Universität zu Köln

Velikkakam James, Geo (2014). Method development and application of Next Generation Sequencing in forward genetics. PhD thesis, Universität zu Köln.

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Abstract

Forward genetic screens remain one of the main genetic tools to characterize gene functions in plants. Recent advances in Next Generation Sequencing (NGS) technology have greatly reduced the time required for mutant identification in forward genetic screening. The major advantage of NGS enabled mapping, known as mapping-by-sequencing, is the simultaneous marker identification and genotyping and identification of the genomic loci causing phenotypes. We have been among the first to show that mapping-by-sequencing can be performed even within the same genetic background using mutagen-induced changes as segregating markers. As a proof of this concept, we mapped a previously unknown suppressor of like heterochromatin protein1 (lhp1) mutant. We developed a computational pipeline for the same and integrated it into an existing mapping-by-sequencing pipeline called SHOREmap. Though mapping-by-sequencing is now being routinely used, less effort has been put in optimizing the experimental set-up. Therefore, we developed new computational pipeline called Pop-Seq simulator that can simulate different mapping populations and sequencing experiments. It simulates recombinant genomes by following empirical determined recombination frequency and landscape, which make simulations close to reality. Using Pop-Seq simulator we simulated different mapping-by-sequencing scenarios and created guidelines for mapping-by-sequencing experiments in Arabidopsis. Although mapping-by-sequencing has already become a standard method in Arabidopsis, the application in crops is hindered by the large genome sizes and the lack of complete reference genomes. Therefore, we have used the Pop-Seq simulator to extend our analysis on the experimental design of mapping- by-sequencing to two crop model species, rice and barley, in which next generation sequencing-based mapping becomes tangible reality. Besides, we have developed a reference-free method called NIKS (needle in the k-stack) that enables mapping-by- sequencing in species without pre-assembled reference sequence, gene annotation, or genetic map. NIKS directly compares genomes using k-mers from whole genome sequencing data to identify homozygous mutations and extend the sequence associated with mutation site by local de novo assembly. We have used ab initio gene structural prediction to annotate the effect of mutations, which led us to the identification of causal mutation. This method will facilitate mapping-by-sequencing in non-model species.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Velikkakam James, Geo geovjames@gmail.com UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-55418
Date:	1 April 2014
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	Natural sciences and mathematics
Uncontrolled Keywords:	Keywords Language Bioinformatics, mapping-by-sequencing, Next generation sequencing English
Date of oral exam:	17 January 2014
Referee:	Name Academic Title Coupland, George Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/5544