Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a ...model system for addressing important challenges facing high-throughput whole genome amplification (WGA) and complete genome sequencing of individual cells.
We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA), and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs.
The methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.
RNA-seq is an effective method for studying the transcriptome, but it can be difficult to apply to scarce or degraded RNA from fixed clinical samples, rare cell populations or cadavers. Recent ...studies have proposed several methods for RNA-seq of low-quality and/or low-quantity samples, but the relative merits of these methods have not been systematically analyzed. Here we compare five such methods using metrics relevant to transcriptome annotation, transcript discovery and gene expression. Using a single human RNA sample, we constructed and sequenced ten libraries with these methods and compared them against two control libraries. We found that the RNase H method performed best for chemically fragmented, low-quality RNA, and we confirmed this through analysis of actual degraded samples. RNase H can even effectively replace oligo(dT)-based methods for standard RNA-seq. SMART and NuGEN had distinct strengths for measuring low-quantity RNA. Our analysis allows biologists to select the most suitable methods and provides a benchmark for future method development.
Massively parallel DNA sequencing technologies are revolutionizing genomics by making it possible to generate billions of relatively short (approximately 100-base) sequence reads at very low cost. ...Whereas such data can be readily used for a wide range of biomedical applications, it has proven difficult to use them to generate high-quality de novo genome assemblies of large, repeat-rich vertebrate genomes. To date, the genome assemblies generated from such data have fallen far short of those obtained with the older (but much more expensive) capillary-based sequencing approach. Here, we report the development of an algorithm for genome assembly, ALLPATHS-LG, and its application to massively parallel DNA sequence data from the human and mouse genomes, generated on the Illumina platform. The resulting draft genome assemblies have good accuracy, short-range contiguity, long-range connectivity, and coverage of the genome. In particular, the base accuracy is high (≥99.95%) and the scaffold sizes (N50 size = 11.5 Mb for human and 7.2 Mb for mouse) approach those obtained with capillary-based sequencing. The combination of improved sequencing technology and improved computational methods should now make it possible to increase dramatically the de novo sequencing of large genomes. The ALLPATHS-LG program is available at http://www.broadinstitute.org/science/programs/genome-biology/crd.
Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid ...Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol.
Exceptionally accurate genome reference sequences have proven to be of great value to microbial researchers. Thus, to date, about 1800 bacterial genome assemblies have been "finished" at great ...expense with the aid of manual laboratory and computational processes that typically iterate over a period of months or even years. By applying a new laboratory design and new assembly algorithm to 16 samples, we demonstrate that assemblies exceeding finished quality can be obtained from whole-genome shotgun data and automated computation. Cost and time requirements are thus dramatically reduced.
To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The ...slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences.
The 2013–2015 West African epidemic of Ebola virus disease (EVD) reminds us of how little is known about biosafety level 4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever ...with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples. We show that whereas the 2013–2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections mainly result from reservoir-to-human infections. We elucidated the spread of LASV across West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translational efficiency. By investigating intrahost evolution, we found that mutations accumulate in epitopes of viral surface proteins, suggesting selection for immune escape. This catalog will serve as a foundation for the development of vaccines and diagnostics.
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•Lassa virus is a life-threatening pathogen that is endemic in West Africa•Lassa virus has diverse and ancient origins in Nigeria•Viral strains from Nigeria and Sierra Leone differ in their translation efficiency•The virus evolves within hosts to evade immune-determined selection pressures
Sequencing analysis of ∼200 Lassa virus genomes reveals its ancient origins and distinct evolutionary features compared to the Ebola virus.
Comparative Functional Genomics of the Fission Yeasts Rhind, Nicholas; Chen, Zehua; Yassour, Moran ...
Science (American Association for the Advancement of Science),
05/2011, Letnik:
332, Številka:
6032
Journal Article
Recenzirano
Odprti dostop
The fission yeast clade—comprising Schizosaccharomyces pombe, S. octosporus, S. cryophilus, and S. japonicus—occupies the basal branch of Ascomycete fungi and is an important model of eukaryote ...biology. A comparative annotation of these genomes identified a near extinction of transposons and the associated innovation of transposon-free centromeres. Expression analysis established that meiotic genes are subject to antisense transcription during vegetative growth, which suggests a mechanism for their tight regulation. In addition, trans-acting regulators control new genes within the context of expanded functional modules for meiosis and stress response. Differences in gene content and regulation also explain why, unlike the budding yeast of Saccharomycotina, fission yeasts cannot use ethanol as a primary carbon source. These analyses elucidate the genome structure and gene regulation of fission yeast and provide tools for investigation across the Schizosaccharomyces clade.
Objective
The purpose of this study was to estimate the time to recovery of command‐following and associations between hypoxemia with time to recovery of command‐following.
Methods
In this ...multicenter, retrospective, cohort study during the initial surge of the United Statesʼ pandemic (March–July 2020) we estimate the time from intubation to recovery of command‐following, using Kaplan Meier cumulative‐incidence curves and Cox proportional hazard models. Patients were included if they were admitted to 1 of 3 hospitals because of severe coronavirus disease 2019 (COVID‐19), required endotracheal intubation for at least 7 days, and experienced impairment of consciousness (Glasgow Coma Scale motor score <6).
Results
Five hundred seventy‐one patients of the 795 patients recovered command‐following. The median time to recovery of command‐following was 30 days (95% confidence interval CI = 27–32 days). Median time to recovery of command‐following increased by 16 days for patients with at least one episode of an arterial partial pressure of oxygen (PaO2) value ≤55 mmHg (p < 0.001), and 25% recovered ≥10 days after cessation of mechanical ventilation. The time to recovery of command‐following was associated with hypoxemia (PaO2 ≤55 mmHg hazard ratio HR = 0.56, 95% CI = 0.46–0.68; PaO2 ≤70 HR = 0.88, 95% CI = 0.85–0.91), and each additional day of hypoxemia decreased the likelihood of recovery, accounting for confounders including sedation. These findings were confirmed among patients without any imagining evidence of structural brain injury (n = 199), and in a non‐overlapping second surge cohort (N = 427, October 2020 to April 2021).
Interpretation
Survivors of severe COVID‐19 commonly recover consciousness weeks after cessation of mechanical ventilation. Long recovery periods are associated with more severe hypoxemia. This relationship is not explained by sedation or brain injury identified on clinical imaging and should inform decisions about life‐sustaining therapies. ANN NEUROL 2022;91:740–755
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