Browsing by Subject "Next generation sequencing"

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Open Access
Characterization of short tandem repeats using local assembly
(2017-03) Demir, Gülfem
Tandem repeats are pieces of DNA where a pattern has multiple consecutive copies adjacent to itself. If the repeat unit (pattern) consists of 2 to 6 nucleotides, it can be referred to as a short tandem repeat or a microsatellite. There are many genetic diseases (such as huntington disease and Fragile-X syndrome) linked with STR expansions and because tandem repeats make up 3% of the sequenced human genome, STR detection research is significant. STR variations have always been a challenge for genome assembly and sequence alignment due to their repetitive nature, sequencing errors, short read lengths, and high incidence of polymerase slippage at STR regions. Despite the information they carry being very valuable, STR variations have not gained enough attention to be a permanent step in genome sequence analysis pipelines. After the 1000 Genomes Project, which aimed to establish the most detailed genetic variation catalogue for humans, the consortium released only two STR prediction sets which are identified by two STR caller tools, lobSTR and RepeatSeq. Many other large research efforts have failed to shed light on STR variations. The main aim of this study is to use sequence assembly methods for regions where we know that there is an STR, based on reference genome, and release a complete pipeline from sample's reads to STR genotype. The assembly problem we are dealing with in the scope of this thesis can be considered as local assembly, which is the assembly procedure of reads that maps to a small part of the genome. We will be focusing on two general assembly approaches that make use of graph data structures: de Bruijn graph (DBG) based methods that rely on a variant of kmer graph, overlap-layout-consensus (OLC) methods that are based on an overlap graph. Even though sequence assembly is a well studied problem, there is not any work that uses assembly algorithms to characterize STRs. We demonstrate that using sequence assembly on STR regions increases the true positive rate compared to state-of-art tools. We evaluated the performance of three different local assembly methods on three different experimental settings: focusing on (i) genotype based performance, (ii) coverage impact, and (iii) evaluating pre-processing and including anking regions. All these experiments supported our initial expectations on using assembly. Besides, we show that OLC based assembly methods bring much higher sensitivity to STR variant calling when compared to DBG based approach. This concludes that assembly with OLC is a better way for genotyping STRs according to our experiments.
Open Access
Determining the origin of synchronous multifocal bladder cancer by exome sequencing
(BioMed Central Ltd., 2015) Acar, Ö.; Özkurt, E.; Demir, G.; Saraç, H.; Alkan C.; Esen, T.; Somel, M.; Lack, N. A.
Background: Synchronous multifocal tumours are commonly observed in urothelial carcinomas of the bladder. The origin of these physically independent tumours has been proposed to occur by either intraluminal migration (clonal) or spontaneous transformation of multiple cells by carcinogens (field effect). It is unclear which model is correct, with several studies supporting both hypotheses. A potential cause of this uncertainty may be the small number of genetic mutations previously used to quantify the relationship between these tumours. Methods: To better understand the genetic lineage of these tumours we conducted exome sequencing of synchronous multifocal pTa urothelial bladder cancers at a high depth, using multiple samples from three patients. Results: Phylogenetic analysis of high confidence single nucleotide variants (SNV) demonstrated that the sequenced multifocal bladder cancers arose from a clonal origin in all three patients (bootstrap value 100 %). Interestingly, in two patients the most common type of tumour-associated SNVs were cytosine mutations of TpC*dinucleotides (Fisher's exact test p < 10-41), likely caused by APOBEC-mediated deamination. Incorporating these results into our clonal model, we found that TpC*type mutations occurred 2-5× more often among SNVs on the ancestral branches than in the more recent private branches (p < 10-4) suggesting that TpC*mutations largely occurred early in the development of the tumour. Conclusions: These results demonstrate that synchronous multifocal bladder cancers frequently arise from a clonal origin. Our data also suggests that APOBEC-mediated mutations occur early in the development of the tumour and may be a driver of tumourigenesis in non-muscle invasive urothelial bladder cancer.
Open Access
Mutations in RAD21 disrupt regulation of apob in patients with chronic intestinal pseudo-obstruction
(W.B. Saunders, 2015) Bonora, E.; Bianco, F.; Cordeddu, L.; Bamshad, M.; Francescatto, L.; Dowless, D.; Stanghellini, V.; Cogliandro, R. F.; Lindberg, G.; Mungan, Z.; Cefle, K.; Ozcelik, T.; Palanduz, S.; Ozturk, S.; Gedikbasi, A.; Gori, A.; Pippucci, T.; Graziano, C.; Volta, U.; Caio, G.; Barbara, G.; D'Amato, M.; Seri, M.; Katsanis, N.; Romeo, G.; De Giorgio, R.
Background Aims Chronic intestinal pseudo-obstruction (CIPO) is characterized by severe intestinal dysmotility that mimics a mechanical subocclusion with no evidence of gut obstruction. We searched for genetic variants associated with CIPO to increase our understanding of its pathogenesis and to identify potential biomarkers. Methods We performed whole-exome sequencing of genomic DNA from patients with familial CIPO syndrome. Blood and lymphoblastoid cells were collected from patients and controls (individuals without CIPO); levels of messenger RNA (mRNA) and proteins were analyzed by quantitative reverse-transcription polymerase chain reaction, immunoblot, and mobility shift assays. Complementary DNAs were transfected into HEK293 cells. Expression of rad21 was suppressed in zebrafish embryos using a splice-blocking morpholino (rad21a). Gut tissues were collected and analyzed. Results We identified a homozygous mutation (p.622, encodes Ala>Thr) in RAD21 in patients from a consanguineous family with CIPO. Expression of RUNX1, a target of RAD21, was reduced in cells from patients with CIPO compared with controls. In zebrafish, suppression of rad21a reduced expression of runx1; this phenotype was corrected by injection of human RAD21 mRNA, but not with the mRNA from the mutated p.622 allele. rad21a Morpholino zebrafish had delayed intestinal transit and greatly reduced numbers of enteric neurons, similar to patients with CIPO. This defect was greater in zebrafish with suppressed expression of ret and rad21, indicating their interaction in the regulation of gut neurogenesis. The promoter region of APOB bound RAD21 but not RAD21 p.622 Ala>Thr; expression of wild-type RAD21 in HEK293 cells repressed expression of APOB, compared with control vector. The gut-specific isoform of APOB (APOB48) is overexpressed in sera from patients with CIPO who carry the RAD21 mutation. APOB48 also is overexpressed in sporadic CIPO in sera and gut biopsy specimens. Conclusions Some patients with CIPO carry mutations in RAD21 that disrupt the ability of its product to regulate genes such as RUNX1 and APOB. Reduced expression of rad21 in zebrafish, and dysregulation of these target genes, disrupts intestinal transit and the development of enteric neurons.
Open Access
Revisiting the complex architecture of ALS in Turkey: expanding genotypes, shared phenotypes, molecular networks, and a public variant database
(John Wiley and Sons, 2020) Tunca, C.; Şeker, T.; Akçimen, F.; Coşkun, C.; Bayraktar, E.; Palvadeau, R.; Zor, S.; Koçoğlu, C.; Kartal, E.; Şen, N. E.; Hamzeiy, H.; Özoğuz-Erimiş, A.; Norman, Utku; Karakahya, Oğuzhan; Olgun, Gülden; Akgün, T.; Durmuş, H.; Şahin, E.; Çakar, A.; Başar-Gürsoy, E.; Babacan-Yıldız, G.; İşak, B.; Uluç, K.; Hanağası, H.; Bilgiç, B.; Turgut, N.; Aysal, F.; Ertaş, M.; Boz, C.; Kotan, D.; İdrisoğlu, H.; Soysal, A.; Uzun-Adatepe, N.; Akalın, M. A.; Koç, F.; Tan, E.; Oflazer, P.; Deymeer, F.; Taştan, Ö.; Çiçek, A. Ercüment; Kavak, E.; Parman, Y.; Başak, A. N.
The last decade has proven that amyotrophic lateral sclerosis (ALS) is clinically and genetically heterogeneous, and that the genetic component in sporadic cases might be stronger than expected. This study investigates 1,200 patients to revisit ALS in the ethnically heterogeneous yet inbred Turkish population. Familial ALS (fALS) accounts for 20% of our cases. The rates of consanguinity are 30% in fALS and 23% in sporadic ALS (sALS). Major ALS genes explained the disease cause in only 35% of fALS, as compared with ~70% in Europe and North America. Whole exome sequencing resulted in a discovery rate of 42% (53/127). Whole genome analyses in 623 sALS cases and 142 population controls, sequenced within Project MinE, revealed well‐established fALS gene variants, solidifying the concept of incomplete penetrance in ALS. Genome‐wide association studies (GWAS) with whole genome sequencing data did not indicate a new risk locus. Coupling GWAS with a coexpression network of disease‐associated candidates, points to a significant enrichment for cell cycle‐ and division‐related genes. Within this network, literature text‐mining highlights DECR1, ATL1, HDAC2, GEMIN4, and HNRNPA3 as important genes. Finally, information on ALS‐related gene variants in the Turkish cohort sequenced within Project MinE was compiled in the GeNDAL variant browser (www.gendal.org).