Avaliação da tecnologia Oxford Nanopore para análise de identidade genética de clones de cana-de-açúcar (Saccharum spp.)

• Autor(a) principal: Borges, Sâmella de Souza
• Data de Publicação: 2020
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UFG
• Texto Completo: http://repositorio.bc.ufg.br/tede/handle/tede/10664
• topic: Oxford Nanopore technologies; Genotipagem por sequenciamento; Identificação clonal; Oxford Nanopore technologies; Genotyping by sequencing; Clonal identification; CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL

Brazil is currently the world's largest producer of sugarcane, which is the raw material for two important products for the Brazilian economy: sugar and ethanol. Aiming both to assess the Oxford Nanopore technology for genotyping applications for sequencing (Genotyping By Sequencing - GBS), with the differential to obtain a high sequencing coverage (> 1,000x), how to develop a GBS platform to to identify sugarcane clones, safely and conveniently, this work used the MinION platform to perform genotyping based on the sequencing of 48 individuals. For this, using the genome of Sorghum bicolor as reference and a set of libraries of sugarcane transcripts, 20 pairs of primers were designed, which were used to obtain amplicons, in which SNPs molecular markers were identified (Single Nucleotide Polymorphisms). Six sequencing libraries were built, the first two being used in pilot trials. The alignment of the sequences obtained in the reference genome was performed using the BWA program. The identification and genotyping of the SNPs was performed using the SAMtools software. The identification of the sugarcane clones was done by calculating the genetic distance between individuals. The cluster analysis was performed using a script written in software R. The sequencing resulted in approximately 841 thousand sequences. The average size of the amplicons was 1.6 kb. High sequencing coverage (average 10,498X / amplicon) was obtained. Nine amplicons were selected, in which 356 SNPs sites were identified. The percentage of mismatches between the obtained and the reference sequences varied from 8% to 20% and the percentage of indels remained homogeneous (~ 6%). The duplicates of the same individual used as biological control formed a knot with a consistency of 94% in the obtained dendrogram, however they did not present perfect genetic identity between them. It is suggested that this fact is mainly associated with the high rate of sequencing error of the Oxford Nanopore sequencing technology, evidencing the difficulty of its use in applications that require a genetic identification with a high degree of security, as occurs in problems involving clonal identification in sugar cane.