Microfluidic platform for next-generation sequencing library preparation with low-input samples. Microfluidic platform for next-generation sequencing library preparation with low-input samples

Advances in Next Generation Sequencing (NGS) have made available a wealth of information that had previously been inaccessible to researchers and clinicians. NGS has been applied to understand genomic, transcriptomic, and epigenomic changes and gained traction as a significant tool capable of accelerating diagnosis, prognosis, and biomarker discovery. However, these NGS assays have yet to be practical methods for patient stratification or diagnosis because of the gap between the tiny quantities of biomaterials provided by a clinical sample and the large DNA input required by most of these assays. Current library preparation methodologies typically require large input amounts of DNA and a long and complicated manual process. Here we present a microfluidic reactor system for NGS library preparation, capable of reducing the number of pipetting steps significantly, automating much of the process, while supporting extremely low DNA input requirement (10 pg per library). This largely automated technology will allow for low-input preparations of 8 libraries simultaneously while reducing batch to batch variation and operator hands-on time. Overall design: We examined library preparation efficiency using various ChIP DNA amounts looking at genome wide H3K4me3 profiles of GM12878 cell line. The system was further validated using ChIP DNA samples prepared from prefrontal cortex (PFC) of 10-week old male CD-1 mice, exploring different histone modifications: H3K4me3, H3K9me3, H3K36me3, and H3K79me2.

下一代测序（Next Generation Sequencing，NGS）技术的进步为研究人员与临床医师带来了此前难以获取的海量研究与临床信息。NGS已被广泛应用于解析基因组、转录组及表观基因组的变化，并作为能够加速诊断、预后评估与生物标志物发现的重要工具得到学界广泛认可。然而，临床样本所能提供的生物材料体量极小，而大多数此类NGS检测方法均需大量DNA输入量，二者间存在显著的体量不匹配问题，导致这类检测尚未能成为用于患者分层或临床诊断的实用手段。当前的NGS文库制备方法通常需要大量DNA输入量，且操作流程冗长复杂。本文介绍一种用于NGS文库制备的微流体反应器系统，该系统可显著减少移液操作步骤，实现流程的高度自动化，同时支持极低的DNA输入需求（每个文库仅需10 pg）。这项高度自动化的技术可同时完成8个文库的低起始量制备，同时有效降低批次间的实验差异与操作人员的实操耗时。
整体实验设计：我们通过设置不同体量的染色质免疫沉淀（Chromatin Immunoprecipitation，ChIP）DNA输入量，检测GM12878细胞系的全基因组H3K4me3修饰谱，以此评估该系统的文库制备效率。本系统进一步使用从10周龄雄性CD-1小鼠前额叶皮层（prefrontal cortex，PFC）中制备的ChIP DNA样本进行验证，探究了H3K4me3、H3K9me3、H3K36me3及H3K79me2等多种组蛋白修饰的检测效果。