Biyoteknolojinin Güncel Uygulamalarının Su Ürünleri Genetik Alanında Kullanılması: Yeni Nesil Dizileme Teknolojileri

Geride bıraktığımız elli yıllık süreçte DNA dizi bilgisinin belirlenmesine yönelik muazzam çaba gösterilmiştir. Geliştirilen teknikler sayesinde kısa oligonukleotidlerden milyonlarca nükleotidlik tüm genom dizilemelerini tek reaksiyonda okuyabilen platformlara geçilmiştir. Bu ilerlemeler, Yeni Nesil Dizileme (YND) teknolojilerinin piyasaya sürülmesi ile gerçekleşmiştir. Kullanılan yöntemler, temelde bir genomun indirgenmiş temsilini oluşturan rastgele kütüphaneler (RADseq, ddRADseq, 2bRADseq, CROPS ve RRL) ile belli bir bölgeyi hedef alan kütüphaneler (RNAseq) olmak üzere ikiye ayrılırlar. Örneklerin hazırlanma süreci kısaca, DNA dizisi çıkarılması hedeflenen türün genomunun restriksiyon ya da sonikasyon yöntemi ile parçalara ayrılarak bir DNA kütüphanesinin oluşturulması ve ardından yüksek üretim hacmine sahip dizileme ekipmanları ile yeni sentezlenen DNA parçalarının yüksek kapasitede (paralel olarak) dizilenmesi, takiben de tüm bu dizilerin bir araya getirilmesi (assembly making) şeklinde özetlenebilir. Bu derlemede, literatürde en fazla kullanılan ve restriksiyon temelli yöntemlerden olan RADseq ve ddRADseq yöntemleri odaklı örneklerin hazırlanması ve biyoinformatik analizleri ele alınmıştır. Ülkemizde potansiyeli henüz keşfedilmemiş olan YND teknolojilerinin su ürünleri genetik literatüründeki kullanım alanları: (i) referans genom haritaları oluşturma (fiziksel), (ii) genetik bağlantı haritalamaları (QTL haritalama), (iii) popülasyon genetiği ve filogeni, (iv) TNP chip dizaynında, (v) verifikasyon ve validasyon çalışmalarında, (vi) ıslah amaçlı genotipleme ile (vii) sürdürülebilir su ürünleri yetiştiriciliği ve çevresel etkinin en aza indirilmesi noktasında bilgilendirici genetik izlenebilirlik alt başlıklarında derlenmiştir.

Using current applications of biotechnology in aquaculture genetics: Next Generation Sequencing Technologies

There have been enormous attempts for determining DNA sequences within the last fifty years. Advances in technology have enabled the shift from the sequencing of short oligonucleotides to whole genome sequencing of millions of bases within a single reaction. Such advances have been attained with the launch of Next Generation Sequencing platforms. Techniques used involve two fundamental sections as generating a reduced representation of the genome of interest through random fragmentation based libraries (RADseq, ddRADseq, 2bRADseq, CROPS ve RRL) and target specific libraries (RNA seq). Briefly, library preparation involves fragmentation of the genomic DNA to be sequenced by using restriction digestion or sonication and then sequence massively in parallel via high-throughput sequencers and make assembly of short fragments. In the present review, RADseq and ddRADseq, the most commonly used techniques of NGS in the literature, have been focused on an explanation of library preparation and bioinformatics analyses. The potential that NGS technologies hold has not been fully understood in our country yet, the applications in aquaculture genetics are: (i) reference genome projects (physical), (ii) genetic linkage mapping (i.e. QTL mapping), (iii) population genetics and phylogeny, (iv) SNP chip design, (v) verification and validation studies, (vi) genotyping for selective breeding as well as (vii) genetic traceability studies for sustainable aquaculture and minimize environmental impacts.

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