A cost effective alternative method to ddRADseq library construction during size selection

Münevver Oral

doi:10.12714/egejfas.40.1.03

TR EN

ddRADseq kütüphanesi oluşturma işlemi fragman seçiminde uygun fiyatlı bir alternatif yöntem

Öz

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.

Anahtar Kelimeler

A cost effective alternative method to ddRADseq library construction during size selection

Abstract

Next generation sequencing (NGS) technologies constitute the most powerful scientific advance of 21^st 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.

Keywords

Supporting Institution

ISEM, University of Montpellier

Thanks

Münevver Oral has received funding from Montpellier University of Excellence (MUSE) an Initiative for Science, Innovation, Territories, and Economy (I-SITE) of the French Investment for the Future Program under Explore#2 international mobility grant. Entire wet-lab work involved in the manuscript was performed at the genomics facility of ISEM the LabEx CeMEB (Centre Méditerranéen pour l’Environnement et la Biodiversité, Montpellier). This study was designed based on the experience of the author gathered working under guidelines of John B. Taggart, Stirling, Scotland during her PhD thesis. Author would like to thank Prof. Dr. Davut Turan for his help and guideline during fieldwork.

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Details

Primary Language

English

Subjects

Structural Biology

Journal Section

Research Article

Authors

Münevver Oral ^*
0000-0001-7318-6641
Türkiye

Publication Date

March 15, 2023

Submission Date

August 16, 2022

Acceptance Date

November 30, 2022

Published in Issue

Year 2023 Volume: 40 Number: 1

DOI

https://doi.org/10.12714/egejfas.40.1.03

IZ

https://izlik.org/JA42TC48NF

Cite

RIS / Bibtex

APA

Oral, M. (2023). A cost effective alternative method to ddRADseq library construction during size selection. Ege Journal of Fisheries and Aquatic Sciences, 40(1), 20-26. https://doi.org/10.12714/egejfas.40.1.03

Cited By

The trouts of the Marmara and Aegean Sea drainages in Türkiye, with the description of a new species (Teleostei, Salmonidae)

Zoosystematics and Evolution

https://doi.org/10.3897/zse.100.112557