A multiplex primer design algorithm for target amplification of continuous genomic regions

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Tezin Türü: Doktora

Tezin Yürütüldüğü Kurum: Orta Doğu Teknik Üniversitesi, Mühendislik Fakültesi, Bilgisayar Mühendisliği Bölümü, Türkiye

Tezin Onay Tarihi: 2016

Öğrenci: AHMET RAŞİT ÖZTÜRK

Danışman: TOLGA CAN

Özet:

Targeted Next Generation Sequencing (NGS) assays are cost-efficient and reliable alternatives to Sanger sequencing. For sequencing of very large set of genes, the target enrichment approach is suitable. However, for smaller genomic regions, the target amplification method is more efficient than both the target enrichment method and Sanger sequencing. The major difficulty of the target amplification method is the preparation of amplicons, regarding required time, equipment, and labor. Multiplex PCR (MPCR) is a good solution for the mentioned problems. However, finding compatible multiplex pairs is an example of a clique decision problem in graph theory and it’s NP-complete by nature. We propose a novel method to design MPCR primers for a continuous genomic region, following the best practices of clinically reliable PCR design processes. On an experimental setup with 48 different combinations of factors, we have shown that multiple parameters might effect finding the first feasible solution. Increasing the length of the initial primer candidate selection sequence gives better results, whereas waiting for a longer time to find the first feasible solution does not have a significant impact. We generated MPCR primer design for the MEFV whole gene; and, our benchmarking experiments show that the proposed MPCR approach is able to produce reliable NGS assay primers for a given sequence in a reasonable amount of time.