A new strategy for genome assembly using short sequence reads and reduced representation libraries

We have developed a novel approach for using massively parallel short-read sequencing to generate fast and inexpensive de novo genomic assemblies comparable to those generated by capillary-based methods. The ultrashort (<100 base) sequences generated by this technology pose specific biological and computational challenges for de novo assembly of large genomes. To account for this, we devised a method for experimentally partitioning the genome using reduced representation (RR) libraries prior to assembly. We use two restriction enzymes independently to create a series of overlapping fragment libraries, each containing a tractable subset of the genome. Together, these libraries allow us to reassemble the entire genome without the need of a reference sequence. As proof of concept, we applied this approach to sequence and assembled the majority of the 125-Mb Drosophila melanogaster genome. We subsequently demonstrate the accuracy of our assembly method with meaningful comparisons against the current available D. melanogaster reference genome (dm3). The ease of assembly and accuracy for comparative genomics suggest that our approach will scale to future mammalian genome-sequencing efforts, saving both time and money without sacrificing quality.Genomes are the fundamental unit by which a species can be defined and form the foundation for deciphering how an organism develops, lives, dies, and is affected by disease. In addition, comparisons of genomes from related species have become a powerful method for finding functional sequences (Eddy 2005; Xie et al. 2005, 2007; Pennacchio et al. 2006; The ENCODE Project Consortium 2007). However, the high cost and effort needed to produce draft genomes with capillary-based sequencing technologies have limited genome-based biological exploration and evolutionary sequence comparisons to dozens of species (Margulies et al. 2007; Stark et al. 2007). This limitation is particularly true for mammalian-sized genomes, which are gigabases in size.With the advent of massively parallel short-read sequencing technologies (Bentley et al. 2008), the cost of sequencing DNA has been reduced by orders of magnitude, now making it possible to sequence hundreds or thousands of genomes. However, the reduced length of the sequence reads, compared with capillary-based approaches, poses new challenges in genome assembly. Here, we sought to address those experimental and bioinformatics hurdles by combining classical biochemical methodologies with new algorithms specifically tailored to handle massive quantities of short-read sequences.To date, the whole-genome shotgun (WGS) approach using massively parallel short-read sequencing has shown significant promise in silico (Butler et al. 2008) and has been applied to de novo sequencing and assembly of small genomes that do not contain an overabundance of low-complexity repetitive sequence (Hernandez et al. 2008). This presents a challenge when scaled to larger more complex genomes, where the information contained in a single short read cannot unambiguously place that read in the genome. Additionally, de novo assembly from WGS short-read sequencing currently requires large computational resources, on the order of hundreds of gigabytes of RAM, when scaled to larger genomes. As a compromise, current mammalian genomic analyses utilizing short-read sequencing technology either use alignments of individual reads against a reference genome (Ley et al. 2008; Wang et al. 2008) or require elaborate parallelization schemes across a large compute farm (Simpson et al. 2009) for assembly. Regardless of computational improvements, effectively handling repetitive sequences in a whole-genome assembly still remains a challenge.Our goal was to establish wet-lab and bioinformatics methods to rapidly sequence and assemble mammalian-sized genomes in a de novo fashion. Importantly, we wanted an approach that (1) did not rely on existing reference assemblies, (2) could be accomplished using commodity computational hardware, and (3) would yield functional assemblies useful for comparative sequence analyses at a fraction of the time and cost of existing capillary-based methods. To accomplish this, we propose a generic genome partitioning approach to solve both the biological and computational challenges of short-read assembly.Traditionally, partitioning of genomic libraries was accomplished through the use of clonal libraries of bacterial artificial chromosomes (BACs) (or yeast artificial chromosomes). This method accurately partitions genomes into more manageable subregions for sequencing and assembly. However, the high financial cost and overhead associated with creating and maintaining these libraries make this method unattractive for scaling to hundreds of genomes. In addition, a single BAC clone, which contains ∼200 kb of sequence, is not large enough to leverage the amount of sequence obtained from a single lane of Illumina data (currently ∼2.5 Gb of sequence), requiring the need for various pooling or indexing strategies (Meyer et al. 2007). Furthermore, virtually all BAC libraries exhibit some degree of variable cloning bias, with some regions overrepresented and others not at all. In silico studies have investigated the cost-saving potential of using a randomized BAC clone library with short-read sequencing (Sundquist et al. 2007); however, even with this shortcut, the clonal library concept does not lend itself to fast and cheap whole-genome partitioning.We propose a novel partitioning approach using restriction enzymes to create a series of reduced representation (RR) libraries by size fractionation. This method was originally described for single nucleotide polymorphism (SNP) discovery using Sanger-based sequencing methods on AB377 machines (Altshuler et al. 2000) and was subsequently used with massively parallel short-read sequencing (Van Tassell et al. 2008). Importantly, this method allows for the selection of a smaller reproducible subset of the genome for assembly. We extended this concept to create a series of distinct RR libraries consisting of similarly sized restriction fragments. Individually, these libraries represent a tractable subset of the genome for sequencing and assembly; when taken together, they represent virtually the entire genome. Using two separate restriction enzymes generates overlapping libraries, which allow for assembly of the genome without using a reference sequence.As proof of concept, we present here a de novo Drosophila melanogaster genomic assembly, equivalent in utility to a comparative grade assembly (Blakesley et al. 2004). Two enzymes were used to create a total of eight libraries. Short reads (∼36 bp) from each library were sequenced on the Illumina Genome Analyzer and assembled using the short-read assembler Velvet (Zerbino and Birney 2008). Contigs assembled from each library were merged into a single nonoverlapping meta assembly using the lightweight assembly program Minimus (Sommer et al. 2007). Furthermore, we sequenced genomic paired-end libraries with short and long inserts to order and orient the contigs into larger genomic scaffolds. When compared with a whole-genome shotgun assembly of the same data, we produce a higher quality assembly more rapidly by reducing the biological complexity and computational cost to assemble each library. Finally, we compare this assembly to the dm3 fly reference to highlight the accuracy of our assembly and utility for comparative sequence analyses. Our results demonstrate that this method is a rapid and cost-effective means to generate high-quality de novo assemblies of large genomes.     

Adapting to the changing paradigm of management of colon injuries

BACKGROUND: Based on the evolution that the management of colonic trauma has undergone since the early 1990s, we hypothesized that the use of diversion has decreased at our institution over the last decade. METHODS: A retrospective review was performed of all patients who presented to our trauma center with colon injuries between 1995 and 2006. RESULTS: A total of 81 patients were analyzed. Twenty-five patients (31%) were treated with diversion and 56 patients (69%) underwent primary repair or resection with anastomosis. The rate of diversion in the first half of the study period as well as the second half of the study period was 31%. There was no difference in the complication rates. CONCLUSIONS: The usage of diversion remains higher than current literature would indicate. As a result, we are implementing a program that will actively encourage our trauma surgeons to improve the quality of patient care by incorporating evidence-based medicine into clinical practice.     

Life-history traits drive the evolutionary rates of mammalian coding and noncoding genomic elements

A comprehensive phylogenetic framework is indispensable for investigating the evolution of genomic features in mammals as a whole, and particularly in humans. Using the ENCODE sequence data, we estimated mammalian neutral evolutionary rates and selective pressures acting on conserved coding and noncoding elements. We show that neutral evolutionary rates can be explained by the generation time (GT) hypothesis. Accordingly, primates (especially humans), having longer GTs than other mammals, display slower rates of neutral evolution. The evolution of constrained elements, particularly of nonsynonymous sites, is in agreement with the expectations of the nearly neutral theory of molecular evolution. We show that rates of nonsynonymous substitutions (dN) depend on the population size of a species. The results are robust to the exclusion of hypermutable CpG prone sites. The average rate of evolution in conserved noncoding sequences (CNCs) is 1.7 times higher than in nonsynonymous sites. Despite this, CNCs evolve at similar or even lower rates than nonsynonymous sites in the majority of basal branches of the eutherian tree. This observation could be the result of an overall gradual or, alternatively, lineage-specific relaxation of CNCs. The latter hypothesis was supported by the finding that 3 of the 20 longest CNCs displayed significant relaxation of individual branches. This observation may explain why the evolution of CNCs fits the expectations of the nearly neutral theory less well than the evolution of nonsynonymous sites.     

Residual functional connectivity in the split-brain revealed with resting-state functional MRI

Split-brain patients present a unique opportunity to address controversies regarding subcortical contributions to interhemispheric coordination. We characterized residual functional connectivity in a complete commissurotomy patient by examining patterns of low-frequency BOLD functional MRI signal. Using independent components analysis and region-of-interest-based functional connectivity analyses, we demonstrate bilateral resting state networks in a patient lacking all major cerebral commissures. Compared with a control group, the patient's interhemispheric correlation scores fell within the normal range for two out of three regions examined. Thus, we provide evidence for bilateral resting state networks in a patient with complete commissurotomy. Such continued interhemispheric interaction suggests that, at least in part, cortical networks in the brain can be coordinated by subcortical mechanisms.     

Noninvasive Doppler ultrasonography for assessing cardiac function: can it replace the Swan-Ganz catheter?

Background

Cardiac function, including cardiac index (CI), traditionally has been measured by a pulmonary artery catheter (PAC). A noninvasive alternative for measuring cardiac function would offer obvious advantages.

Methods

A prospective study of trauma and nontrauma patients was performed in a surgical intensive care unit over a 3-month period. CI was determined using both a standard PAC and a continuous-wave Doppler ultrasound (UTS). The study had 2 phases: phase I was nonblinded and phase II was blinded; the correlation between UTS- and PAC-derived CI was assessed.

Results

A total of 120 paired measurements of CI were observed in 31 patients. The UTS-derived CI measurements showed agreement with PAC measurements in both phase I and phase II of the study with a bias of .06 L/min/m² ± .4 L/min/m². Paired measurements correlated well in both phase I (r = .97, R2 = .95, P < .0001) and phase II (r = .93, R2 = .86, P < .0001) of the study.

Conclusions

Doppler UTS correlates well with PAC measurements of CI. This noninvasive modality is an accurate and safe alternative to PAC.     

Time-dependent association between metabolic syndrome and risk of CKD in Korean men without hypertension or diabetes

BACKGROUND: The time-dependent association between metabolic syndrome and risk of chronic kidney disease (CKD) is not clear. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: The study cohort was composed of 10,685 healthy men without CKD, hypertension, or diabetes who participated in a health-checkup program at a large work site. PREDICTOR: Metabolic syndrome. OUTCOMES & MEASUREMENTS: CKD was defined as an estimated glomerular filtration rate (GFR) less than 60 mL/min/1.73 m(2). A standard Cox proportional hazards model and a time-dependent Cox model were used to calculate adjusted hazard ratios (HRs) in the CKD model. RESULTS: During 40,616.8 person-years of follow-up, 291 incident cases of CKD developed; 787 patients (7.4%) had metabolic syndrome at baseline and 1,444 (14.4%) developed incident metabolic syndrome during follow-up. After adjustment for age, baseline GFR, gamma-glutamyltransferase level, and uric acid level, metabolic syndrome at baseline was associated with a significantly increased risk of CKD (HR, 1.99; 95% confidence interval, 1.46 to 2.73). Metabolic syndrome over time as a time-dependent variable also predicted the development of CKD (HR, 1.83; [corrected] 95% confidence interval, 1.34 to 2.49) [corrected] The relationship between metabolic syndrome and incident CKD remained significant, even after further adjustment for the homeostasis model assessment of insulin resistance, high-sensitivity C-reactive protein level, current smoking, alcohol consumption, or regular exercise. In addition, there were graded relationships between number of metabolic syndrome traits or quintile of homeostasis model assessment of insulin resistance over time as a time-dependent variable and risk of CKD. Both increased triglyceride and low high-density lipoprotein cholesterol levels among metabolic syndrome traits were associated with significantly increased risk of CKD. These results were effectively unchanged, even after additional adjustment for incident hypertension and incident diabetes. LIMITATIONS: Estimated GFR was used instead of a directly measured GFR to define CKD. CONCLUSION: Metabolic syndrome is an independent risk factor for the development of CKD in Korean men without hypertension or diabetes, even with changes in status of metabolic syndrome over time.     

The evolution of the COVID-19 pandemic in paediatric patients with sickle cell disease: From Alpha to Omicron

We compare the impact of SARS-CoV-2 variants on healthcare utilization and clinical presentation in paediatric patients with sickle cell disease (SCD). One hundred and ninety-one unique patients with SCD and positive SARS-CoV-2 polymerase chain reactions were identified between March 2020 and January 2022. Hospitalizations, which accounted for 42% (N = 81) of cases, were highest during the Delta dominant era (48%) and lowest during Omicron (36%) (p = 0.285). The most common SCD-related complication was vaso-occlusive pain (37%, N = 71), which accounted for 51% of all hospital admissions (N = 41), and acute chest was highest in the Alpha variant era (N = 15). Overall, COVID-19 remained mild in clinical severity within most paediatric SCD patients.