Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 470

Search results for: whole exome sequencing

470 Efficient Reuse of Exome Sequencing Data for Copy Number Variation Callings

Authors: Chen Wang, Jared Evans, Yan Asmann


With the quick evolvement of next-generation sequencing techniques, whole-exome or exome-panel data have become a cost-effective way for detection of small exonic mutations, but there has been a growing desire to accurately detect copy number variations (CNVs) as well. In order to address this research and clinical needs, we developed a sequencing coverage pattern-based method not only for copy number detections, data integrity checks, CNV calling, and visualization reports. The developed methodologies include complete automation to increase usability, genome content-coverage bias correction, CNV segmentation, data quality reports, and publication quality images. Automatic identification and removal of poor quality outlier samples were made automatically. Multiple experimental batches were routinely detected and further reduced for a clean subset of samples before analysis. Algorithm improvements were also made to improve somatic CNV detection as well as germline CNV detection in trio family. Additionally, a set of utilities was included to facilitate users for producing CNV plots in focused genes of interest. We demonstrate the somatic CNV enhancements by accurately detecting CNVs in whole exome-wide data from the cancer genome atlas cancer samples and a lymphoma case study with paired tumor and normal samples. We also showed our efficient reuses of existing exome sequencing data, for improved germline CNV calling in a family of the trio from the phase-III study of 1000 Genome to detect CNVs with various modes of inheritance. The performance of the developed method is evaluated by comparing CNV calling results with results from other orthogonal copy number platforms. Through our case studies, reuses of exome sequencing data for calling CNVs have several noticeable functionalities, including a better quality control for exome sequencing data, improved joint analysis with single nucleotide variant calls, and novel genomic discovery of under-utilized existing whole exome and custom exome panel data.

Keywords: bioinformatics, computational genetics, copy number variations, data reuse, exome sequencing, next generation sequencing

Procedia PDF Downloads 177
469 A Pipeline for Detecting Copy Number Variation from Whole Exome Sequencing Using Comprehensive Tools

Authors: Cheng-Yang Lee, Petrus Tang, Tzu-Hao Chang


Copy number variations (CNVs) have played an important role in many kinds of human diseases, such as Autism, Schizophrenia and a number of cancers. Many diseases are found in genome coding regions and whole exome sequencing (WES) is a cost-effective and powerful technology in detecting variants that are enriched in exons and have potential applications in clinical setting. Although several algorithms have been developed to detect CNVs using WES and compared with other algorithms for finding the most suitable methods using their own samples, there were not consistent datasets across most of algorithms to evaluate the ability of CNV detection. On the other hand, most of algorithms is using command line interface that may greatly limit the analysis capability of many laboratories. We create a series of simulated WES datasets from UCSC hg19 chromosome 22, and then evaluate the CNV detective ability of 19 algorithms from OMICtools database using our simulated WES datasets. We compute the sensitivity, specificity and accuracy in each algorithm for validation of the exome-derived CNVs. After comparison of 19 algorithms from OMICtools database, we construct a platform to install all of the algorithms in a virtual machine like VirtualBox which can be established conveniently in local computers, and then create a simple script that can be easily to use for detecting CNVs using algorithms selected by users. We also build a table to elaborate on many kinds of events, such as input requirement, CNV detective ability, for all of the algorithms that can provide users a specification to choose optimum algorithms.

Keywords: whole exome sequencing, copy number variations, omictools, pipeline

Procedia PDF Downloads 236
468 Whole Exome Sequencing Data Analysis of Rare Diseases: Non-Coding Variants and Copy Number Variations

Authors: S. Fahiminiya, J. Nadaf, F. Rauch, L. Jerome-Majewska, J. Majewski


Background: Sequencing of protein coding regions of human genome (Whole Exome Sequencing; WES), has demonstrated a great success in the identification of causal mutations for several rare genetic disorders in human. Generally, most of WES studies have focused on rare variants in coding exons and splicing-sites where missense substitutions lead to the alternation of protein product. Although focusing on this category of variants has revealed the mystery behind many inherited genetic diseases in recent years, a subset of them remained still inconclusive. Here, we present the result of our WES studies where analyzing only rare variants in coding regions was not conclusive but further investigation revealed the involvement of non-coding variants and copy number variations (CNV) in etiology of the diseases. Methods: Whole exome sequencing was performed using our standard protocols at Genome Quebec Innovation Center, Montreal, Canada. All bioinformatics analyses were done using in-house WES pipeline. Results: To date, we successfully identified several disease causing mutations within gene coding regions (e.g. SCARF2: Van den Ende-Gupta syndrome and SNAP29: 22q11.2 deletion syndrome) by using WES. In addition, we showed that variants in non-coding regions and CNV have also important value and should not be ignored and/or filtered out along the way of bioinformatics analysis on WES data. For instance, in patients with osteogenesis imperfecta type V and in patients with glucocorticoid deficiency, we identified variants in 5'UTR, resulting in the production of longer or truncating non-functional proteins. Furthermore, CNVs were identified as the main cause of the diseases in patients with metaphyseal dysplasia with maxillary hypoplasia and brachydactyly and in patients with osteogenesis imperfecta type VII. Conclusions: Our study highlights the importance of considering non-coding variants and CNVs during interpretation of WES data, as they can be the only cause of disease under investigation.

Keywords: whole exome sequencing data, non-coding variants, copy number variations, rare diseases

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467 Accurate HLA Typing at High-Digit Resolution from NGS Data

Authors: Yazhi Huang, Jing Yang, Dingge Ying, Yan Zhang, Vorasuk Shotelersuk, Nattiya Hirankarn, Pak Chung Sham, Yu Lung Lau, Wanling Yang


Human leukocyte antigen (HLA) typing from next generation sequencing (NGS) data has the potential for applications in clinical laboratories and population genetic studies. Here we introduce a novel technique for HLA typing from NGS data based on read-mapping using a comprehensive reference panel containing all known HLA alleles and de novo assembly of the gene-specific short reads. An accurate HLA typing at high-digit resolution was achieved when it was tested on publicly available NGS data, outperforming other newly-developed tools such as HLAminer and PHLAT.

Keywords: human leukocyte antigens, next generation sequencing, whole exome sequencing, HLA typing

Procedia PDF Downloads 428
466 Whole Exome Sequencing in Characterizing Mysterious Crippling Disorder in India

Authors: Swarkar Sharma, Ekta Rai, Ankit Mahajan, Parvinder Kumar, Manoj K Dhar, Sushil Razdan, Kumarasamy Thangaraj, Carol Wise, Shiro Ikegawa M.D., K.K. Pandita M.D.


Rare disorders are poorly understood hence, remain uncharacterized or patients are misdiagnosed and get poor medical attention. A rare mysterious skeletal disorder that remained unidentified for decades and rendered many people physically challenged and disabled for life has been reported in an isolated remote village ‘Arai’ of Poonch district of Jammu and Kashmir. This village is located deep in mountains and the population residing in the region is highly consanguineous. In our survey of the region, 70 affected people were reported, showing similar phenotype, in the village with a population of approximately 5000 individuals. We were able to collect samples from two multi generational extended families from the village. Through Whole Exome sequencing (WES), we identified a rare variation NM_003880.3:c.156C>A NP_003871.1:p.Cys52Ter, which results in introduction of premature stop codon in WISP3 gene. We found this variation perfectly segregating with the disease in one of the family. However, this variation was absent in other family. Interestingly, a novel splice site mutation at position c.643+1G>A of WISP3 gene, perfectly segregating with the disease was observed in the second family. Thus, exploiting WES and putting different evidences together (familial histories and genetic data, clinical features, radiological and biochemical tests and findings), the disease has finally been diagnosed as a very rare recessive hereditary skeletal disease “Progressive Pseudorheumatoid Arthropathy of Childhood” (PPAC) also known as “Spondyloepiphyseal Dysplasia Tarda with Progressive Arthropathy” (SEDT-PA). This genetic characterization and identification of the disease causing mutations will aid in genetic counseling, critically required to curb this rare disorder and to prevent its appearance in future generations in the population. Further, understanding of the role of WISP3 gene the biological pathways should help in developing treatment for the disorder.

Keywords: whole exome sequencing, Next Generation Sequencing, rare disorders

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465 Identification of New Familial Breast Cancer Susceptibility Genes: Are We There Yet?

Authors: Ian Campbell, Gillian Mitchell, Paul James, Na Li, Ella Thompson


The genetic cause of the majority of multiple-case breast cancer families remains unresolved. Next generation sequencing has emerged as an efficient strategy for identifying predisposing mutations in individuals with inherited cancer. We are conducting whole exome sequence analysis of germ line DNA from multiple affected relatives from breast cancer families, with the aim of identifying rare protein truncating and non-synonymous variants that are likely to include novel cancer predisposing mutations. Data from more than 200 exomes show that on average each individual carries 30-50 protein truncating mutations and 300-400 rare non-synonymous variants. Heterogeneity among our exome data strongly suggest that numerous moderate penetrance genes remain to be discovered, with each gene individually accounting for only a small fraction of families (~0.5%). This scenario marks validation of candidate breast cancer predisposing genes in large case-control studies as the rate-limiting step in resolving the missing heritability of breast cancer. The aim of this study is to screen genes that are recurrently mutated among our exome data in a larger cohort of cases and controls to assess the prevalence of inactivating mutations that may be associated with breast cancer risk. We are using the Agilent HaloPlex Target Enrichment System to screen the coding regions of 168 genes in 1,000 BRCA1/2 mutation-negative familial breast cancer cases and 1,000 cancer-naive controls. To date, our interim analysis has identified 21 genes which carry an excess of truncating mutations in multiple breast cancer families versus controls. Established breast cancer susceptibility gene PALB2 is the most frequently mutated gene (13/998 cases versus 0/1009 controls), but other interesting candidates include NPSR1, GSN, POLD2, and TOX3. These and other genes are being validated in a second cohort of 1,000 cases and controls. Our experience demonstrates that beyond PALB2, the prevalence of mutations in the remaining breast cancer predisposition genes is likely to be very low making definitive validation exceptionally challenging.

Keywords: predisposition, familial, exome sequencing, breast cancer

Procedia PDF Downloads 408
464 CMPD: Cancer Mutant Proteome Database

Authors: Po-Jung Huang, Chi-Ching Lee, Bertrand Chin-Ming Tan, Yuan-Ming Yeh, Julie Lichieh Chu, Tin-Wen Chen, Cheng-Yang Lee, Ruei-Chi Gan, Hsuan Liu, Petrus Tang


Whole-exome sequencing focuses on the protein coding regions of disease/cancer associated genes based on a priori knowledge is the most cost-effective method to study the association between genetic alterations and disease. Recent advances in high throughput sequencing technologies and proteomic techniques has provided an opportunity to integrate genomics and proteomics, allowing readily detectable mutated peptides corresponding to mutated genes. Since sequence database search is the most widely used method for protein identification using Mass spectrometry (MS)-based proteomics technology, a mutant proteome database is required to better approximate the real protein pool to improve disease-associated mutated protein identification. Large-scale whole exome/genome sequencing studies were launched by National Cancer Institute (NCI), Broad Institute, and The Cancer Genome Atlas (TCGA), which provide not only a comprehensive report on the analysis of coding variants in diverse samples cell lines but a invaluable resource for extensive research community. No existing database is available for the collection of mutant protein sequences related to the identified variants in these studies. CMPD is designed to address this issue, serving as a bridge between genomic data and proteomic studies and focusing on protein sequence-altering variations originated from both germline and cancer-associated somatic variations.

Keywords: TCGA, cancer, mutant, proteome

Procedia PDF Downloads 482
463 Familial Exome Sequencing to Decipher the Complex Genetic Basis of Holoprosencephaly

Authors: Artem Kim, Clara Savary, Christele Dubourg, Wilfrid Carre, Houda Hamdi-Roze, Valerie Dupé, Sylvie Odent, Marie De Tayrac, Veronique David


Holoprosencephaly (HPE) is a rare congenital brain malformation resulting from the incomplete separation of the two cerebral hemispheres. It is characterized by a wide phenotypic spectrum and a high degree of locus heterogeneity. Genetic defects in 16 genes have already been implicated in HPE, but account for only 30% of cases, suggesting that a large part of genetic factors remains to be discovered. HPE has been recently redefined as a complex multigenic disorder, requiring the joint effect of multiple mutational events in genes belonging to one or several developmental pathways. The onset of HPE may result from accumulation of the effects of multiple rare variants in functionally-related genes, each conferring a moderate increase in the risk of HPE onset. In order to decipher the genetic basis of HPE, unconventional patterns of inheritance involving multiple genetic factors need to be considered. The primary objective of this study was to uncover possible disease causing combinations of multiple rare variants underlying HPE by performing trio-based Whole Exome Sequencing (WES) of familial cases where no molecular diagnosis could be established. 39 families were selected with no fully-penetrant causal mutation in known HPE gene, no chromosomic aberrations/copy number variants and without any implication of environmental factors. As the main challenge was to identify disease-related variants among a large number of nonpathogenic polymorphisms detected by WES classical scheme, a novel variant prioritization approach was established. It combined WES filtering with complementary gene-level approaches: transcriptome-driven (RNA-Seq data) and clinically-driven (public clinical data) strategies. Briefly, a filtering approach was performed to select variants compatible with disease segregation, population frequency and pathogenicity prediction to identify an exhaustive list of rare deleterious variants. The exome search space was then reduced by restricting the analysis to candidate genes identified by either transcriptome-driven strategy (genes sharing highly similar expression patterns with known HPE genes during cerebral development) or clinically-driven strategy (genes associated to phenotypes of interest overlapping with HPE). Deeper analyses of candidate variants were then performed on a family-by-family basis. These included the exploration of clinical information, expression studies, variant characteristics, recurrence of mutated genes and available biological knowledge. A novel bioinformatics pipeline was designed. Applied to the 39 families, this final integrated workflow identified an average of 11 candidate variants per family. Most of candidate variants were inherited from asymptomatic parents suggesting a multigenic inheritance pattern requiring the association of multiple mutational events. The manual analysis highlighted 5 new strong HPE candidate genes showing recurrences in distinct families. Functional validations of these genes are foreseen.

Keywords: complex genetic disorder, holoprosencephaly, multiple rare variants, whole exome sequencing

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462 Exploring an Exome Target Capture Method for Cross-Species Population Genetic Studies

Authors: Benjamin A. Ha, Marco Morselli, Xinhui Paige Zhang, Elizabeth A. C. Heath-Heckman, Jonathan B. Puritz, David K. Jacobs


Next-generation sequencing has enhanced the ability to acquire massive amounts of sequence data to address classic population genetic questions for non-model organisms. Targeted approaches allow for cost effective or more precise analyses of relevant sequences; although, many such techniques require a known genome and it can be costly to purchase probes from a company. This is challenging for non-model organisms with no published genome and can be expensive for large population genetic studies. Expressed exome capture sequencing (EecSeq) synthesizes probes in the lab from expressed mRNA, which is used to capture and sequence the coding regions of genomic DNA from a pooled suite of samples. A normalization step produces probes to recover transcripts from a wide range of expression levels. This approach offers low cost recovery of a broad range of genes in the genome. This research project expands on EecSeq to investigate if mRNA from one taxon may be used to capture relevant sequences from a series of increasingly less closely related taxa. For this purpose, we propose to use the endangered Northern Tidewater goby, Eucyclogobius newberryi, a non-model organism that inhabits California coastal lagoons. mRNA will be extracted from E. newberryi to create probes and capture exomes from eight other taxa, including the more at-risk Southern Tidewater goby, E. kristinae, and more divergent species. Captured exomes will be sequenced, analyzed bioinformatically and phylogenetically, then compared to previously generated phylogenies across this group of gobies. This will provide an assessment of the utility of the technique in cross-species studies and for analyzing low genetic variation within species as is the case for E. kristinae. This method has potential applications to provide economical ways to expand population genetic and evolutionary biology studies for non-model organisms.

Keywords: coastal lagoons, endangered species, non-model organism, target capture method

Procedia PDF Downloads 90
461 An Integrative Computational Pipeline for Detection of Tumor Epitopes in Cancer Patients

Authors: Tanushree Jaitly, Shailendra Gupta, Leila Taher, Gerold Schuler, Julio Vera


Genomics-based personalized medicine is a promising approach to fight aggressive tumors based on patient's specific tumor mutation and expression profiles. A remarkable case is, dendritic cell-based immunotherapy, in which tumor epitopes targeting patient's specific mutations are used to design a vaccine that helps in stimulating cytotoxic T cell mediated anticancer immunity. Here we present a computational pipeline for epitope-based personalized cancer vaccines using patient-specific haplotype and cancer mutation profiles. In the workflow proposed, we analyze Whole Exome Sequencing and RNA Sequencing patient data to detect patient-specific mutations and their expression level. Epitopes including the tumor mutations are computationally predicted using patient's haplotype and filtered based on their expression level, binding affinity, and immunogenicity. We calculate binding energy for each filtered major histocompatibility complex (MHC)-peptide complex using docking studies, and use this feature to select good epitope candidates further.

Keywords: cancer immunotherapy, epitope prediction, NGS data, personalized medicine

Procedia PDF Downloads 180
460 BingleSeq: A User-Friendly R Package for Single-Cell RNA-Seq Data Analysis

Authors: Quan Gu, Daniel Dimitrov


BingleSeq was developed as a shiny-based, intuitive, and comprehensive application that enables the analysis of single-Cell RNA-Sequencing count data. This was achieved via incorporating three state-of-the-art software packages for each type of RNA sequencing analysis, alongside functional annotation analysis and a way to assess the overlap of differential expression method results. At its current state, the functionality implemented within BingleSeq is comparable to that of other applications, also developed with the purpose of lowering the entry requirements to RNA Sequencing analyses. BingleSeq is available on GitHub and will be submitted to R/Bioconductor.

Keywords: bioinformatics, functional annotation analysis, single-cell RNA-sequencing, transcriptomics

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459 Clinical Impact of Ultra-Deep Versus Sanger Sequencing Detection of Minority Mutations on the HIV-1 Drug Resistance Genotype Interpretations after Virological Failure

Authors: S. Mohamed, D. Gonzalez, C. Sayada, P. Halfon


Drug resistance mutations are routinely detected using standard Sanger sequencing, which does not detect minor variants with a frequency below 20%. The impact of detecting minor variants generated by ultra-deep sequencing (UDS) on HIV drug-resistance (DR) interpretations has not yet been studied. Fifty HIV-1 patients who experienced virological failure were included in this retrospective study. The HIV-1 UDS protocol allowed the detection and quantification of HIV-1 protease and reverse transcriptase variants related to genotypes A, B, C, E, F, and G. DeepChek®-HIV simplified DR interpretation software was used to compare Sanger sequencing and UDS. The total time required for the UDS protocol was found to be approximately three times longer than Sanger sequencing with equivalent reagent costs. UDS detected all of the mutations found by population sequencing and identified additional resistance variants in all patients. An analysis of DR revealed a total of 643 and 224 clinically relevant mutations by UDS and Sanger sequencing, respectively. Three resistance mutations with > 20% prevalence were detected solely by UDS: A98S (23%), E138A (21%) and V179I (25%). A significant difference in the DR interpretations for 19 antiretroviral drugs was observed between the UDS and Sanger sequencing methods. Y181C and T215Y were the most frequent mutations associated with interpretation differences. A combination of UDS and DeepChek® software for the interpretation of DR results would help clinicians provide suitable treatments. A cut-off of 1% allowed a better characterisation of the viral population by identifying additional resistance mutations and improving the DR interpretation.

Keywords: HIV-1, ultra-deep sequencing, Sanger sequencing, drug resistance

Procedia PDF Downloads 244
458 Genomics of Adaptation in the Sea

Authors: Agostinho Antunes


The completion of the human genome sequencing in 2003 opened a new perspective into the importance of whole genome sequencing projects, and currently multiple species are having their genomes completed sequenced, from simple organisms, such as bacteria, to more complex taxa, such as mammals. This voluminous sequencing data generated across multiple organisms provides also the framework to better understand the genetic makeup of such species and related ones, allowing to explore the genetic changes underlining the evolution of diverse phenotypic traits. Here, recent results from our group retrieved from comparative evolutionary genomic analyses of selected marine animal species will be considered to exemplify how gene novelty and gene enhancement by positive selection might have been determinant in the success of adaptive radiations into diverse habitats and lifestyles.

Keywords: marine genomics, evolutionary bioinformatics, human genome sequencing, genomic analyses

Procedia PDF Downloads 522
457 Risk Association of RANKL and OPG Gene Polymorphism with Breast to Bone Metastasis

Authors: Najeeb Ullah Khan


Background: The receptor activator NF-κβ ligand (RANKL) and Osteoprotegerin (OPG) polymorphisms have been associated with the progression of breast cancer to bone metastasis. Here, we aimed to investigate the association of RANKL and OPG gene polymorphism with breast to bone metastasis in the Pashtun population, Pakistan. Methods: Genomic DNA was obtained from all the study subjects (106 breast cancer, 58 breast to bone metastasis, and 51 healthy controls). RANKL (rs9533156) and OPG (rs2073618, rs3102735) polymorphisms were genotyped using Tetra-ARMS PCR. Results: Our results indicated that the frequencies of OPG (rs3102735) risk allele and genotypes carrying risk allele in breast cancer vs healthy control (C- p=0.005; CC- p=0.0208; TC- p=0.0181), bone metastasis vs healthy control (C- p=0.0211; CC- p=0.0153; TC- p=0.0775), and breast cancer vs breast to bone metastasis (C- p=0.0001; CC- p=0.0001; TC- p=0.001) were found significantly associated with disease risk. However, there was no significant association observed for OPG (rs2073618) risk allele and risk allele containing genotypes in all study groups. Similarly, RANKL (rs9533156) risk alleles and corresponding genotypes in breast cancer vs healthy control (C- p=0.0001; CC- p=0.0001; TC- p=0.0084), bone metastasis vs healthy control (C- p=0.0001; CC- p=0.0001; TC- p=0.5593), and breast cancer vs breast to bone metastasis (C- p=0.0185; CC- p=0.6077; TC- p=0.1436) showed significant association except for the risk allele carrying genotypes in breast cancer to bone metastasis (TC, p=0.1436; CC, p=0.6077). Conclusion: OPG (rs3102735) and RANKL (rs9533156) showed significant association with breast to bone metastasis, while OPG (rs2073618) didn’t show a significant association with breast to bone metastasis in Pashtun population of Pakistan. However, more investigation will be required to disseminate the results while gene sequencing or whole-exome sequencing.

Keywords: breast cancer, bone metastasis, OPG, RANKL, polymorphism

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456 A Clustering-Sequencing Approach to the Facility Layout Problem

Authors: Saeideh Salimpour, Sophie-Charlotte Viaux, Ahmed Azab, Mohammed Fazle Baki


The Facility Layout Problem (FLP) is key to the efficient and cost-effective operation of a system. This paper presents a hybrid heuristic- and mathematical-programming-based approach that divides the problem conceptually into those of clustering and sequencing. First, clusters of vertically aligned facilities are formed, which are later on sequenced horizontally. The developed methodology provides promising results in comparison to its counterparts in the literature by minimizing the inter-distances for facilities which have more interactions amongst each other and aims at placing the facilities with more interactions at the centroid of the shop.

Keywords: clustering-sequencing approach, mathematical modeling, optimization, unequal facility layout problem

Procedia PDF Downloads 248
455 MSIpred: A Python 2 Package for the Classification of Tumor Microsatellite Instability from Tumor Mutation Annotation Data Using a Support Vector Machine

Authors: Chen Wang, Chun Liang


Microsatellite instability (MSI) is characterized by high degree of polymorphism in microsatellite (MS) length due to a deficiency in mismatch repair (MMR) system. MSI is associated with several tumor types and its status can be considered as an important indicator for tumor prognostic. Conventional clinical diagnosis of MSI examines PCR products of a panel of MS markers using electrophoresis (MSI-PCR) which is laborious, time consuming, and less reliable. MSIpred, a python 2 package for automatic classification of MSI was released by this study. It computes important somatic mutation features from files in mutation annotation format (MAF) generated from paired tumor-normal exome sequencing data, subsequently using these to predict tumor MSI status with a support vector machine (SVM) classifier trained by MAF files of 1074 tumors belonging to four types. Evaluation of MSIpred on an independent 358-tumor test set achieved overall accuracy of over 98% and area under receiver operating characteristic (ROC) curve of 0.967. These results indicated that MSIpred is a robust pan-cancer MSI classification tool and can serve as a complementary diagnostic to MSI-PCR in MSI diagnosis.

Keywords: microsatellite instability, pan-cancer classification, somatic mutation, support vector machine

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454 Massively Parallel Sequencing Improved Resolution for Paternity Testing

Authors: Xueying Zhao, Ke Ma, Hui Li, Yu Cao, Fan Yang, Qingwen Xu, Wenbin Liu


Massively parallel sequencing (MPS) technologies allow high-throughput sequencing analyses with a relatively affordable price and have gradually been applied to forensic casework. MPS technology identifies short tandem repeat (STR) loci based on sequence so that repeat motif variation within STRs can be detected, which may help one to infer the origin of the mutation in some cases. Here, we report on one case with one three-step mismatch (D18S51) in family trios based on both capillary electrophoresis (CE) and MPS typing. The alleles of the alleged father (AF) are [AGAA]₁₇AGAG[AGAA]₃ and [AGAA]₁₅. The mother’s alleles are [AGAA]₁₉ and [AGAA]₉AGGA[AGAA]₃. The questioned child’s (QC) alleles are [AGAA]₁₉ and [AGAA]₁₂. Given that the sequence variants in repeat regions of AF and mother are not observed in QC’s alleles, the QC’s allele [AGAA]₁₂ was likely inherited from the AF’s allele [AGAA]₁₅ by loss of three repeat [AGAA]. Besides, two new alleles of D18S51 in this study, [AGAA]₁₇AGAG[AGAA]₃ and [AGAA]₉AGGA[AGAA]₃, have not been reported before. All the results in this study were verified using Sanger-type sequencing. In summary, the MPS typing method can offer valuable information for forensic genetics research and play a promising role in paternity testing.

Keywords: family trios analysis, forensic casework, ion torrent personal genome machine (PGM), massively parallel sequencing (MPS)

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453 Evolutionary Genomic Analysis of Adaptation Genomics

Authors: Agostinho Antunes


The completion of the human genome sequencing in 2003 opened a new perspective into the importance of whole genome sequencing projects, and currently multiple species are having their genomes completed sequenced, from simple organisms, such as bacteria, to more complex taxa, such as mammals. This voluminous sequencing data generated across multiple organisms provides also the framework to better understand the genetic makeup of such species and related ones, allowing to explore the genetic changes underlining the evolution of diverse phenotypic traits. Here, recent results from our group retrieved from comparative evolutionary genomic analyses of varied species will be considered to exemplify how gene novelty and gene enhancement by positive selection might have been determinant in the success of adaptive radiations into diverse habitats and lifestyles.

Keywords: adaptation, animals, evolution, genomics

Procedia PDF Downloads 342
452 Removal of Nitrogen Compounds from Industrial Wastewater Using Sequencing Batch Reactor: The Effects of React Time

Authors: Ali W. Alattabi, Khalid S. Hashim, Hassnen M. Jafer, Ali Alzeyadi


This study was performed to optimise the react time (RT) and study its effects on the removal rates of nitrogen compounds in a sequencing batch reactor (SBR) treating synthetic industrial wastewater. The results showed that increasing the RT from 4 h to 10, 16 and 22 h significantly improved the nitrogen compounds’ removal efficiency, it was increased from 69.5% to 95%, 75.7 to 97% and from 54.2 to 80.1% for NH3-N, NO3-N and NO2-N respectively. The results obtained from this study showed that the RT of 22 h was the optimum for nitrogen compounds removal efficiency.

Keywords: ammonia-nitrogen, retention time, nitrate, nitrite, sequencing batch reactor, sludge characteristics

Procedia PDF Downloads 243
451 Automatic Reporting System for Transcriptome Indel Identification and Annotation Based on Snapshot of Next-Generation Sequencing Reads Alignment

Authors: Shuo Mu, Guangzhi Jiang, Jinsa Chen


The analysis of Indel for RNA sequencing of clinical samples is easily affected by sequencing experiment errors and software selection. In order to improve the efficiency and accuracy of analysis, we developed an automatic reporting system for Indel recognition and annotation based on image snapshot of transcriptome reads alignment. This system includes sequence local-assembly and realignment, target point snapshot, and image-based recognition processes. We integrated high-confidence Indel dataset from several known databases as a training set to improve the accuracy of image processing and added a bioinformatical processing module to annotate and filter Indel artifacts. Subsequently, the system will automatically generate data, including data quality levels and images results report. Sanger sequencing verification of the reference Indel mutation of cell line NA12878 showed that the process can achieve 83% sensitivity and 96% specificity. Analysis of the collected clinical samples showed that the interpretation accuracy of the process was equivalent to that of manual inspection, and the processing efficiency showed a significant improvement. This work shows the feasibility of accurate Indel analysis of clinical next-generation sequencing (NGS) transcriptome. This result may be useful for RNA study for clinical samples with microsatellite instability in immunotherapy in the future.

Keywords: automatic reporting, indel, next-generation sequencing, NGS, transcriptome

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450 Language Shapes Thought: An Experimental Study on English and Mandarin Native Speakers' Sequencing of Size

Authors: Hsi Wei


Does the language we speak affect the way we think? This question has been discussed for a long time from different aspects. In this article, the issue is examined with an experiment on how speakers of different languages tend to do different sequencing when it comes to the size of general objects. An essential difference between the usage of English and Mandarin is the way we sequence the size of places or objects. In English, when describing the location of something we may say, for example, ‘The pen is inside the trashcan next to the tree at the park.’ In Mandarin, however, we would say, ‘The pen is at the park next to the tree inside the trashcan.’ It’s clear that generally English use the sequence of small to big while Mandarin the opposite. Therefore, the experiment was conducted to test if the difference of the languages affects the speakers’ ability to do the different sequencing. There were two groups of subjects; one consisted of English native speakers, another of Mandarin native speakers. Within the experiment, three nouns were showed as a group to the subjects as their native languages. Before they saw the nouns, they would first get an instruction of ‘big to small’, ‘small to big’, or ‘repeat’. Therefore, the subjects had to sequence the following group of nouns as the instruction they get or simply repeat the nouns. After completing every sequencing and repetition in their minds, they pushed a button as reaction. The repetition design was to gather the mere reading time of the person. As the result of the experiment showed, English native speakers reacted more quickly to the sequencing of ‘small to big’; on the other hand, Mandarin native speakers reacted more quickly to the sequence ‘big to small’. To conclude, this study may be of importance as a support for linguistic relativism that the language we speak do shape the way we think.

Keywords: language, linguistic relativism, size, sequencing

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449 Genomics of Aquatic Adaptation

Authors: Agostinho Antunes


The completion of the human genome sequencing in 2003 opened a new perspective into the importance of whole genome sequencing projects, and currently multiple species are having their genomes completed sequenced, from simple organisms, such as bacteria, to more complex taxa, such as mammals. This voluminous sequencing data generated across multiple organisms provides also the framework to better understand the genetic makeup of such species and related ones, allowing to explore the genetic changes underlining the evolution of diverse phenotypic traits. Here, recent results from our group retrieved from comparative evolutionary genomic analyses of selected marine animal species will be considered to exemplify how gene novelty and gene enhancement by positive selection might have been determinant in the success of adaptive radiations into diverse habitats and lifestyles.

Keywords: comparative genomics, adaptive evolution, bioinformatics, phylogenetics, genome mining

Procedia PDF Downloads 441
448 The Role and Importance of Genome Sequencing in Prediction of Cancer Risk

Authors: M. Sadeghi, H. Pezeshk, R. Tusserkani, A. Sharifi Zarchi, A. Malekpour, M. Foroughmand, S. Goliaei, M. Totonchi, N. Ansari–Pour


The role and relative importance of intrinsic and extrinsic factors in the development of complex diseases such as cancer still remains a controversial issue. Determining the amount of variation explained by these factors needs experimental data and statistical models. These models are nevertheless based on the occurrence and accumulation of random mutational events during stem cell division, thus rendering cancer development a stochastic outcome. We demonstrate that not only individual genome sequencing is uninformative in determining cancer risk, but also assigning a unique genome sequence to any given individual (healthy or affected) is not meaningful. Current whole-genome sequencing approaches are therefore unlikely to realize the promise of personalized medicine. In conclusion, since genome sequence differs from cell to cell and changes over time, it seems that determining the risk factor of complex diseases based on genome sequence is somewhat unrealistic, and therefore, the resulting data are likely to be inherently uninformative.

Keywords: cancer risk, extrinsic factors, genome sequencing, intrinsic factors

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447 A Study on the Treatment of Municipal Waste Water Using Sequencing Batch Reactor

Authors: Bhaven N. Tandel, Athira Rajeev


Sequencing batch reactor process is a suspended growth process operating under non-steady state conditions which utilizes a fill and draw reactor with complete mixing during the batch reaction step (after filling) and where the subsequent steps of aeration and clarification occur in the same tank. All sequencing batch reactor systems have five steps in common, which are carried out in sequence as follows, (1) fill (2) react (3) settle (sedimentation/clarification) (4) draw (decant) and (5) idle. The study was carried out in a sequencing batch reactor of dimensions 44cmx30cmx70cm with a working volume of 40 L. Mechanical stirrer of 100 rpm was used to provide continuous mixing in the react period and oxygen was supplied by fish tank aerators. The duration of a complete cycle of sequencing batch reactor was 8 hours. The cycle period was divided into different phases in sequence as follows-0.25 hours fill phase, 6 hours react period, 1 hour settling phase, 0.5 hours decant period and 0.25 hours idle phase. The study consisted of two runs, run 1 and run 2. Run 1 consisted of 6 hours aerobic react period and run 2 consisted of 3 hours aerobic react period followed by 3 hours anoxic react period. The influent wastewater used for the study had COD, BOD, NH3-N and TKN concentrations of 308.03±48.94 mg/L, 100.36±22.05 mg/L, 14.12±1.18 mg/L, and 24.72±2.21 mg/L respectively. Run 1 had an average COD removal efficiency of 41.28%, BOD removal efficiency of 56.25%, NH3-N removal efficiency of 86.19% and TKN removal efficiency of 54.4%. Run 2 had an average COD removal efficiency of 63.19%, BOD removal efficiency of 73.85%, NH3-N removal efficiency of 90.74% and TKN removal efficiency of 65.25%. It was observed that run 2 gave better performance than run 1 in the removal of COD, BOD and TKN.

Keywords: municipal waste water, aerobic, anoxic, sequencing batch reactor

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446 De Novo Assembly and Characterization of the Transcriptome during Seed Development, and Generation of Genic-SSR Markers in Pomegranate (Punica granatum L.)

Authors: Ozhan Simsek, Dicle Donmez, Burhanettin Imrak, Ahsen Isik Ozguven, Yildiz Aka Kacar


Pomegranate (Punica granatum L.) is known to be one of the oldest edible fruit tree species, with a wide geographical global distribution. Fruits from the two defined varieties (Hicaznar and 33N26) were taken at intervals after pollination and fertilization at different sizes. Seed samples were used for transcriptome sequencing. Primary sequencing was produced by Illumina Hi-Seq™ 2000. Firstly, we had raw reads, and it was subjected to quality control (QC). Raw reads were filtered into clean reads and aligned to the reference sequences. De novo analysis was performed to detect genes expressed in seeds of pomegranate varieties. We performed downstream analysis to determine differentially expressed genes. We generated about 27.09 gb bases in total after Illumina Hi-Seq sequencing. All samples were assembled together, we got 59,264 Unigenes, the total length, average length, N50, and GC content of Unigenes are 84.547.276 bp, 1.426 bp, 2,137 bp, and 46.20 %, respectively. Unigenes were annotated with 7 functional databases, finally, 42.681(NR: 72.02%), 39.660 (NT: 66.92%), 30.790 (Swissprot: 51.95%), 20.212 (COG: 34.11%), 27.689 (KEGG: 46.72%), 12.328 (GO: 20.80%), and 33,833 (Interpro: 57.09%) Unigenes were annotated. With functional annotation results, we detected 42.376 CDS, and 4.999 SSR distribute on 16.143 Unigenes.

Keywords: next generation sequencing, SSR, RNA-Seq, Illumina

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445 Genome Sequencing, Assembly and Annotation of Gelidium Pristoides from Kenton-on-Sea, South Africa

Authors: Sandisiwe Mangali, Graeme Bradley


Genome is complete set of the organism's hereditary information encoded as either deoxyribonucleic acid or ribonucleic acid in most viruses. The three different types of genomes are nuclear, mitochondrial and the plastid genome and their sequences which are uncovered by genome sequencing are known as an archive for all genetic information and enable researchers to understand the composition of a genome, regulation of gene expression and also provide information on how the whole genome works. These sequences enable researchers to explore the population structure, genetic variations, and recent demographic events in threatened species. Particularly, genome sequencing refers to a process of figuring out the exact arrangement of the basic nucleotide bases of a genome and the process through which all the afore-mentioned genomes are sequenced is referred to as whole or complete genome sequencing. Gelidium pristoides is South African endemic Rhodophyta species which has been harvested in the Eastern Cape since the 1950s for its high economic value which is one motivation for its sequencing. Its endemism further motivates its sequencing for conservation biology as endemic species are more vulnerable to anthropogenic activities endangering a species. As sequencing, mapping and annotating the Gelidium pristoides genome is the aim of this study. To accomplish this aim, the genomic DNA was extracted and quantified using the Nucleospin Plank Kit, Qubit 2.0 and Nanodrop. Thereafter, the Ion Plus Fragment Library was used for preparation of a 600bp library which was then sequenced through the Ion S5 sequencing platform for two runs. The produced reads were then quality-controlled and assembled through the SPAdes assembler with default parameters and the genome assembly was quality assessed through the QUAST software. From this assembly, the plastid and the mitochondrial genomes were then sampled out using Gelidiales organellar genomes as search queries and ordered according to them using the Geneious software. The Qubit and the Nanodrop instruments revealed an A260/A280 and A230/A260 values of 1.81 and 1.52 respectively. A total of 30792074 reads were obtained and produced a total of 94140 contigs with resulted into a sequence length of 217.06 Mbp with N50 value of 3072 bp and GC content of 41.72%. A total length of 179281bp and 25734 bp was obtained for plastid and mitochondrial respectively. Genomic data allows a clear understanding of the genomic constituent of an organism and is valuable as foundation information for studies of individual genes and resolving the evolutionary relationships between organisms including Rhodophytes and other seaweeds.

Keywords: Gelidium pristoides, genome, genome sequencing and assembly, Ion S5 sequencing platform

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444 Full Length Transcriptome Sequencing and Differential Expression Gene Analysis of Hybrid Larch under PEG Stress

Authors: Zhang Lei, Zhao Qingrong, Wang Chen, Zhang Sufang, Zhang Hanguo


Larch is the main afforestation and timber tree species in Northeast China, and drought is one of the main factors limiting the growth of Larch and other organisms in Northeast China. In order to further explore the mechanism of Larch drought resistance, PEG was used to simulate drought stress. The full-length sequencing of Larch embryogenic callus under PEG simulated drought stress was carried out by combining Illumina-Hiseq and SMRT-seq. A total of 20.3Gb clean reads and 786492 CCS reads were obtained from the second and third generation sequencing. The de-redundant transcript sequences were predicted by lncRNA, 2083 lncRNAs were obtained, and the target genes were predicted, and a total of 2712 target genes were obtained. The de-redundant transcripts were further screened, and 1654 differentially expressed genes (DEGs )were obtained. Among them, different DEGs respond to drought stress in different ways, such as oxidation-reduction process, starch and sucrose metabolism, plant hormone pathway, carbon metabolism, lignin catabolic/biosynthetic process and so on. This study provides basic full-length sequencing data for the study of Larch drought resistance, and excavates a large number of DEGs in response to drought stress, which helps us to further understand the function of Larch drought resistance genes and provides a reference for in-depth analysis of the molecular mechanism of Larch drought resistance.

Keywords: larch, drought stress, full-length transcriptome sequencing, differentially expressed genes

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443 Mixed Model Sequencing in Painting Production Line

Authors: Unchalee Inkampa, Tuanjai Somboonwiwat


Painting process of automobiles and automobile parts, which is a continuous process based on EDP (Electrode position paint, EDP). Through EDP, all work pieces will be continuously sent to the painting process. Work process can be divided into 2 groups based on the running time: Painting Room 1 and Painting Room 2. This leads to continuous operation. The problem that arises is waiting for workloads onto Painting Room. The grading process EDP to Painting Room is a major problem. Therefore, this paper aim to develop production sequencing method by applying EDP to painting process. It also applied fixed rate launching for painting room and earliest due date (EDD) for EDP process and swap pairwise interchange for waiting time to a minimum of machine. The result found that the developed method could improve painting reduced waiting time, on time delivery, meeting customers wants and improved productivity of painting unit.

Keywords: sequencing, mixed model lines, painting process, electrode position paint

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442 In silico Analysis of a Causative Mutation in Cadherin-23 Gene Identified in an Omani Family with Hearing Loss

Authors: Mohammed N. Al Kindi, Mazin Al Khabouri, Khalsa Al Lamki, Tommasso Pappuci, Giovani Romeo, Nadia Al Wardy


Hereditary hearing loss is a heterogeneous group of complex disorders with an overall incidence of one in every five hundred newborns presented as syndromic and non-syndromic forms. Cadherin-related 23 (CDH23) is one of the listed deafness causative genes. CDH23 is found to be expressed in the stereocilia of hair cells and the retina photoreceptor cells. Defective CDH23 has been associated mostly with prelingual severe-to-profound sensorineural hearing loss (SNHL) in either syndromic (USH1D) or non-syndromic SNHL (DFNB12). An Omani family diagnosed clinically with severe-profound sensorineural hearing loss was genetically analysed by whole exome sequencing technique. A novel homozygous missense variant, c.A7451C (p.D2484A), in exon 53 of CDH23 was detected. One hundred and thirty control samples were analysed where all were negative for the detected variant. The variant was analysed in silico for pathogenicity verification using several mutation prediction software. The variant proved to be a pathogenic mutation and is reported for the first time in Oman and worldwide. It is concluded that in silico mutation prediction analysis might be used as a useful molecular diagnostics tool benefiting both genetic counseling and mutation verification. The aspartic acid 2484 alanine missense substitution might be the main disease-causing mutation that damages CDH23 function and could be used as a genetic hearing loss marker for this particular Omani family.

Keywords: Cdh23, d2484a, in silico, Oman

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441 Enzymatic Repair Prior To DNA Barcoding, Aspirations, and Restraints

Authors: Maxime Merheb, Rachel Matar


Retrieving ancient DNA sequences which in return permit the entire genome sequencing from fossils have extraordinarily improved in recent years, thanks to sequencing technology and other methodological advances. In any case, the quest to search for ancient DNA is still obstructed by the damage inflicted on DNA which accumulates after the death of a living organism. We can characterize this damage into three main categories: (i) Physical abnormalities such as strand breaks which lead to the presence of short DNA fragments. (ii) Modified bases (mainly cytosine deamination) which cause errors in the sequence due to an incorporation of a false nucleotide during DNA amplification. (iii) DNA modifications referred to as blocking lesions, will halt the PCR extension which in return will also affect the amplification and sequencing process. We can clearly see that the issues arising from breakage and coding errors were significantly decreased in recent years. Fast sequencing of short DNA fragments was empowered by platforms for high-throughput sequencing, most of the coding errors were uncovered to be the consequences of cytosine deamination which can be easily removed from the DNA using enzymatic treatment. The methodology to repair DNA sequences is still in development, it can be basically explained by the process of reintroducing cytosine rather than uracil. This technique is thus restricted to amplified DNA molecules. To eliminate any type of damage (particularly those that block PCR) is a process still pending the complete repair methodologies; DNA detection right after extraction is highly needed. Before using any resources into extensive, unreasonable and uncertain repair techniques, it is vital to distinguish between two possible hypotheses; (i) DNA is none existent to be amplified to begin with therefore completely un-repairable, (ii) the DNA is refractory to PCR and it is worth to be repaired and amplified. Hence, it is extremely important to develop a non-enzymatic technique to detect the most degraded DNA.

Keywords: ancient DNA, DNA barcodong, enzymatic repair, PCR

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