Advancements in Next-Generation Sequencing Levy, Shawn E; Myers, Richard M
Annual review of genomics and human genetics,
08/2016, Letnik:
17, Številka:
1
Journal Article
Recenzirano
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The term next-generation sequencing is almost a decade old, but it remains the colloquial way to describe highly parallel or high-output sequencing methods that produce data at or beyond the genome ...scale. Since the introduction of these technologies, the number of applications and methods that leverage the power of genome-scale sequencing has increased at an exponential pace. This review highlights recent concepts, technologies, and methods from next-generation sequencing to illustrate the breadth and depth of the applications and research areas that are driving progress in genomics.
Lineage mapping has identified both proliferative and quiescent intestinal stem cells, but the molecular circuitry controlling stem cell quiescence is incompletely understood. By lineage mapping, we ...show Lrig1, a pan-ErbB inhibitor, marks predominately noncycling, long-lived stem cells that are located at the crypt base and that, upon injury, proliferate and divide to replenish damaged crypts. Transcriptome profiling of Lrig1+ colonic stem cells differs markedly from the profiling of highly proliferative, Lgr5+ colonic stem cells; genes upregulated in the Lrig1+ population include those involved in cell cycle repression and response to oxidative damage. Loss of Apc in Lrig1+ cells leads to intestinal adenomas, and genetic ablation of Lrig1 results in heightened ErbB1-3 expression and duodenal adenomas. These results shed light on the relationship between proliferative and quiescent intestinal stem cells and support a model in which intestinal stem cell quiescence is maintained by calibrated ErbB signaling with loss of a negative regulator predisposing to neoplasia.
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► Lrig1, an ErbB negative regulator, marks quiescent intestinal stem cells ► Lrig1 and Lgr5 transcriptome profiles are distinct ► Loss of APC in Lrig1+ cells results in intestinal adenomas ► Functionally null Lrig1 mice develop duodenal adenomas
Loss of calibrated EGFR/ErbB signaling in quiescent intestinal stem cells has neoplastic consequences, generating advanced colon adenomas. Although Lrig1+ cells are distinct from proliferating Lgr5+ cells, they are located at the base of the crypt.
Bacteria behave differently in space, as indicated by reports of reduced lag phase, higher final cell counts, enhanced biofilm formation, increased virulence, and reduced susceptibility to ...antibiotics. These phenomena are theorized, at least in part, to result from reduced mass transport in the local extracellular environment, where movement of molecules consumed and excreted by the cell is limited to diffusion in the absence of gravity-dependent convection. However, to date neither empirical nor computational approaches have been able to provide sufficient evidence to confirm this explanation. Molecular genetic analysis findings, conducted as part of a recent spaceflight investigation, support the proposed model. This investigation indicated an overexpression of genes associated with starvation, the search for alternative energy sources, increased metabolism, enhanced acetate production, and other systematic responses to acidity-all of which can be associated with reduced extracellular mass transport.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Pancreatic islet endocrine cell and endothelial cell (EC) interactions mediated by vascular endothelial growth factor-A (VEGF-A) signaling are important for islet differentiation and the formation of ...highly vascularized islets. To dissect how VEGF-A signaling modulates intra-islet vasculature, islet microenvironment, and β cell mass, we transiently increased VEGF-A production by β cells. VEGF-A induction dramatically increased the number of intra-islet ECs but led to β cell loss. After withdrawal of the VEGF-A stimulus, β cell mass, function, and islet structure normalized as a result of a robust, but transient, burst in proliferation of pre-existing β cells. Bone marrow-derived macrophages (MΦs) recruited to the site of β cell injury were crucial for the β cell proliferation, which was independent of pancreatic location and circulating factors such as glucose. Identification of the signals responsible for the proliferation of adult, terminally differentiated β cells will improve strategies aimed at β cell regeneration and expansion.
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•Increasing β cell VEGF-A activates islet endothelial cells but leads to β cell loss•VEGF-A-modulated islet microenvironment plays an integral part in β cell regeneration•Islet VEGF-A induction leads to the recruitment of bone marrow-derived macrophages•Macrophages directly or with islet endothelial cells induce β cell proliferation
Brissova et al. show that transiently increasing β cell VEGF-A activates islet endothelial cells but, surprisingly, leads to β cell loss. After removal of the VEGF-A stimulus, β cells proliferated, leading to islet regeneration. The regeneration required bone marrow-derived macrophages and was independent of the pancreatic location or circulating factors.
Next-Generation Sequencing Strategies Levy, Shawn E; Boone, Braden E
Cold Spring Harbor perspectives in medicine,
07/2019, Letnik:
9, Številka:
7
Journal Article
Recenzirano
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More than a decade ago, the term "next-generation" sequencing was coined to describe what was, at the time, revolutionary new methods to sequence RNA and DNA at a faster pace and cheaper cost than ...could be performed by standard bench-top protocols. Since then, the field of DNA sequencing has evolved at a rapid pace, with new breakthroughs allowing capacity to exponentially increase and cost to dramatically decrease. As genome-scale sequencing has become routine, a paradigm shift is occurring in genomics, which uses the power of high-throughput, rapid sequencing power with large-scale studies. These new approaches to genetic discovery will provide direct impact to fields such as personalized medicine, evolution, and biodiversity. This work reviews recent technology advances and methods in next-generation sequencing and highlights current large-scale sequencing efforts driving the evolution of the genomics space.
Systems biology is an approach to comprehensively study complex interactions within a biological system. Most published systems vaccinology studies have utilized whole blood or peripheral blood ...mononuclear cells (PBMC) to monitor the immune response after vaccination. Because human blood is comprised of multiple hematopoietic cell types, the potential for masking responses of under-represented cell populations is increased when analyzing whole blood or PBMC. To investigate the contribution of individual cell types to the immune response after vaccination, we established a rapid and efficient method to purify human T and B cells, natural killer (NK) cells, myeloid dendritic cells (mDC), monocytes, and neutrophils from fresh venous blood. Purified cells were fractionated and processed in a single day. RNA-Seq and quantitative shotgun proteomics were performed to determine expression profiles for each cell type prior to and after inactivated seasonal influenza vaccination. Our results show that transcriptomic and proteomic profiles generated from purified immune cells differ significantly from PBMC. Differential expression analysis for each immune cell type also shows unique transcriptomic and proteomic expression profiles as well as changing biological networks at early time points after vaccination. This cell type-specific information provides a more comprehensive approach to monitor vaccine responses.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier ...to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole-exome sequencing and transcriptome sequencing in a cohort of 1,001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.
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•Exome sequencing in 1,001 DLBCL patients comprehensively identifies 150 driver genes•Unbiased CRISPR screen in DLBCL cell lines identifies essential oncogenes•Integrative analysis connects genomics, CRISPR hits, and clinical outcome•A genomic risk model of survival outperforms existing risk-assessment methods
An integrative analysis in 1,001 newly diagnosed DLBCL patients identifies 150 genetic drivers with functional characterization using an unbiased CRISPR screen in DLBCL cell lines and connects with clinical outcome.
Many patients with type 1 diabetes (T1D) have residual β cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D ...diagnosis. The features of these residual β cells and α cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant β cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D α cells was markedly reduced, and these cells had alterations in transcription factors constituting α and β cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of α-to-β cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.
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•T1D β cells appear to maintain several aspects of regulated insulin secretion•T1D α cells have impaired glucagon secretion and altered gene expression•Unlike in rodents, α-to-β cell conversion in human T1D is a very rare event•T1D α cell identity factors improve in a non-autoimmune, normoglycemic environment
Brissova et al. find that β cells in the type 1 diabetic (T1D) pancreas maintain several functional and molecular features, but α cells have impaired glucagon secretion and an altered gene expression profile. These findings provide insight into the mechanism of α cell dysfunction in T1D.
Sequencing errors are key confounding factors for detecting low-frequency genetic variants that are important for cancer molecular diagnosis, treatment, and surveillance using deep next-generation ...sequencing (NGS). However, there is a lack of comprehensive understanding of errors introduced at various steps of a conventional NGS workflow, such as sample handling, library preparation, PCR enrichment, and sequencing. In this study, we use current NGS technology to systematically investigate these questions.
By evaluating read-specific error distributions, we discover that the substitution error rate can be computationally suppressed to 10
to 10
, which is 10- to 100-fold lower than generally considered achievable (10
) in the current literature. We then quantify substitution errors attributable to sample handling, library preparation, enrichment PCR, and sequencing by using multiple deep sequencing datasets. We find that error rates differ by nucleotide substitution types, ranging from 10
for A>C/T>G, C>A/G>T, and C>G/G>C changes to 10
for A>G/T>C changes. Furthermore, C>T/G>A errors exhibit strong sequence context dependency, sample-specific effects dominate elevated C>A/G>T errors, and target-enrichment PCR led to ~ 6-fold increase of overall error rate. We also find that more than 70% of hotspot variants can be detected at 0.1 ~ 0.01% frequency with the current NGS technology by applying in silico error suppression.
We present the first comprehensive analysis of sequencing error sources in conventional NGS workflows. The error profiles revealed by our study highlight new directions for further improving NGS analysis accuracy both experimentally and computationally, ultimately enhancing the precision of deep sequencing.
To evaluate evidence for de novo etiologies in schizophrenia, we sequenced at high coverage the exomes of families recruited from two populations with distinct demographic structures and history. We ...sequenced a total of 795 exomes from 231 parent-proband trios enriched for sporadic schizophrenia cases, as well as 34 unaffected trios. We observed in cases an excess of de novo nonsynonymous single-nucleotide variants as well as a higher prevalence of gene-disruptive de novo mutations relative to controls. We found four genes (LAMA2, DPYD, TRRAP and VPS39) affected by recurrent de novo events within or across the two populations, which is unlikely to have occurred by chance. We show that de novo mutations affect genes with diverse functions and developmental profiles, but we also find a substantial contribution of mutations in genes with higher expression in early fetal life. Our results help define the genomic and neural architecture of schizophrenia.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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