Scientists in China and the UK recently published an open-access optimized protocol for RAD-seq in the Theoretical and Applied Genetics journal. The method is targeted at large studies of plants and enables users to specify sequence coverage parameters.
From lead author Ning Jiang and collaborators, “A highly robust and optimized sequence-based approach for genetic polymorphism discovery and genotyping in large plant populations” offers a step-by-step protocol. “This optimized approach provides both a computational tool and a library construction protocol, which can maximize the number of genomic sequence reads that uniformly cover a plant genome and minimize the number of sequence reads representing chloroplast DNA and rRNA genes,” the scientists write.
The challenge with using existing RAD-seq protocols for plants, according to the authors, is that chloroplast and rRNA genes can account for the majority of sequence reads in an experiment if scientists don’t adjust for them, making this process inefficient for plant population genotyping.
In the new protocol, the team employed two size selection steps using the Pippin Prep. The workflow looks like this: digestion; ligating barcoded adapters; Pippin Prep sizing; more digestion; PCR amplification; and another size-selection step. (For details, check out this workflow graphic.)
The team validated the method through analysis of six sequencing libraries “for parental lines and their segregating offspring of both diploid and tetraploid Arabidopsis and potato,” they report. They saw balanced sequence representation across the samples. “Sequence data from the optimized RAD-seq experiments shows that the undesirable chloroplast and rRNA contributed sequence reads can be controlled at 3–10 %,” they note.
For pooling, the scientists recommend a maximum of 12 samples per sequencing library to reduce the variation in
number of sequence reads per plant.