Application of next generation sequencing in genetic and genomic studies
Author: Wang, Jingwen
Date: 2016-09-09
Location: 9Q Månen, Alfred Nobels Allé 8, Karolinska Institutet, Huddinge
Time: 09.30
Department: Inst för biovetenskaper och näringslära / Dept of Biosciences and Nutrition
Abstract
Genetic variants that spread along the human genome play vital roles in determining our
traits, affecting development and potentially causing disorders. Most common disorders have
complex underlying mechanisms involving genetic or environmental factors and the
interaction between them. Over the past decade, genome-wide association studies (GWAS)
have identified thousands of common variants that contribute to complex disorders and
partially explain the heritability. However, there is still a large portion that is unexplained and
the missing heritability may be caused by several factors, such as rare or low-frequency
variants with high effect that are not covered by GWAS and linkage analysis. With the
development of next generation sequencing (NGS), it is possible to rapidly detect large
amount of novel rare and low-frequency variants simultaneously at a low cost. This new
technology provides vast information on studying the association of genetic variations and
complex disorders. Once the susceptibility gene is mapped, model organisms such as
zebrafish (Danio rerio) are popular for further investigating the possible function of diseaseassociated gene in determining the phenotype. However, the genome annotation of zebrafish
is not complete, which affects the characterization of gene functions. Accordingly, highthroughput RNA sequencing can be employed for identifying new transcripts.
In our studies, pooled DNA samples were used for whole genome sequencing (WGS) and exome sequencing. In Paper I, we evaluated minor allele frequency (MAF) estimates using three variant detection tools with two sets of pooled exome sequencing and one set of pooled WGS data. The MAFs from the pooled sequencing data demonstrated high concordance (r = 0.88-0.94) with those from the individual genotyping data. In Paper II, exome sequencing implementing pooling strategy was performed on 100 idiopathic scoliosis (IS) patients for mapping susceptibility genes. After validating 20 candidate single nucleotide variants (SNVs), we did not find associations between them and IS. However, the previously reported common variant rs11190870 near LBX1 was validated in a large Scandinavian cohort. In Paper III, we analyzed WGS of pooled DNA samples performed on 19 affected individuals who shared a phenotype-linked haplotype in a dyslexic Finish family. Two of the individuals were sequenced for the whole genome individually as well. The screen for causative variants was narrowed down to a rare SNV, which might affect the binding affinity of LHX2 that regulated dyslexia associated gene ROBO1. In Paper IV, RNA sequencing (RNA-seq) data were analyzed for identifying novel transcripts in zebrafish early development using an inhouse pipeline. We discovered 152 novel transcribed regions (NTRs), validated more than 10 NTRs and quantified their expression in early developmental stages.
In our studies, we evaluated and applied a pooling approach for identifying variants susceptible to disease using high-throughput DNA sequencing. Based on RNA sequencing data, we provided new information for genome annotation on model organism zebrafish, which is valuable for studying the function of disease causative genes. In summary, the whole series of studies demonstrate how NGS can be applied in studying the genetic basis of complex disorders and assisting in follow-up functional studies in model organisms.
In our studies, pooled DNA samples were used for whole genome sequencing (WGS) and exome sequencing. In Paper I, we evaluated minor allele frequency (MAF) estimates using three variant detection tools with two sets of pooled exome sequencing and one set of pooled WGS data. The MAFs from the pooled sequencing data demonstrated high concordance (r = 0.88-0.94) with those from the individual genotyping data. In Paper II, exome sequencing implementing pooling strategy was performed on 100 idiopathic scoliosis (IS) patients for mapping susceptibility genes. After validating 20 candidate single nucleotide variants (SNVs), we did not find associations between them and IS. However, the previously reported common variant rs11190870 near LBX1 was validated in a large Scandinavian cohort. In Paper III, we analyzed WGS of pooled DNA samples performed on 19 affected individuals who shared a phenotype-linked haplotype in a dyslexic Finish family. Two of the individuals were sequenced for the whole genome individually as well. The screen for causative variants was narrowed down to a rare SNV, which might affect the binding affinity of LHX2 that regulated dyslexia associated gene ROBO1. In Paper IV, RNA sequencing (RNA-seq) data were analyzed for identifying novel transcripts in zebrafish early development using an inhouse pipeline. We discovered 152 novel transcribed regions (NTRs), validated more than 10 NTRs and quantified their expression in early developmental stages.
In our studies, we evaluated and applied a pooling approach for identifying variants susceptible to disease using high-throughput DNA sequencing. Based on RNA sequencing data, we provided new information for genome annotation on model organism zebrafish, which is valuable for studying the function of disease causative genes. In summary, the whole series of studies demonstrate how NGS can be applied in studying the genetic basis of complex disorders and assisting in follow-up functional studies in model organisms.
List of papers:
I. Wang J, Skoog T, Einarsdottir E, Kaartokallio T, Laivuori H, Grauers A, Gerdhem P, Hytönen M, Lohi H, Kere J, Jiao H. Investigation of rare and low-frequency variants using high-throughput sequencing with pooled DNA samples. [Submitted]
II. Grauers A, Wang J, Einarsdottir E, Simony A, Danielsson A, Åkesson K, Ohlin A, Halldin K, Grabowski P, Tenne M, Laivuori H, Dahlman I, Andersen M, Christensen SB, Karlsson MK, Jiao H, Kere J, Gerdhem P. Candidate gene analysis and exome sequencing confirm LBX1 as a susceptibility gene for idiopathic scoliosis. Spine J. 2015 Oct 1;15(10):2239-46.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Massinen S, Wang J, Laivuori K, Bieder A, Paez IT, Jiao H, Kere J. Genomic sequencing of a dyslexia susceptibility haplotype encompassing ROBO1. J Neurodev Disord. 2016 Jan 27;8:4.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. Wang J, Vesterlund L, Kere J, Jiao H. Identification of novel transcribed regions in zebrafish (Danio rerio) using RNA-sequencing [Submitted]
I. Wang J, Skoog T, Einarsdottir E, Kaartokallio T, Laivuori H, Grauers A, Gerdhem P, Hytönen M, Lohi H, Kere J, Jiao H. Investigation of rare and low-frequency variants using high-throughput sequencing with pooled DNA samples. [Submitted]
II. Grauers A, Wang J, Einarsdottir E, Simony A, Danielsson A, Åkesson K, Ohlin A, Halldin K, Grabowski P, Tenne M, Laivuori H, Dahlman I, Andersen M, Christensen SB, Karlsson MK, Jiao H, Kere J, Gerdhem P. Candidate gene analysis and exome sequencing confirm LBX1 as a susceptibility gene for idiopathic scoliosis. Spine J. 2015 Oct 1;15(10):2239-46.
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Massinen S, Wang J, Laivuori K, Bieder A, Paez IT, Jiao H, Kere J. Genomic sequencing of a dyslexia susceptibility haplotype encompassing ROBO1. J Neurodev Disord. 2016 Jan 27;8:4.
Fulltext (DOI)
Pubmed
View record in Web of Science®
IV. Wang J, Vesterlund L, Kere J, Jiao H. Identification of novel transcribed regions in zebrafish (Danio rerio) using RNA-sequencing [Submitted]
Institution: Karolinska Institutet
Supervisor: Jiao, Hong
Issue date: 2016-07-07
Rights:
Publication year: 2016
ISBN: 978-91-7676-321-6
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