Capillary array electrophoresis (CAE) microplates that can analyze 96 samples in less than 8 min have been produced by bonding 10-cm-diameter micromachined glass wafers to form a glass sandwich structure. The microplate has 96 sample wells and 48 separation channels with an injection unit that permits the serial analysis of two different samples on each capillary. An elastomer sheet with an 8 by 12 array of holes is placed on top of the glass sandwich structure to define the sample wells. Samples are addressed with an electrode array that makes up the third layer of the assembly. Detection of all lanes with high temporal resolution was achieved by using a laser-excited confocal f luorescence scanner. To demonstrate the functionality of these microplates, electrophoretic separation and f luorescence detection of a restriction fragment marker for the diagnosis of hereditary hemochromatosis were performed. CAE microplates will facilitate all types of high-throughput genetic analysis because their high assay speed provides a throughput that is 50 to 100 times greater than that of conventional slab gels.
Bacterial whole genome sequencing holds promise as a disruptive technology in clinical microbiology, but it has not yet been applied systematically or comprehensively within a clinical context. Here, over the course of one year, we performed prospective collection and whole genome sequencing of nearly all bacterial isolates obtained from a tertiary care hospital’s intensive care units (ICUs). This unbiased collection of 1,229 bacterial genomes from 391 patients enables detailed exploration of several features of clinical pathogens. A sizable fraction of isolates identified as clinically relevant corresponded to previously undescribed species: 12% of isolates assigned a species-level classification by conventional methods actually qualified as distinct, novel genomospecies on the basis of genomic similarity. Pan-genome analysis of the most frequently encountered pathogens in the collection revealed substantial variation in pan-genome size (1,420 to 20,432 genes) and the rate of gene discovery (1 to 152 genes per isolate sequenced). Surprisingly, although potential nosocomial transmission of actively surveilled pathogens was rare, 8.7% of isolates belonged to genomically related clonal lineages that were present among multiple patients, usually with overlapping hospital admissions, and were associated with clinically significant infection in 62% of patients from which they were recovered. Multi-patient clonal lineages were particularly evident in the neonatal care unit, where seven separate Staphylococcus epidermidis clonal lineages were identified, including one lineage associated with bacteremia in 5/9 neonates. Our study highlights key differences in the information made available by conventional microbiological practices versus whole genome sequencing, and motivates the further integration of microbial genome sequencing into routine clinical care.
Large-scale bacterial genome sequencing efforts to date have provided limited information on the most prevalent category of disease: sporadically acquired infections caused by common pathogenic bacteria. Here, we performed whole-genome sequencing and de novo assembly of 312 blood-or urine-derived isolates of extraintestinal pathogenic (ExPEC) Escherichia coli, a common agent of sepsis and community-acquired urinary tract infections, obtained during the course of routine clinical care at a single institution. We find that ExPEC E. coli are highly genomically heterogeneous, consistent with pangenome analyses encompassing the larger species. Investigation of differential virulence factor content and antibiotic resistance phenotypes reveals markedly different profiles among lineages and among strains infecting different body sites. We use high-resolution molecular epidemiology to explore the dynamics of infections at the level of individual patients, including identification of possible person-to-person transmission. Notably, a limited number of discrete lineages caused the majority of bloodstream infections, including one subclone (ST131-H30) responsible for 28% of bacteremic E. coli infections over a 3-yr period. We additionally use a microbial genome-wide-association study (GWAS) approach to identify individual genes responsible for antibiotic resistance, successfully recovering known genes but notably not identifying any novel factors. We anticipate that in the near future, whole-genome sequencing of microorganisms associated with clinical disease will become routine. Our study reveals what kind of information can be obtained from sequencing clinical isolates on a large scale, even well-characterized organisms such as E. coli, and provides insight into how this information might be utilized in a healthcare setting.
Background Cystic fibrosis (CF) is a genetic disease which carries high morbidity and mortality from lung-function decline. Monitoring disease progression and treatment response in young patients is desirable, but serial imaging via CT is often considered prohibitive, and detailed functional information cannot be obtained using conventional imaging techniques. Hyperpolarized 129Xe magnetic resonance imaging (MRI) can depict and quantify regional ventilation, but has not been investigated in pediatrics. We hypothesized that 129Xe MRI is feasible and would demonstrate ventilation defects in mild CF lung disease with greater sensitivity than FEV1. Methods 11 healthy controls (age 6–16 years) and 11 patients with mild CF (age 8–16 years, Forced Expiratory Volume (FEV1) percent predicted >70%) were recruited for this study. Nine CF patients had an FEV1>85%. Each subject was imaged via hyperpolarized 129Xe MRI, and the ventilation defect percentage (VDP) was measured. Recent FEV1 and VDP were compared between the groups. Results FEV1 for controls was 100.3%±8.5% (mean ± sd) and for CF patients was 97.9%±16.0% (p=0.67). VDP was 6.4%±2.8% for controls and 18.3%±8.6% for CF (p<0.001). When considering the 9 CF patients with normal FEV1 (>85%), the mean FEV1 was 103.1%±12.3% (p=0.57 compared to controls) and VDP was 15.4% ± 6.3% (p=0.002). Conclusions Hyperpolarized 129Xe MRI demonstrated ventilation defects in CF patients with normal FEV1 and more effectively discriminated CF from controls than FEV1. Thus 129Xe may be a useful outcome measure to detect mild CF lung disease and to investigate regional lung function in pediatric lung diseases and to follow disease progression.
The PRDM9 gene encodes a protein with a highly variable tandem-repeat zinc finger (ZF) DNA-binding domain that plays a key role in determining sequence-specific hotspots of meiotic recombination genome-wide. Here we survey the diversity of the PRDM9 ZF domain by sequencing this region in 64 primates from 18 species, revealing 68 unique alleles across all groups. We report ubiquitous positive selection at nucleotide positions corresponding to DNA contact residues and the expansion of ZFs within clades, which confirms the rapid evolution of the ZF domain throughout the primate lineage. Alignment of Neanderthal and Denisovan sequences suggests that PRDM9 in archaic hominins was closely related to present-day human alleles that are rare and specific to African populations. In the context of its role in reproduction, our results are consistent with variation in PRDM9 contributing to speciation events in primates.
Upper airway obstruction during sleep is characterized by inspiratory airflow limitation and reductions in maximal inspiratory airflow (VImax). To determine how mechanical factors modulate VImax, we analyzed pressure-flow relationships obtained in the isolated upper airway of paralyzed cats. VImax and its determinants, the pharyngeal critical pressure (Pcrit) and the nasal resistance (Rn) upstream to the flow-limiting site (FLS), were measured as caudal tracheal displacement, neck position, and airway length were systematically varied. As the proximal tracheal stump was displaced caudally, graded increases in VImax from 145.3 +/- 90.8 (SD) to 285.9 +/- 117.5 ml/s (P < 0.02) and decreases in Pcrit from -3.0 +/- 3.0 to -9.5 +/- 3.4 cmH2O (P < 0.002) were seen without any significant change in Rn. During neck flexion, significant decreases in VImax from 192.1 +/- 68.5 to 87.2 +/- 48.4 ml/s (P = 0.001), increases in Pcrit from -5.3 +/- 2.03 cmH2O to -1.6 +/- 1.4 cmH2O (P < 0.001), and decreases in Rn from 29.7 +/- 12.2 cmH2O.l-1.s to 16.2 +/- 8.9 cmH2O.l-1.s (P < 0.001) were noted compared with the neutral or extended neck position. Relative to the neutral airway length, upper airway length was found to decrease by 1.15 +/- 0.14 cm during neck flexion and to lengthen by 0.45 +/- 0.12 cm during neck extension. When tracheal displacement and neck position were altered, VImax and Rn correlated directly and Pcrit correlated inversely with airway length (P < 0.001). We conclude that alterations in airflow mechanics with caudal tracheal displacement and changes in neck positions are primarily due to alterations in airway length.
We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated fourcolor confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect. We present experimental evidence consistent with the predictions of the theory.
Ultrashort echo-time MRI detected structural lung disease in very young patients with CF and provided imaging data that correlated well with CT. By quantifying early CF lung disease without using ionizing radiation, ultrashort echo-time MRI appears well suited for pediatric patients requiring longitudinal imaging for clinical care or research studies. Clinical Trial registered with www.clinicaltrials.gov (NCT01832519).
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