PO.MCB08.05 · 分子与细胞生物学
一套采用新型文库制备靶向富集方案、可全自动无人值守运行的系统，用于检测液体活检和肿瘤样本中的超低频突变
A fully automated walkaway system programmed with a novel library prep target enrichment protocol for detection of ultra-low frequency mutations in liquid biopsy and tumor samples
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作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
液体活检通过高灵敏度检测循环游离DNA（cfDNA）中的基因组改变，已经变革了肿瘤学领域。然而灵敏度、PCR偏倚、污染、可重复性以及冗长繁琐的工作流程等问题尚未得到解决。在此，我们对一种新型文库制备与靶向富集探针技术Agilent Avida的工作流程实现了全自动化。该技术经过高度优化以适配循环肿瘤DNA（ctDNA），从而应对现有的部分挑战。我们采用预分装试剂的自动化方案是在Agilent Magnis NGS Prep System上开发的，适用于宽范围的DNA投入量（1-100 ng）以及多种样本类型（完整DNA、FFPE DNA和cfDNA）。利用该平台，我们构建的文库具有高样本回收率，且回收率与投入量呈线性关系，无需预富集PCR即可实现高灵敏度变异检测，最大限度降低了GC偏倚。我们的自动化方案可在约8小时内生成多达8个靶向富集、可直接用于Illumina测序的DNA文库，全程无需任何用户干预，从而将潜在污染降至最低。我们使用Avida目录panel和定制panel（覆盖广泛的靶标大小范围）生成数据，以确定检测的灵敏度。使用这些探针，我们能够利用充分表征的cfDNA、甲醛受损参考标准品以及真实ctDNA样本，可靠地检测出跨越关键癌基因的癌症相关基因组改变，包括SNV、indel、CNV和易位。例如，使用15 ng的SeraCare V4 ctDNA样本，以Avida DNA Onco LB panel（1.17 Mb，覆盖164个泛癌相关基因）进行富集，并按1亿读段对的预算进行测序，我们能够检测出低至0.25%的SNP频率。此外，在我们的自动化运行中，8个技术重复之间获得了高度可重复性。例如，对于使用Avida DNA Onco LB panel、每次运行采用不同样本类型和投入量的5次独立Magnis运行（40个样本），靶向命中率的变异系数（CV）百分比为0.8%至2.3%，文库复杂度（分子标签UMI回收率）为1.9%至8.2%，碱基覆盖度为1%至3.2%，均一性为0.03%至0.09%。
查看英文原文 English abstract
Liquid biopsy has transformed oncology through high sensitivity detection of genomic alterations from circulating cell-free DNA (cfDNA). Issues such as sensitivity, PCR bias, contamination, reproducibility, and long and laborious workflows are yet to be resolved. Here, we have fully automated the workflow for a novel library preparation and target enrichment probe technology, Agilent Avida, that tackles some of the existing challenges by being highly optimized to work with circulating tumor DNA (ctDNA). Our automated protocol using pre-aliquoted reagents, was developed on the Agilent Magnis NGS Prep System for a wide range of DNA inputs (1-100 ng) and various sample types (intact, FFPE DNA, and cfDNA). Using this platform, we construct libraries with a high sample recovery that scales linearly with the input amount and is capable of high sensitivity variant detection without a need for pre-enrichment PCR, minimizing the GC bias. Our automated protocol generates up to eight target-enriched Illumina sequencing-ready DNA libraries in about 8 hours without the need of any user intervention, thus minimizing potential contamination. We have generated data using both Avida catalog and custom panels covering a broad range of target sizes to determine assay sensitivity. Using these probes, we were able to reliably detect cancer-associated genomic alterations, including SNVs, indels, CNVs, and translocations across key oncogenes using well characterized cfDNA and formalin compromised reference standards as well as real ctDNA samples. For example, using 15 ng of SeraCare V4 ctDNA sample enriched with Avida DNA Onco LB panel (1.17 Mb covering 164 pan-cancer-associated genes) and sequenced with a budget of 100 M read pairs, we were able to detect SNP frequencies down to 0.25%. Furthermore, in our automated runs, we obtained high reproducibility across 8 technical replicates. For example, for 5 independent Magnis runs (40 samples) of various sample types and inputs per run using the Avida DNA Onco LB panel, the % Coefficient of Variation (CV) ranged from 0.8 to 2.3% for on-target rate, 1.9 to 8.2% for library complexity (unique molecular identifier UMI recovery), 1 to 3.2% for base coverage, and 0.03 to 0.09% for uniformity.
利益披露 Disclosure
S. Bigdeli, None..
J. Godoski, None..
B. Buehler, None..
B. Arezi, None..
B. Clark, None..
D. Rhodes, None..
S. castaneda, None..
B. Lam, None..
Y. Bao, None..
J. zhu, None..
G. Amparo, None..
N. Mehendale, None..
K. Win, None..
K. Chapman, None.