A quantitative Reduced Representation Sequencing (qRRS) of genomes; a paradigm shift in NGS-based genotyping

Next Generation Sequencing (NGS) is an extensively used tool for massive parallel sequencing of genomes. However, several applications such as NGS-based genotyping still suffer from pitfalls that limit their accuracy and utility. To mitigate these pitfalls, we have developed a significant advancement in short-read NGS library preparation. Presented here is an inexpensive quantitative reduced representation sequencing (qRRS) approach for dosage-sensitive genotyping and quantitative strain-level metagenome/microbiome profiling. The scalable, ligation-free and double-stranded DNA-protection assay eliminates off-target annealing temperature-dependent hybridization. This is achieved by using single-stranded barcoded adapters for isothermal strand displacement of double-stranded DNA templates with restriction site overhangs as the only priming site. As much as 9,216 samples can be multiplexed. Novel features in this protocol include a paradigm shift in adapter design that prevents chimeric reads and barcode swapping, a flow cell cluster enhancer that generates about 50% more yields, and consistent high-quality base calling scores. The library preparation workflow is optimized for ease-of-use and can be completed in one to two days. To accommodate these novel features during data pre-processing and downstream analytics, we have developed bioinformatic and analytical pipelines for empirical-based NGS data quality filtering (ngsComposer), haplotype-based variant calling and filtering (GBSapp), and quantitative metagenomic alignment and taxonomic exact matching (Qmatey). The qRRS approach establishes new standards in high-fidelity quantitative genotyping, minimizes missing data and allelic dropout, and makes functional microbiome studies more accessible. Compared to 16S amplicon sequencing, which uses a single gene for microbiome profiling, qRRS provides multiple genome-wide sequences for strain-level taxonomic delineation and quantification. We are now exploring its utility as a diagnostic tool for scoring multiple diseases (and disease complexes) based on titer levels of pathogens in a single assay. We envision that the enhanced quality and quantity of qRRS-derived markers will improve genomic-assisted breeding efforts.

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Author(s): Bode A. Olukolu, G. Craig Yencho

Publication: Tree Improvement and Genetics - Southern Forest Tree Improvement Conference - 2021