Abstract
Next Generation Sequencing (NGS) is driving rapid advancement in biological understanding and RNA-sequencing (RNA-seq) has become an indispensable tool for biology and medicine. There is a growing need for access to these technologies although preparation of NGS libraries remains a bottleneck to wider adoption. Here we report a novel method for the production of strand specific RNA-seq libraries utilizing the terminal breathing of double-stranded cDNA to capture and incorporate a sequencing adapter. Breath Adapter Directional sequencing (BrAD-seq) reduces sample handling and requires far fewer enzymatic steps than most available methods to produce high quality strand-specific RNA-seq libraries. The method we present is optimized for 3-prime Digital Gene Expression (DGE) libraries and can easily extend to full transcript coverage shotgun (SHO) type strand-specific libraries and is modularized to accommodate a diversity of RNA and DNA input materials. BrAD-seq offers a highly streamlined and inexpensive option for RNA-seq libraries.
Highlights
Generation Sequencing (NGS) technologies have rapidly become foundational tools of genomics research (Koboldt et al, 2013)
Library Enrichment as a matter of procedure we don’t typically quantify mRNA concentration prior to library synthesis to maintain higher throughput, when beginning experiments with unfamiliar materials it can be of utility to have some idea how many enrichment cycles would be reasonable to try
To ascertain the relationship between the input mRNA concentration and the number of enrichment cycles chosen, 22 mRNA samples which were used for Digital Gene Expression (DGE) library synthesis were quantified on a Bioanalyzer using the RNA 6000 Pico kit (Agilent Technologies)
Summary
Generation Sequencing (NGS) technologies have rapidly become foundational tools of genomics research (Koboldt et al, 2013). RNA-sequencing (RNA-seq) has transformed gene expression analyses and promoted the study of non-model organisms at an unprecedented level of detail with the ability to generate transcriptome assemblies for virtually any species (Sémon, 2014). On the most commonly used Illumina platform the ability to sequence a large number of biological samples requires the creation of libraries from nucleic acid samples with specified sequence “adapters” at the termini of the molecules. There are a variety of methods available to generate adapter-added libraries from nucleic acid samples from a variety of source materials, the process still remains technically challenging, laborious, and expensive, thereby limiting widespread access to the technology. The method is optimized to create strand specific 3-prime Digital Gene Expression (DGE—providing readout from the 3′ end of the mRNA) and can be adapted for strand-specific non-DGE shotgun type (SHO) and more
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