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Shelley, Nolan

University of British Columbia

In the past decade, the decrease in cost, and increase in accuracy of third-generation sequencing has enabled the assembly of highly contiguous, chromosome-length scaffolds of complex eukaryotic genomes, particularly when used in conjunction with Hi-C or optical mapping. While the use of linkage mapping in the improvement of eukaryotic genomes has fallen out of favour because of this, it has the added benefit of simultaneously providing a snapshot of the recombination landscape of the organism of interest. Despite this, there does not exist an automated pipeline that uses linkage data to make updates to an existing reference genome based on statistically significant deviations from the linkage expected based on true contiguity. In this thesis, a protocol is described which uses genotyped haploid offspring from a single pair of parents to reassemble a reference genome in precisely this way. It is unique in its ability to properly account for samples of unknown tetrad origin, uncertainties in genotype due to contamination and low sequencing depth, a lack of training data for variant position filtration and linkage between pairs of chromosomes caused by structural variation in the parents. We used the protocol to reassemble the v5 reference genome of the unicellular green alga, Chlamydomonas reinhardtii, by genotyping 481 low depth (~1X) offspring from the mating of the laboratory strain, 4286, with the highly divergent natural strain, J356. The results were compared to the recently published v6 reference genome, assembled using long reads from the PacBio Sequel platform. All changes made were identical to those in v6, except in the centromere of chromosome 15, which was unable to be reassembled due to a lack of recombination. Our genome was also compared to a new, zero-gap assembly of the CC1690 strain. Offspring reads aligned to this CC1690 assembly were used to identify crossover events. Discrepancies in crossover distribution between chromosomes 3 and 12, and the rest of the genome, combined with the identification of a central, linked region between this pair of chromosomes, suggested the presence of a large translocation between chromosomes 3 and 12 in the parents, to be confirmed in a follow-up study.

Text; Dataset

2024-06-06

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/88413

Botany

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/