Molecular biology based strategies to aid assembly in de novo genome projects

This thesis presents and critically assesses work undertaken and published between 2009 and 2018. It evaluates the benefits, limitations and impact of novel approaches to next generation sequencing library construction for de novo genome projects developed by the author. Since the first fully sequenced genome was published in 1978, DNA sequencing technology has advanced rapidly and costs reduced significantly. Next generation sequencers capable of sequencing millions of DNA molecules in parallel revolutionised the genomics industry. Today, if the right strategies are adopted, prokaryotic genomes can be fully sequenced in a matter of hours for a few hundred pounds and a high degree of contiguity achieved in even the most challenging eukaryotic genomes within a few weeks for tens of thousands of pounds. Chapter 2 describes the design and application of a bespoke, high throughput bacterial artificial chromosome sequencing pipeline designed to sequence complex eukaryotic genomes harbouring a wide variety of repeat structures. Chapter 3 focuses on novel approaches to optimise insert size in amplification-free, paired-end library construction and Chapter 4 discusses innovative solutions to construct large insert, highly complex long mate pair libraries which have much tighter insert size distributions than previously published methods. Chapter 5 demonstrates the application of the methods discussed in earlier chapters in wheat de novo genome projects, highlighting the benefits the author’s approaches bring to sequencing a complex polyploid plant genome. The presented methods establish new ways of thinking about next generation sequencing library construction, pushing the boundaries of complexity and maximising spatial information. Keywords: Genome assembly, next generation sequencing, DNA, de novo, amplification-free paired-end libraries, long mate pair libraries, bacterial artificial chromosomes