (GIs) and plasmids promote bacterial pathogen emergence. Excision is an early step in GI mobilization, producing a circular GI and a deletion site in the chromosome; circular forms are also known for some bacterial insertion sequences (ISs). The recombinant sequence at the junctions of such circles and deletions can be detected sensitively in high-throughput sequencing data, using new computational methods that enable empirical discovery of mobile DNAs. For the rich mobilome of a hospital Klebsiella pneumoniae strain, circularization junctions (CJs) were detected for six GIs and seven IS types. Our methods revealed differential biology of multiple mobile DNAs, imprecision of integrases and transposases, and differential activity among identical IS copies for IS26, ISKpn18 and ISKpn21. Using the resistance of circular dsDNA molecules to exonuclease, internally calibrated with the native plasmids, showed that not all molecules bearing GI CJs were circular. Transpositions were also detected, revealing replicon preference (ISKpn18 prefers a conjugative IncA/C2 plasmid), local action (IS26), regional preferences, selection (against capsule synthesis) and IS polarity inversion. Efficient discovery and global characterization of numerous mobile elements per experiment improves accounting for the new gene combinations that arise in emerging pathogens.

Ray, Debjit R.; Schoeniger, Joseph S.; Williams, Kelly P.; Hamblin, Rachelle H.; Sinha, Anupama S.; Hudson, Corey H.; Branda, Steven B.

Abstract not provided.

Genome Announcements

D'haeseleer, Patrik; Johnson, Shannon L.; Davenport, Karen W.; Chain, Patrick S.; Schoeniger, Joseph S.; Ray, Debjit R.; Sinha, Anupama S.; Williams, Kelly P.; Peña, José; Branda, Steven B.; El-Etr, Sahar

Here, we present the draft genome sequence of Burkholderia pseudomallei PHLS 6, a virulent clinical strain isolated from a melioidosis patient in Bangladesh in 1960. The draft genome consists of 39 contigs and is 7,322,181 bp long.

Jebrail, Mais J.; Sinha, Anupama S.; Renzi, Ronald F.; Van De Vreugde, James L.; Gondhalekar, Carmen G.; Schoeniger, Joseph S.; Branda, Steven B.

Abstract not provided.

Infection and Immunity

Poorey, Kunal N.; Sinha, Anupama S.; Curtis, Deanna J.; Williams, Kelly P.; Branda, Steven B.; Meagher, Robert M.

Yersinia enterocolitica is typically considered an extracellular pathogen; however, during the course of an infection, a significant number of bacteria are stably maintained within host cell vacuoles. Little is known about this population and the role it plays during an infection. To address this question and to elucidate the spatially and temporally dynamic gene expression patterns of Y. enterocoliticabiovar 1B through the course of an in vitro infection, transcriptome sequencing and differential gene expression analysis of bacteria infecting murine macrophage cells were performed under four distinct conditions. Bacteria were first grown in a nutrient-rich medium at 26°C to establish a baseline of gene expression that is unrelated to infection. The transcriptomes of these bacteria were then compared to bacteria grown in a conditioned cell culture medium at 37°C to identify genes that were differentially expressed in response to the increased temperature and medium but not in response to host cells. Infections were then performed, and the transcriptomes of bacteria found on the extracellular surface and intracellular compartments were analyzed individually. The upregulated genes revealed potential roles for a variety of systems in promoting intracellular virulence, including the Ysa type III secretion system, the Yts2 type II secretion system, and the Tad pilus. It was further determined that mutants of each of these systems had decreased virulence while infecting macrophages. Overall, these results reveal the complete set of genes expressed by Y. enterocolitica in response to infection and provide the groundwork for future virulence studies.

PLoS ONE

Bartsch, Michael B.; Edwards, Harrison S.; Lee, Daniel; Moseley, Caroline E.; Tew, Karen E.; Renzi, Ronald F.; Van De Vreugde, James L.; Kim, Hanyoup; Knight, Daniel L.; Sinha, Anupama S.; Branda, Steven B.; Patel, Kamlesh P.

Advances in molecular biology, microfluidics, and laboratory automation continue to expand the accessibility and applicability of these methods beyond the confines of conventional, centralized laboratory facilities and into point of use roles in clinical, military, forensic, and field-deployed applications. As a result, there is a growing need to adapt the unit operations of molecular biology (e.g., aliquoting, centrifuging, mixing, and thermal cycling) to compact, portable, low-power, and automation-ready formats. Here we present one such adaptation, the rotary zone thermal cycler (RZTC), a novel wheel-based device capable of cycling up to four different fixed-temperature blocks into contact with a stationary 4-microliter capillarybound sample to realize 1-3 second transitions with steady state heater power of less than 10 W. We demonstrate the utility of the RZTC for DNA amplification as part of a highly integrated rotary zone PCR (rzPCR) system that uses low-volume valves and syringe-based fluid handling to automate sample loading and unloading, thermal cycling, and between-run cleaning functionalities in a compact, modular form factor. In addition to characterizing the performance of the RZTC and the efficacy of different online cleaning protocols, we present preliminary results for rapid single-plex PCR, multiplex short tandem repeat (STR) amplification, and second strand cDNA synthesis.

Lab on a Chip

Jebrail, Mais J.; Renzi, Ronald F.; Sinha, Anupama S.; Van De Vreugde, James L.; Gondhalekar, Carmen; Ambriz, Cesar; Meagher, Robert M.; Branda, Steven B.

Digital microfluidics (DMF) is a powerful technique for sample preparation and analysis for a broad range of biological and chemical applications. In many cases, it is desirable to carry out DMF on an open surface, such that the matrix surrounding the droplets is ambient air. However, the utility of the air-matrix DMF format has been severely limited by problems with droplet evaporation, especially when the droplet-based biochemical reactions require high temperatures for long periods of time. We present a simple solution for managing evaporation in air-matrix DMF: just-in-time replenishment of the reaction volume using droplets of solvent. We demonstrate that this solution enables DMF-mediated execution of several different biochemical reactions (RNA fragmentation, first-strand cDNA synthesis, and PCR) over a range of temperatures (4-95°C) and incubation times (up to 1 h or more) without use of oil, humidifying chambers, or off-chip heating modules. Reaction volumes and temperatures were maintained roughly constant over the course of each experiment, such that the reaction kinetics and products generated by the air-matrix DMF device were comparable to those of conventional benchscale reactions. This simple yet effective solution for evaporation management is an important advance in developing air-matrix DMF for a wide variety of new, high-impact applications, particularly in the biomedical sciences.

Branda, Steven B.; Jebrail, Mais J.; Sinha, Anupama S.; Renzi, Ronald F.; Bartsch, Michael B.; Van De Vreugde, James L.; Gondhalekar, Carmen G.; Amriz, Cesar A.; Schoeniger, Joseph S.; Meagher, Robert M.; Patel, Kamlesh P.

Abstract not provided.

Poorey, Kunal N.; Sinha, Anupama S.; Curtis, Deanna J.; Meagher, Robert M.

Abstract not provided.

Sinha, Anupama S.; Curtis, Deanna J.; Branda, Steven B.; Meagher, Robert M.

Abstract not provided.

Jebrail, Mais J.; Sinha, Anupama S.; Renzi, Ronald F.; Patel, Kamlesh P.; Branda, Steven B.

Abstract not provided.

Sinha, Anupama S.; Curtis, Deanna J.; Branda, Steven B.

Abstract not provided.

PLoS ONE

Kim, Hanyoup; Jebrail, Mais J.; Sinha, Anupama S.; Bent, Zachary B.; Solberg, Owen D.; Williams, Kelly P.; Langevin, Stanley A.; Renzi, Ronald F.; Van De Vreugde, James L.; Meagher, Robert M.; Schoeniger, Joseph S.; Lane, Todd L.; Branda, Steven B.; Bartsch, Michael B.; Patel, Kamlesh D.

Next-generation sequencing (NGS) is emerging as a powerful tool for elucidating genetic information for a wide range of applications. Unfortunately, the surging popularity of NGS has not yet been accompanied by an improvement in automated techniques for preparing formatted sequencing libraries. To address this challenge, we have developed a prototype microfluidic system for preparing sequencer-ready DNA libraries for analysis by Illumina sequencing. Our system combines droplet-based digital microfluidic (DMF) sample handling with peripheral modules to create a fully-integrated, sample-in library-out platform. In this report, we use our automated system to prepare NGS libraries from samples of human and bacterial genomic DNA. E. coli libraries prepared on-device from 5 ng of total DNA yielded excellent sequence coverage over the entire bacterial genome, with >99% alignment to the reference genome, even genome coverage, and good quality scores. Furthermore, we produced a de novo assembly on a previously unsequenced multi-drug resistant Klebsiella pneumoniae strain BAA-2146 (KpnNDM). The new method described here is fast, robust, scalable, and automated. Our device for library preparation will assist in the integration of NGS technology into a wide variety of laboratories, including small research laboratories and clinical laboratories. © 2013 Kim et al.

RNA Biology

Langevin, Stanley A.; Bent, Zachary B.; Solberg, Owen D.; Curtis, Deanna J.; Lane, Pamela L.; Williams, Kelly P.; Schoeniger, Joseph S.; Lane, Todd L.; Sinha, Anupama S.

Abstract not provided.

Journal of Visualized Experiments

Sinha, Anupama S.; Jebrail, Mais J.; Patel, Kamlesh P.

Abstract not provided.

Sinha, Anupama S.; Bent, Zachary B.

Biological weapons of mass destruction and emerging infectious diseases represent a serious and growing threat to our national security. Effective response to a bioattack or disease outbreak critically depends upon efficient and reliable distinguishing between infected vs healthy individuals, to enable rational use of scarce, invasive, and/or costly countermeasures (diagnostics, therapies, quarantine). Screening based on direct detection of the causative pathogen can be problematic, because culture- and probe-based assays are confounded by unanticipated pathogens (e.g., deeply diverged, engineered), and readily-accessible specimens (e.g., blood) often contain little or no pathogen, particularly at pre-symptomatic stages of disease. Thus, in addition to the pathogen itself, one would like to detect infection-specific host response signatures in the specimen, preferably ones comprised of nucleic acids (NA), which can be recovered and amplified from tiny specimens (e.g., fingerstick draws). Proof-of-concept studies have not been definitive, however, largely due to use of sub-optimal sample preparation and detection technologies. For purposes of pathogen detection, Sandia has developed novel molecular biology methods that enable selective isolation of NA unique to, or shared between, complex samples, followed by identification and quantitation via Second Generation Sequencing (SGS). The central hypothesis of the current study is that variations on this approach will support efficient identification and verification of NA-based host response signatures of infectious disease. To test this hypothesis, we re-engineered Sandia's sophisticated sample preparation pipelines, and developed new SGS data analysis tools and strategies, in order to pioneer use of SGS for identification of host NA correlating with infection. Proof-of-concept studies were carried out using specimens drawn from pathogen-infected non-human primates (NHP). This work provides a strong foundation for large-scale, highly-efficient efforts to identify and verify infection-specific host NA signatures in human populations.

Jebrail, Mais J.; Sinha, Anupama S.; Kim, Hanyoup; Branda, Steven B.

Abstract not provided.