New Technology Development for Next-Generation Sequencing
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Next-Generation Sequencing (NGS) technologies have been evolving at an unparalleled pace. The ability to generate millions of base pairs of data in a short time and at lower cost than previously has led to a dramatic expansion of technologies within the field. This dissertation discusses the development and validation of new methods for assessing genomic variation, dynamic changes in gene expression, high-accuracy sequencing, and analysis of recombination events. By reducing the cost of analyzing many samples for genetic divergence by genotyping the same region of the genome in multiple samples, researchers can pursue investigations on a larger scale. Next-RAD (Nextera fragmentation with Restriction-Associated Digestion) allows analysis of a uniform subset of loci between organisms for comparison of populations by genetic differences with reduced burdens of cost and data analysis. This method was applied to the Anopheles darlingi mosquito to identify three distinct species that were thought to be a uniform population. The lowering cost of large-scale sequencing investigations allows for massively parallel analysis of genomic function in a single assay. Regulation of gene expression in response to stress is a complex process which can only be understood by analyzing many pathways in tandem. A novel method is described which quantifies on a genome-wide scale the expression of millions of randomer tags driven by associated transcriptional enhancers. This method provides novel data in the form of high-resolution analysis of gene regulation. Aside from generating novel data types, another force behind development of new technologies is to improve data quality. One limitation of NGS is the inherent error rate. PELE-Seq (Paired End Low Error Sequencing) was developed to address this problem, by employing completely overlapping paired-end reads as well as a dual barcoding strategy to eliminate incorrect sequences resulting from final library amplification. This new tool improves data quality dramatically. Finally, the rapid expansion of tools necessitates the identification of new applications for these technologies. To this end, 10x Genomics Linked-Read sequencing was employed to identify recombination events in multiple species. The haplotype-resolved nature of the data generated from such assays has many promising applications.