Robust DNA Isolation and High-Throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 hours, with only 8 hours of active hands-on time with minimal modifications.

植物标本馆（Herbarium）是可支撑多种生物学研究的珍贵植物材料资源库。利用植物标本馆标本开展研究时往往面临多重挑战，包括样本保存质量不均、DNA降解严重，以及对珍稀标本实施破坏性采样的问题。为了在大规模测序项目中更高效地利用植物标本馆材料，亟需开发一种可靠且可扩展的DNA提取与文库制备方案。本研究报道了一套适用于植物标本馆标本的稳健全流程DNA提取及高通量文库构建方法，该方案无需针对单个样本进行个性化调整。此方案专为低质量干燥植物材料优化，通过优化组织研磨流程、调整文库片段筛选策略，并为低产出文库引入可选的重扩增步骤，对现有技术方法进行了针对性改良。对低产出DNA文库进行重扩增，可挽救来自不可替代且具有潜在研究价值的植物标本馆标本的样本，无需额外开展破坏性采样，且不会在常见系统发育分析应用中引入可检测的测序偏差。该方案已在数百种禾本科植物样本上完成验证测试，经进一步确认后有望适配其他植物类群的研究需求，不过该方案仍存在一定局限性：当DNA片段极度降解无法获取目标长度范围的片段时，或当部分植物材料中含有干扰DNA纯净提取的次生代谢产物时，其应用可能受到限制。总体而言，本方案提供了一种快速且全面的方法，可在13小时内完成24个样本的DNA提取与文库制备，仅需8小时的手动操作时长，且仅需极少量个性化调整。