Approaches to variant discovery for conifer transcriptome sequencing

There is a wide diversity of bioinformatic tools available for the assembly of next generation sequence and subsequence variant calling to identify genetic markers at scale. Integration of genomics tools such as genomic selection, association studies, pedigree analysis and analysis of genetic diversity, into operational breeding is a goal for New Zealand’s most widely planted exotic tree species, Pinus radiata. In the absence of full reference genomes for large megagenomes such as in conifers, RNA sequencing in a range of genotypes and tissue types, offers a rich source of genetic markers for downstream application. We compared nine different assembler and variant calling software combinations in a single transcriptomic library and found that Single Nucleotide Polymorphism (SNPs) discovery could vary by as much as an order of magnitude (8,061 SNPs up to 86,815 SNPs). The assembler with the best realignment of the packages trialled, Trinity, in combination with several variant callers was then applied to a much larger multi-genotype, multi-tissue transcriptome and identified 683,135 in silico SNPs across a predicted 449,951 exons when mapped to the Pinus taeda ver 1.01e reference.当前已有大量多样化的生物信息学工具，可用于下一代测序序列组装与亚序列变异检出，以规模化识别遗传标记。将基因组选择、关联分析、系谱分析及遗传多样性分析等基因组学工具整合至生产性育种流程中，是新西兰种植最广泛的外来针叶树种——辐射松（Pinus radiata）的育种目标之一。鉴于针叶树等巨型基因组物种尚无完整参考基因组，对多种基因型与组织类型开展RNA测序，可为下游应用提供丰富的遗传标记资源。本研究在单个转录组文库中对比了9种不同的组装软件与变异检出软件组合，结果显示单核苷酸多态性（Single Nucleotide Polymorphism, SNPs）的检出数量差异可达一个数量级（范围为8061个SNPs至86815个SNPs）。在本次测试的软件包中，重比对效果最佳的组装软件Trinity与多款变异检出工具联用，随后被应用于规模更大的多基因型、多组织转录组数据集；当比对至火炬松（Pinus taeda）ver 1.01e参考基因组时，共在预测的449951个外显子中识别出683135个虚拟SNPs。