Exploiting Genome Variation to Improve Next-Generation Sequencing Data Analysis and CRISPR gRNA Design in Populus tremula x alba 717-1B4

The genetic diversity of Populus species makes the genus a rich resource for functional genomic studies and agronomic trait improvement. However, sequence variation between the reference (P. trichocarpa) genome and that of the routinely transformed genotype (P. tremula x alba 717-1B4) poses significant challenges for research.  Techniques such as quantitative PCR, next-generation sequencing data analysis, and guide RNA (gRNA) design in CRISPR (clustered regularly interspaced short palindromic repeats)-based genome editing all rely on high sequence specificity. We generated more than 94M paired-end reads (~40X) of 717 genomic sequences, but only half of them mapped onto the reference genome.  As de novo genome assembly is impractical, we applied established variant calling pipelines to process genomic and transcriptomic sequences, and identified over 10 million SNPs and small indels.  By substituting the variants into the reference genome, we generated a custom 717 genome (Pta717s). When applied to RNA-Seq data analysis, the fraction of uniquely mapped reads increased by 10-20% relative to that obtained with the P. trichocarpa reference genome, depending on read length and sequence type (single read or paired end.  The increased mapping rates enabled detection of 500-1500 more genes. For single-end, 50 bp ChIP-Seq data, the mapping rate increased by 10%. A website was developed for querying and searching the Pta717s genome by gene model ID or sequence. The custom genome has been instrumental in the design of CRISPR gRNAs for efficient genome editing in 717. The same approach should be applicable to other outcrossing species with a single sequenced genome.