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Sequenciamento direto dos genes SIX3, SHH, TGIF1, ZIC2 e array-CGH no estudo de pacientes com holoprosencefalia; Direct sequencing of the genes SIX3, SHH, TGIF1, ZIC2 and array-CGH on the study of patients with holoprosencephaly

Rocha, Ana Laís Bignotto da
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 21/08/2013 PT
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Objetivos: Analisar por meio da técnica de sequenciamento direto a presença de alterações moleculares nos genes SHH, SIX3, ZIC2 e TGIF1 em indivíduos com diagnóstico clínico de HPE. Analisar por meio da técnica de CGH-array a presença de alterações moleculares em indivíduos com diagnóstico clínico de HPE previamente submetidos à análise por sequenciamento direto. Local: Laboratório de Genética e Citogenética Humana HRAC/USP, Bauru-SP. Casuística e metodologia: Foram selecionados 50 indivíduos, de ambos os sexos com idades entre 03 meses a 50 anos com diagnóstico clínico para HPE. Todos foram analisados por meio da técnica de sequenciamento direto para os genes SHH e TGIF1 completamente e para os genes ZIC2 e SIX3 parcialmente. Dentre os indivíduos que não apresentaram alterações na técnica de sequenciamento oito indivíduos com fenótipo mais grave foram selecionados para a análise por CGH-array. Resultados e discussão: Foram analisados 50 indivíduos por meio da técnica de sequenciamento direto dos gene SHH e TGIF1, foram encontradas duas variantes patogênicas na análise do gene SHH, no caso 1 a variante p.24G>P foi identificada, e no caso 2 foi identificada a variante c.1031del C. No gene TGIF1 foram encontrados cinco polimorfismos já descritos na literatura. Foi identificada uma nova variante silenciosa no éxon 1 do gene ZIC2 p.Q46Q (c. 431 G>A) e um polimorfismo já descrito na literatura em dois indivíduos no gene SIX3. A análise por CGH-array revelou a presença de uma microdeleção no caso 37...

Comparison of HPV genotyping by type-specific PCR and sequencing

Carvalho,Nara de Oliveira; del Castillo,Dora Méndez; Perone,Carlos; Januário,José Nélio; Melo,Victor Hugo de; Brasileiro Filho,Geraldo
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/02/2010 EN
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Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide and there is a strong link between certain high-risk viral types and cervical carcinogenesis. Although there are several typing methods, it is still unclear which test is the best. This study compared the effectiveness of type-specific PCR (TS-PCR) and sequencing, with a focus on their clinical application. A total of 260 cervical samples from HPV-positive patients were tested for types 6, 11, 16, 18, 31, 33 and 35 using TS-PCR and sequencing. The genotype was identified in 36% of cases by TS-PCR and in 75% by sequencing. Sequencing was four times more likely to identify the viral type in positive samples than TS-PCR (p = 0.00). Despite being more effective for virus genotyping, sequencing was unable to identify viral types in multiple infections. Combining both techniques resulted in highly sensitive detection (87% of cases), showing that they are complementary methods. HPV genotyping is an important step in HPV management, helping to identify patients with a higher risk of developing cervical cancer and contributing to the development of type-specific vaccines.

P49-S Recent Improvements in SOLiD Sequencing Chemistry

Nutter, B.
Fonte: The Association of Biomolecular Resource Facilities Publicador: The Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /02/2007 EN
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The sequencing by oligonucleotide ligation and detection (SOLiD) system being commercialized by Applied Biosystems generates sequences of randomly generated fragment or mate-paired DNA molecules utilizing a novel ligation-based chemistry and fluorescent probe pools. Consistent improvements have been made in total sequence throughput per run by enhanced read length and base calls, higher bead densities per array, and increased frequency of assignable reads. In combination, these improvements have allowed progressively deeper sequencing coverage of more complex model organisms. To date, we have sequenced, to varying depths of coverage, the genomes of several bacterial genomes. In addition to whole genome sequencing, we have developed a workflow to allow targeted resequencing and are working with collaborators to develop tag-based sequencing applications. Improvements to the SOLiD sequencing technology and the impact on the increased sequencing throughput as they apply to a number of different applications will be presented.

Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses

Fullwood, Melissa J.; Wei, Chia-Lin; Liu, Edison T.; Ruan, Yijun
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /04/2009 EN
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Comprehensive understanding of functional elements in the human genome will require thorough interrogation and comparison of individual human genomes and genomic structures. Such an endeavor will require improvements in the throughputs and costs of DNA sequencing. Next-generation sequencing platforms have impressively low costs and high throughputs but are limited by short read lengths. An immediate and widely recognized solution to this critical limitation is the paired-end tag (PET) sequencing for various applications, collectively called the PET sequencing strategy, in which short and paired tags are extracted from the ends of long DNA fragments for ultra-high-throughput sequencing. The PET sequences can be accurately mapped to the reference genome, thus demarcating the genomic boundaries of PET-represented DNA fragments and revealing the identities of the target DNA elements. PET protocols have been developed for the analyses of transcriptomes, transcription factor binding sites, epigenetic sites such as histone modification sites, and genome structures. The exclusive advantage of the PET technology is its ability to uncover linkages between the two ends of DNA fragments. Using this unique feature, unconventional fusion transcripts...

Exome Sequencing-Driven Discovery of Coding Polymorphisms Associated with Common Metabolic Phenotypes

Albrechtsen, A.; Grarup, N.; Sparsø, T.; Korneliussen, T.; Nie, C.; Skotte, L.; Ladenvall, C.; Cauchi, S.; Stančáková, A.; Andersen, G.; Astrup, A.; Banasik, K.; Bolund, L.; Charpentier, G.; Doney, A. S. F.; Dorkhan, M.; Forsen, T.; Frayling, T. M.; G
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
EN_US
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Aims/hypothesis: Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. Methods: The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 (kg/m^2) and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p < 0.05) with case–control status, from four selected annotation categories or from loci reported to associate with metabolic traits. These variants were genotyped in 15,989 Danes to search for association with 12 metabolic phenotypes (stage 2). In stage 3, polymorphisms showing potential associations were genotyped in a further 63,896 Europeans. Results: Exome sequencing identified 70,182 polymorphisms with MAF >1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, (p = 8.5 × 10^{−14}))...

Deep Sequencing Analysis of Phage Libraries using Illumina Platform

Matochko, Wadim L.; Chu, Kiki; Jin, Bingjie; Lee, Sam Whan; Whitesides, George McClelland; Derda, Ratmir
Fonte: Elsevier BV Publicador: Elsevier BV
Tipo: Artigo de Revista Científica
EN_US
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This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacteriophage (phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 107 reads in a single run. We applied Illumina sequencing to analyze phage libraries. Using PCR, we isolated the variable regions from M13KE phage vectors from a phage display library. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5′-terminal region. We used this approach to examine how diversity of peptides in phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2 × 107 reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6 bp)...

Comparison of Illumina and 454 Deep Sequencing in Participants Failing Raltegravir-Based Antiretroviral Therapy

Li, Jonathan Z.; Chapman, Brad; Charlebois, Patrick; Hofmann, Oliver; Weiner, Brian; Porter, Alyssa J.; Samuel, Reshmi; Vardhanabhuti, Saran; Zheng, Lu; Eron, Joseph; Taiwo, Babafemi; Zody, Michael C.; Henn, Matthew R.; Kuritzkes, Daniel R.; Hide, Winston
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN_US
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Background: The impact of raltegravir-resistant HIV-1 minority variants (MVs) on raltegravir treatment failure is unknown. Illumina sequencing offers greater throughput than 454, but sequence analysis tools for viral sequencing are needed. We evaluated Illumina and 454 for the detection of HIV-1 raltegravir-resistant MVs. Methods: A5262 was a single-arm study of raltegravir and darunavir/ritonavir in treatment-naïve patients. Pre-treatment plasma was obtained from 5 participants with raltegravir resistance at the time of virologic failure. A control library was created by pooling integrase clones at predefined proportions. Multiplexed sequencing was performed with Illumina and 454 platforms at comparable costs. Illumina sequence analysis was performed with the novel snp-assess tool and 454 sequencing was analyzed with V-Phaser. Results: Illumina sequencing resulted in significantly higher sequence coverage and a 0.095% limit of detection. Illumina accurately detected all MVs in the control library at ≥0.5% and 7/10 MVs expected at 0.1%. 454 sequencing failed to detect any MVs at 0.1% with 5 false positive calls. For MVs detected in the patient samples by both 454 and Illumina, the correlation in the detected variant frequencies was high (R2 = 0.92...

Homozygosity Mapping and Whole Exome Sequencing Reveal a Novel Homozygous COL18A1 Mutation Causing Knobloch Syndrome

Haghighi, Alireza; Tiwari, Amit; Piri, Niloofar; Nürnberg, Gudrun; Saleh-Gohari, Nasrollah; Haghighi, Amirreza; Neidhardt, John; Nürnberg, Peter; Berger, Wolfgang
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN_US
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The aim of this study was to identify the genetic basis of a chorioretinal dystrophy with high myopia of unknown origin in a child of a consanguineous marriage. The proband and ten family members of Iranian ancestry participated in this study. Linkage analysis was carried out with DNA samples of the proband and her parents by using the Human SNP Array 6.0. Whole exome sequencing (WES) was performed with the patients’ DNA. Specific sequence alterations within the homozygous regions identified by whole exome sequencing were verified by Sanger sequencing. Upon genetic analysis, a novel homozygous frameshift mutation was found in exon 42 of the COL18A1 gene in the patient. Both parents were heterozygous for this sequence variation. Mutations in COL18A1 are known to cause Knobloch syndrome (KS). Retrospective analysis of clinical records of the patient revealed surgical removal of a meningocele present at birth. The clinical features shown by our patient were typical of KS with the exception of chorioretinal degeneration which is a rare manifestation. This is the first case of KS reported in a family of Iranian ancestry. We identified a novel disease-causing (deletion) mutation in the COL18A1 gene leading to a frameshift and premature stop codon in the last exon. The mutation was not present in SNP databases and was also not found in 192 control individuals. Its localization within the endostatin domain implicates a functional relevance of endostatin in KS. A combined approach of linkage analysis and WES led to a rapid identification of the disease-causing mutation even though the clinical description was not completely clear at the beginning.

Efficient experimental design and analysis strategies for the detection of differential expression using RNA-Sequencing

Robles, José A; Qureshi, Sumaira E; Stephen, Stuart J; Wilson, Susan R; Burden, Conrad J; Taylor, Jennifer M
Fonte: BioMed Central Publicador: BioMed Central
Tipo: Artigo de Revista Científica Formato: 14 pages
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Background: RNA sequencing (RNA-Seq) has emerged as a powerful approach for the detection of differential gene expression with both high-throughput and high resolution capabilities possible depending upon the experimental design chosen. Multiplex experimental designs are now readily available, these can be utilised to increase the numbers of samples or replicates profiled at the cost of decreased sequencing depth generated per sample. These strategies impact on the power of the approach to accurately identify differential expression. This study presents a detailed analysis of the power to detect differential expression in a range of scenarios including simulated null and differential expression distributions with varying numbers of biological or technical replicates, sequencing depths and analysis methods. Results: Differential and non-differential expression datasets were simulated using a combination of negative binomial and exponential distributions derived from real RNA-Seq data. These datasets were used to evaluate the performance of three commonly used differential expression analysis algorithms and to quantify the changes in power with respect to true and false positive rates when simulating variations in sequencing depth...

Candidate gene discovery in autoimmunity by using extreme phenotypes, next generation sequencing and whole exome capture

Johar, Angad S.; Anaya, Juan-Manuel; Andrews, Dan; Patel, Hardip R.; Field, Matthew; Goodnow, Chris; Arcos-Burgos, Mauricio
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica Formato: 6 pages
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Whole exome sequencing (WES) is a widely used strategy for detection of protein coding and splicing variants associated with inherited diseases. Many studies have shown that the strategy has been broad and proficient due to its ability in detecting a high proportion of disease causing variants, using only a small portion of the genome. In this review we outline the main steps involved in WES, the comprehensive analysis of the massive data obtained including the genomic capture, amplification, sequencing, alignment, curating, filtering and genetic analysis to determine the presence of candidate variants with potential pathogenic/functional effect. Further, we propose that the multiple autoimmune syndrome, an extreme phenotype of autoimmune disorders, is a very well suited trait to tackle genomic variants of major effect underpinning the lost of self-tolerance.

Sequencing error correction without a reference genome

Sleep, J.; Schreiber, A.; Baumann, U.
Fonte: BioMed Central Ltd. Publicador: BioMed Central Ltd.
Tipo: Artigo de Revista Científica
Publicado em //2013 EN
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Background: Next (second) generation sequencing is an increasingly important tool for many areas of molecular biology, however, care must be taken when interpreting its output. Even a low error rate can cause a large number of errors due to the high number of nucleotides being sequenced. Identifying sequencing errors from true biological variants is a challenging task. For organisms without a reference genome this difficulty is even more challenging. Results: We have developed a method for the correction of sequencing errors in data from the Illumina Solexa sequencing platforms. It does not require a reference genome and is of relevance for microRNA studies, unsequenced genomes, variant detection in ultra-deep sequencing and even for RNA-Seq studies of organisms with sequenced genomes where RNA editing is being considered. Conclusions: The derived error model is novel in that it allows different error probabilities for each position along the read, in conjunction with different error rates depending on the particular nucleotides involved in the substitution, and does not force these effects to behave in a multiplicative manner. The model provides error rates which capture the complex effects and interactions of the three main known causes of sequencing error associated with the Illumina platforms.; Julie A Sleep...

Sequenz- und Sequenzierungseffekte bei der Bearbeitung voraussetzungsreicher Aufgaben; Sequence effects and sequencing effects for solving knowledge-rich problems

Scheiter, Katharina
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
DE_DE
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Sequenzeffekte bestehen in Variationen der Problemlöseperformanz bei der Bearbeitung multipler Aufgaben in Abhängigkeit von der Reihenfolge, in der die Aufgaben dargeboten werden. Sequenzierungseffekte ergeben sich dagegen, wenn sich die Performanz von Personen, die Aufgaben in einer vorgegebenen Sequenz bearbeiten, von der Performanz derjenigen Personen unterscheidet, die eine davon abweichende Bearbeitungsreihenfolge wählen. Diese Effekte werden für voraussetzungsreiche Aufgaben untersucht, deren Bearbeitung in hohem Ausmaß Vorwissen voraussetzt. Nach dem im theoretischen Teil der Arbeit entwickelten Rahmenmodell können Sequenzeffekte resultieren, wenn sich Aufgabensequenzen in dem Ausmaß unterscheiden, in dem sie Lernprozesse bei der Aufgabenbearbeitung und/oder Wissenstransferprozesse unterstützen. Lernprozesse werden vor allem gefördert, wenn aufeinander folgende Aufgaben in ihrer Komplexität zunehmen, während ein Wissenstransfer insbesondere durch die sukzessive Bearbeitung strukturell ähnlicher Aufgaben begünstigt wird. Im Unterschied zu voraussetzungsarmen Aufgaben sind Lernprozesse bei der Bearbeitung voraussetzungsreicher Aufgaben vorwissensabhängig und können zu umfangreichen Erweiterungen des Wissensbestands eines Problemlösers führen (z.B. Erwerb von Fallwissen). Weiterhin legen Befunde zum analogen Transfer nahe...

Whole genome analysis of Arabidopsis thaliana using Next Generation Sequencing; Analyse des Genoms der Modellpflanze Arabidopsis thaliana mittels Next Generation Sequencing

Schneeberger, Korbinian
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
EN
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As-of the end of 2010 it has become commonplace that Next Generation Sequencing NGS) has revolutionized biology. Despite this it remains true that the advent of NGS reduced the costs of whole-genome sequencing tremendously. Soon even small labs will be able to afford sequencing of every genome of every species they like. Nonetheless sequencing of genomes might be cheap, resultant sequence reads alone are mostly not informative. It requires an army of new methods to handle the requirements that Next Generation Sequencing data brings along. In this thesis I present parts our efforts of the last four years to implement NGS technologies in plant whole-genome sequencing. First, I will outline how NGS read alignments against multiple reference sequences simultaneously can be performed efficiently and how this affects the outcome of whole-genome sequencing. Afterwards I will show how reference sequence guided assembly can further improve the reconstruction of genomic sequences. And finally I will summarize how we adapted our computational methods to plant genetics to pinpoint genomic disruptions that were causal for previously identified phenotypes.; Gegen Ende des Jahres 2010 ist es schon zum Allgemeinplatz geworden, dass Next Generation Sequencing (NGS) die Biologie revolutioniert hat. Doch das ändert nichts an der Tatsache...

The impact of activity sequencing on the differences between ELT methods: a critical analysis of sample units

Criado S??nchez, Raquel
Fonte: Universidad de Granada Publicador: Universidad de Granada
Tipo: Artigo de Revista Científica
ENG
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One of the methodological variables frequently forgotten by most ELT methods is activity sequencing. However, this is not a secondary variable and it should be intrinsically related to the cognitive built-in process governing knowledge acquisition. Consequently, activity sequencing fully affects the pedagogically based organisation of the teaching materials and the cognitive processes of language acquisition. The aim of this paper is to perform an analysis of the activity sequencing in three different textbooks representative of three main methods in the history of ELT: the Direct Method, the Audiolingual Method and the Communicative Method. The analysis will be undertaken from pedagogical and cognitive perspectives. The results will illustrate a) the degree of agreement or non-agreement detected in each method and textbook; b) that the activity sequencing variable is crucial to determine the nature of such differences or similarities. Since the activity sequencing is similar across the three textbooks, it could be assumed that methodological differences may be of less consequence than usually considered.; Una de las variables que han sido preteridas con frecuencia por casi todos los m??todos para la ense??anza del ingl??s como lengua extranjera es la secuenciaci??n de actividades. Sin embargo...

Aplicação do sequenciamento de nova geração no diagnóstico molecular de cardiomiopatia hipertrófica; Application of next-generation sequencing in the molecular diagnostics of hypertrophic cardiomyopathy

Oliveira, Théo Gremen Mimary de
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 13/07/2015 PT
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Introdução: A cardiomiopatia hipertrófica (CH) é uma doença cardíaca estrutural primária, caracterizada por hipertrofia do ventrículo esquerdo, sem dilatação, geralmente assimétrica e predominantemente septal. Na população geral a prevalência estimada da CH é de 0,2% (1:500), correspondendo a 0,5% de todas as cardiopatias. Atualmente estão descritas mais de 1400 mutações associadas à CH em 20 genes relacionados com os miofilamentos do sarcômero, o disco-Z e o transporte de cálcio, sendo que os três mais associados são os genes MYH7, MYBPC3 e TNNT2, responsáveis por 50% do casos com diagnóstico molecular positivo no Brasil. Dessa forma, o advento de novas tecnologias de sequenciamento de DNA de alta performance promete revolucionar o diagnóstico molecular, tornando mais rápida e barata a identificação de alterações genéticas, impactando positivamente na custo-efetividade do manejo diagnóstico e terapêutico de pacientes e famílias com o diagnóstico de CH. Materiais e Métodos: Noventa e uma amostras de uma casuística de pacientes não relacionados, portadores de CH com diagnóstico molecular prévio para os 3 genes mais associados (19 positivas e 72 negativas) foram utilizadas juntamente com uma amostra referência do HapMap (NA12878) na validação de um pipeline proposto para a identificação de alterações genéticas em um painel com 74 genes associados à cardiomiopatias hereditárias...

An Integrated Data Analysis Suite and Programming Framework for High-Throughput DNA Sequencing; Umfassende Datenanalyselösung sowie Software-Framework für die Hochdurchsatz-DNA-Sequenzierung

Ott, Felix
Fonte: Universität Tübingen Publicador: Universität Tübingen
Tipo: Dissertation; info:eu-repo/semantics/doctoralThesis
EN
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The various parallel DNA sequencing methods that have been introduced since 2005 to complement the established dideoxynucleotide chain termination method have proved a key technology with a wide range of applications in genetic research, but also challenge with a constant ood of data. A multitude of algorithms have been proposed and implemented addressing dierent aspects of sequencing data processing and data analysis for a variety of high-throughput sequencing applications. Routine data analysis however requires a consistently assembled tool chain to ensure smooth end-to-end processing starting from raw sequencing read data up to primary analysis results. The software SHORE aims to be a modular, general-purpose data analysis suite for high-throughput DNA sequencing. With this work, we extend and generalize the concepts of the SHORE analysis pipeline to accommodate increased throughput of sequencing devices and development of novel sequencing protocols such as bar-coded sample multiplexing. To provide universal support of basic features such as data set compression or indexing and query mechanisms, we develop a generic C++ programming framework libshore to form the foundation of all of SHORE's modules. Furthermore, we aim to provide a generic application programming interface facilitating modular design as well as parallelization of high-throughput sequencing data processing and analysis algorithms. A further focus of this work was on the development of data analysis algorithms for chromatin immunoprecipitation and other sequence enrichment and expression proling studies. Through genome-wide binding-site proling for transcription factors...

Recent progress in the methods of genome sequencing

Ning-wei,Zhao
Fonte: Instituto de Tecnologia do Paraná - Tecpar Publicador: Instituto de Tecnologia do Paraná - Tecpar
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/04/2010 EN
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Genome sequencing is a very important tool for the development of genetic diagnosis, drugs of gene engineering, pharmacogenetics, etc. As the HGP comes into people's ears, there is an emerging need for the genome sequencing. During the recent years, there are two different traditional strategies available for this target: shotgun sequencing and hierarchical sequencing. Besides these, many efforts are pursuing new ideas to facilitate fast and cost-effective genome sequencing, including 454 GS system, polony sequencing, single molecular array, nanopore sequencing, with each having different unique characteristics, but remains to be fully developed.

Targeted 'Next-Generation' Sequencing in Anophthalmia and Microphthalmia Patients Confirms SOX2, OTX2 and FOXE3 Mutations

Lopez Jimenez, Nelson; Yahyavi, Mani; Bardakjian, Tanya; Tonkin, Leath; Schneider, Adele; Sherr, Elliott H; Slavotinek, Anne M; Flannick, Jason A.; Li, Jiang
Fonte: BioMed Central Publicador: BioMed Central
Tipo: Artigo de Revista Científica
EN_US
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Background: Anophthalmia/microphthalmia (A/M) is caused by mutations in several different transcription factors, but mutations in each causative gene are relatively rare, emphasizing the need for a testing approach that screens multiple genes simultaneously. We used next-generation sequencing to screen 15 A/M patients for mutations in 9 pathogenic genes to evaluate this technology for screening in A/M. Methods We used a pooled sequencing design, together with custom single nucleotide polymorphism (SNP) calling software. We verified predicted sequence alterations using Sanger sequencing. Results: We verified three mutations - c.542delC in SOX2, resulting in p.Pro181Argfs*22, p.Glu105X in OTX2 and p.Cys240X in FOXE3. We found several novel sequence alterations and SNPs that were likely to be non-pathogenic - p.Glu42Lys in CRYBA4, p.Val201Met in FOXE3 and p.Asp291Asn in VSX2. Our analysis methodology gave one false positive result comprising a mutation in PAX6 (c.1268A > T, predicting p.X423LeuextX*15) that was not verified by Sanger sequencing. We also failed to detect one 20 base pair (bp) deletion and one 3 bp duplication in SOX2. Conclusions: Our results demonstrated the power of next-generation sequencing with pooled sample groups for the rapid screening of candidate genes for A/M as we were correctly able to identify disease-causing mutations. However...

Sintenia genomica entre sorgo e cana-de-açúcar inferida a partir do sequenciamento de um pool de BACs = Genomic synteny between sorghum and sugarcane inferred from a BAC pool sequencing; Genomic synteny between sorghum and sugarcane inferred from a BAC pool sequencing

Vagner Katsumi Okura
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 28/08/2015 PT
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36.48%
O sequenciamento genômico de plantas tem se acelerado nos últimos anos principalmente devido ao avanço das tecnologias de sequenciamento de nova geração, capazes de gerar um grande volume de dados com custo cada vez menor. No entanto, o sequenciamento e a montagem de genomas de plantas ainda continua sendo um grande desafio em função da alta complexidade desses genomas que na sua grande maioria possuem alto grau de ploidia e grande proporção de sequências repetitivas. O sequenciamento de bibliotecas produzidas com DNA genômico de plantas clonados em vetores BACs (bacterial artificial chromosomes) pode ser uma estratégia efetiva para sequenciamento de genomas complexos, por dividir a tarefa de montagem em problemas menores. No geral, bibliotecas de BACs contém fragmentos de DNA de 100 a 200 kilobases, cujo conjunto cobre o genoma clonado várias vezes. Entretanto, mesmo com as novas tecnologias de sequenciamento, o custo de sequenciar bibliotecas de BACs ainda é alto, pois na maioria das vezes o sequenciamento é realizado a partir do DNA isolado de cada BAC individualmente. Uma alternativa seria sequenciar pools contendo centenas de BACs amostrados randomicamente, que dessa forma diminuiria o custo proporcionalmente ao número de BACs do pool. Neste trabalho...

The study of biodiversity in the era of massive sequencing

Escalante,Ana E.; Jardón Barbolla,Lev; Ramírez-Barahona,Santiago; Eguiarte,Luis E.
Fonte: Instituto de Biología Publicador: Instituto de Biología
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2014 EN
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Recent years have witnessed the advent and rapid development of massive sequencing technology, commonly known as Next Generation Sequencing (NGS). This technology allows for rapid, massive and inexpensive sequencing of genome regions or entire genomes, making possible genomic studies of non-model organisms and has seen great progress in metagenomic studies. The promise of this information-rich era is to expand the molecular approach of ecological and evolutionary studies towards urgent issues related with conservation and management of biological diversity in the face of global change. Among the current NGS technologies, there are fundamental differences that impact DNA sequence accuracy, length and range of applications. Key differences among platforms are the procedure for library preparation (when needed) and the sequencing process itself (e.g., pyrosequencing, synthesis). In this review we describe the technical details of commercially available platforms for massive sequencing. We discuss their potential applications for specific biodiversity analyses, from model to non-model organisms, from Single Nucleotide Polymorphism (SNPs) to entire genome analysis and metagenomic approaches of microbial communities, including possible taxonomic...