Next-generation Sequencing Methods for Complex Communities
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Advances in sequencing technology have opened up the possibility of investigating complex communities, but deviations from homogeneity in a sample create challenges in generating and analyzing sequence data. There are two kinds of heterogeneous populations that are addressed in this dissertation: low-frequency sequence variants in a group of largely homogeneous cells and rare members in complex biological communities. It is important to be able to fully characterize the heterogeneity of a sample, as rare genetic variants may provide fuel for selection and rare members of a complex community can play critical roles. Thus, heterogeneity can have important biological roles in everything from ecological community structure to human disease development and progression. In order to assess low-frequency mutations, Paired-End Low Error Sequencing (PELE-Seq) was used. With this method, mutations occurring at frequencies as low as 1 in 10,000 were identified, including some with transcriptional consequences. To investigate rare members of a larger community, an enrichment method was developed to sequence transcripts from host-associated bacteria. Rather than having to sequence the abundant zebrafish host RNA, the enrichment protocol allowed even very minor members of the community to be efficiently sequenced, enabling a first look at the gene expression changes during colonization. This dissertation includes work from previously published co-authored material.