The advantages of SMRT sequencing Roberts, Richard J; Carneiro, Mauricio O; Schatz, Michael C
GenomeBiology.com,
01/2013, Letnik:
14, Številka:
7
Journal Article
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Of the current next-generation sequencing technologies, SMRT sequencing is sometimes overlooked. However, attributes such as long reads, modified base detection and high accuracy make SMRT a useful ...technology and an ideal approach to the complete sequencing of small genomes.
Pacific Biosciences technology provides a fundamentally new data type that provides the potential to overcome some limitations of current next generation sequencing platforms by providing ...significantly longer reads, single molecule sequencing, low composition bias and an error profile that is orthogonal to other platforms. With these potential advantages in mind, we here evaluate the utility of the Pacific Biosciences RS platform for human medical amplicon resequencing projects.
We evaluated the Pacific Biosciences technology for SNP discovery in medical resequencing projects using the Genome Analysis Toolkit, observing high sensitivity and specificity for calling differences in amplicons containing known true or false SNPs. We assessed data quality: most errors were indels (~14%) with few apparent miscalls (~1%). In this work, we define a custom data processing pipeline for Pacific Biosciences data for human data analysis.
Critically, the error properties were largely free of the context-specific effects that affect other sequencing technologies. These data show excellent utility for follow-up validation and extension studies in human data and medical genetics projects, but can be extended to other organisms with a reference genome.
advantages of SMRT sequencing Roberts, Richard J; Carneiro, Mauricio O; Schatz, Michael C
Genome biology,
07/2013, Letnik:
14, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Of the current next-generation sequencing technologies, SMRT sequencing is sometimes overlooked. However, attributes such as long reads, modified base detection and high accuracy make SMRT a useful ...technology and an ideal approach to the complete sequencing of small genomes.
This unit describes how to use BWA and the Genome Analysis Toolkit (GATK) to map genome sequencing data to a reference and produce high-quality variant calls that can be used in downstream analyses. ...The complete workflow includes the core NGS data processing steps that are necessary to make the raw data suitable for analysis by the GATK, as well as the key methods involved in variant discovery using the GATK.
As massively parallel sequencing is increasingly being used for clinical decision making, it has become critical to understand parameters that affect sequencing quality and to establish methods for ...measuring and reporting clinical sequencing standards. In this report, we propose a definition for reduced coverage regions and describe a set of standards for variant calling in clinical sequencing applications.
To enable sequencing centers to assess the regions of poor sequencing quality in their own data, we optimized and used a tool (ExCID) to identify reduced coverage loci within genes or regions of particular interest. We used this framework to examine sequencing data from 500 patients generated in 10 projects at sequencing centers in the National Human Genome Research Institute/National Cancer Institute Clinical Sequencing Exploratory Research Consortium.
This approach identified reduced coverage regions in clinically relevant genes, including known clinically relevant loci that were uniquely missed at individual centers, in multiple centers, and in all centers.
This report provides a process road map for clinical sequencing centers looking to perform similar analyses on their data.
At a time when genomes are being sequenced by the hundreds, much attention has shifted from identifying genes and phenotypes to understanding the networks of interactions among genes. We developed a ...gene network developmental model expanding on previous models of transcription regulatory networks. In our model, each network is described by a matrix representing the interactions between transcription factors, and a vector of continuous values representing the transcription factor expression in an individual.
In this work we used the gene network model to look at the impact of mating as well as insertions and deletions of genes in the evolution of complexity of these networks. We found that the natural process of diploid mating increases the likelihood of maintaining complexity, especially in higher order networks (more than 10 genes). We also show that gene insertion is a very efficient way to add more genes to a network as it provides a much higher chance of developmental stability.
The continuous model affords a more complete view of the evolution of interacting genes. The notion of a continuous output vector also incorporates the reality of gene networks and graded concentrations of gene products.
The recent development of a semiconductor-based, non-optical DNA sequencing technology promises scalable, low-cost and rapid sequence data production. The technology has previously been applied ...mainly to genomic sequencing and targeted re-sequencing. Here we demonstrate the utility of Ion Torrent semiconductor-based sequencing for sensitive, efficient and rapid chromatin immunoprecipitation followed by sequencing (ChIP-seq) through the application of sample preparation methods that are optimized for ChIP-seq on the Ion Torrent platform. We leverage this method for epigenetic profiling of tumour tissues.
By characterizing the geographic and functional spectrum of human genetic variation, the 1000 Genomes Project aims to build a resource to help to understand the genetic contribution to disease. Here ...we describe the genomes of 1,092 individuals from 14 populations, constructed using a combination of low-coverage whole-genome and exome sequencing. By developing methods to integrate information across several algorithms and diverse data sources, we provide a validated haplotype map of 38 million single nucleotide polymorphisms, 1.4 million short insertions and deletions, and more than 14,000 larger deletions. We show that individuals from different populations carry different profiles of rare and common variants, and that low-frequency variants show substantial geographic differentiation, which is further increased by the action of purifying selection. We show that evolutionary conservation and coding consequence are key determinants of the strength of purifying selection, that rare-variant load varies substantially across biological pathways, and that each individual contains hundreds of rare non-coding variants at conserved sites, such as motif-disrupting changes in transcription-factor-binding sites. This resource, which captures up to 98% of accessible single nucleotide polymorphisms at a frequency of 1% in related populations, enables analysis of common and low-frequency variants in individuals from diverse, including admixed, populations.
Medulloblastomas are the most common malignant brain tumours in children. Identifying and understanding the genetic events that drive these tumours is critical for the development of more effective ...diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma on the basis of transcriptional and copy number profiles. Here we use whole-exome hybrid capture and deep sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas have low mutation rates consistent with other paediatric tumours, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were newly identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR and LDB1. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant, but not wild-type, β-catenin. Together, our study reveals the alteration of WNT, hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and within specific subtypes of this disease, and nominates the RNA helicase DDX3X as a component of pathogenic β-catenin signalling in medulloblastoma.
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