TY  - JOUR
T1  - A cost effective alternative method to ddRADseq library construction during size selection
TT  - ddRADseq kütüphanesi oluşturma işlemi fragman seçiminde uygun fiyatlı bir alternatif yöntem
AU  - Oral, Münevver
PY  - 2023
DA  - March
DO  - 10.12714/egejfas.40.1.03
JF  - Ege Journal of Fisheries and Aquatic Sciences
JO  - EgeJFAS
PB  - Ege Üniversitesi
WT  - DergiPark
SN  - 2148-3140
SP  - 20
EP  - 26
VL  - 40
IS  - 1
LA  - en
AB  - Next generation sequencing (NGS) technologies constitute the most powerful scientific advance of 21st century with a promise of fast and cost effective data generation in biology. Yet, up to date NGS studies remain often limited to laboratories with established resources. In the present study, we employed construction of ddRADseq library by using routine lab consumables (agarose gel electrophoresis: AGE thereafter) compared to high-tech NGS consumables (paramagnetic beads) during size selection. The ddRADseq library was constructed for sequencing size selected based on universally used paramagnetic beads, while remaining aliquot was used as a template to assess the feasibility of ddRADseq library construction using AGE for labs with limited resources. Both libraries were optimised for 15 PCR cycles indicating similarity in template intensity. Post-PCR quantification of the libraries was comparable (~10 ng.µL-1). Size distribution assessment revealed a cleaner pick at the ddRADseq library size selected manually based on AGE. Similarly, intercalating agent of Qubit confirmed the quantity of libraries was similar (>3 ng.µL-1). Although being more time consuming due to pre-electrophoresis preparations, serial wash and staining steps, ddRADseq library construction is achievable using routine lab consumables provided to supply the adaptors and PCR primers for the initial wet-lab work. These results manifest the feasibility of ddRADseq library generation for labs with limited resources.
KW  - ddRADseq lab workflow
KW  - next generation sequencing
KW  - Library preparation
KW  - Kütüphane hazırlama
KW  - ddRADseq laboratuvar iş akışı
KW  - yeni nesil dizileme
N2  - Yeni nesil dizileme (YND) teknolojileri, biyolojide hızlı ve uygun maliyetli veri üretimi vaadi ile 21. yüzyılın en güçlü bilimsel ilerlemesini oluşturmaktadır. Yine de, güncel YND çalışmaları genellikle yerleşik kaynaklara sahip laboratuvarlarla sınırlı kalmaktadır. Bu çalışmada, kütüphane fragman seçimi sırasında yüksek teknoloji ürünü YND sarf malzemelerine (paramanyetik boncuklar) kıyasla rutin laboratuvar sarf malzemelerinden (agaroz jel elektroforezi: buradan itibaren AGE) kullanarak ddRADseq kütüphaneleri oluşturuldu. Standart ddRADseq kütüphanesi, evrensel olarak kullanılan paramanyetik boncuklara dayalı olarak seçilen fragmanlarla oluşturulurken, kalan kısım, sınırlı kaynaklara sahip laboratuvarlar için AGE kullanılarak aynı fragman büyüklüğünde ddRADseq kütüphanesi yapılabilirliğini değerlendirmek için bir şablon olarak kullanıldı. Her iki kütüphane de kalıp DNA yoğunluğunda benzerlik gösteren 15 PCR döngüsü için optimize edilmiştir. Kütüphanelerin PCR sonrası yoğunlukları benzerlik gösterdi (~10 ng.µL-1). Boyut dağılımı değerlendirmesi, AGE ile manuel olarak seçilen ddRADseq kütüphane boyutunda daha temiz bir seçim olduğunu ortaya çıkardı. Benzer şekilde, Qubit ölçümleri de kütüphane DNA miktarının yakın olduğunu ortaya koydu (>3 ng.µL-1). Elektroforez öncesi hazırlıklar, seri yıkama ve boyama adımları nedeniyle daha fazla zaman almasına rağmen, ddRADseq kütüphane kurulum işlemi başlangıç için gerekli adaptör ve PCR primerlerinin sağlanması kaydıyla rutin laboratuvar sarf malzemeleri kullanılarak gerçekleştirilebilir. Bu sonuçlar, sınırlı kaynaklara sahip laboratuvarlar için ddRADseq kütüphanesi oluşturmanın uygulanabilirliğini ortaya koymaktadır.
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UR  - https://doi.org/10.12714/egejfas.40.1.03
L1  - https://dergipark.org.tr/tr/download/article-file/2600004
ER  -