Apostle MiniMax 技术

High-throughput sample processing for methylation analysis in an automated, enclosed environment. Alejandro Stark, Thomas R. Pisanic, James G. Herman, Tza-Huei Wang. SLAS Technology Volume 27, Issue 3, June 2022, Pages 172-179. 

作者单位：美国约翰霍普金斯大学、匹兹堡大学联合研究组。

(Figure 4) - "Most notably, the Apostle particles outperformed all others, achieving almost 2-fold higher recovery yields than the particles supplied in the X kit. "

论文第6页图4 结果, " 尤为值得关注的是，Apostle 纳米磁珠表现优于其他所有方法，取得了比 X 试剂中提供的纳米磁珠几乎2倍的回收率。“ 

论文摘要

Variation in methylcytosine is perhaps the most well-studied epigenetic mechanism of gene regulation. Methods that have been developed and implemented for assessing DNA methylation require sample DNA to be extracted, purified and chemically-processed through bisulfite conversion before downstream analysis. While some automated solutions exist for each of these individual process steps, a fully integrated solution for accomplishing the entire process in a high-throughput manner has yet to be demonstrated. Thus, sample processing methods still require numerous manual steps that may reduce sample throughput and precision, while increasing the risk of contamination and human error. In this work, we present an integrated, automated solution for performing the entire sample preparation process, including DNA extraction, purification, bisulfite conversion and PCR plate preparation within in an enclosed environment. The method employs silica-coated magnetic particles that eliminate the need for a centrifuge or vacuum manifold, thereby reducing the complexity and cost of the required automation platform. Toward this end, we also compare commercial DNA extraction and bisulfite conversion kits to identify a protocol suitable for automation to significantly improve genomic and bisulfite-treated DNA yields over manufacturer protocols. Overall, this research demonstrated development of an automated protocol that offers the ability to generate high-quality, bisulfite-treated DNA samples in a high-throughput and clean environment with minimal user intervention and comparable yields to manual processing.

Comparative analysis of cell-free DNA extraction efficiency from plasma.  Beckman Coulter Life Sciences. Indianapolis, IN. Technical Note 2022.

作者单位：美国贝克曼库尔特公司

Results and discussion

Mean cfDNA concentrations in the eluates were determined to be 8.30 ng/μL for samples extracted with the QIAamp kit and 11.15 ng/μL for samples extracted with the Apostle kit when measured with the Qubit assay (Figure 1A). This illustrates a 34.3% higher yield when using the Apostle kit as compared to the QIAamp kit. （此结果显示 Apostle 试剂盒比 X 试剂盒产率高 34.4%。）

When evaluating the contents of specific genes in the eluate, the Apostle purification displayed a higher yield than QIAamp (Figure 1B). 8358 DNAJC5G positive droplets were detected in the Apostle samples compared to 7269 droplets in the QIAamp samples. Similarly, 10849 ACTG1 droplets were detected in the Apostle-extracted samples versus 9463 droplets detected in the QIAamp-extracted samples. This data demonstrates an average of 14.8% higher yield using the Apostle kit.（此结果显示 Apostle 试剂盒比 X 试剂盒产率平均高 14.8%。）

Finally, when evaluating cfDNA fragments via the Agilent bioanalyzer instrument, the Apostle kit demonstrated the highest yield (Figure 1C and D). Mononucleosomal cfDNA concentration in the eluate of Apostle samples was estimated to be 10.64 ng/μL versus 6.26 ng/μL in the eluate of QIAamp samples. The dinucleosomal cfDNA concentration was low for both kits, with 0.13 ng/μL recorded for Apostle samples and 0.24 ng/μL for QIAamp samples. This indicates a slightly better purification of dinucleosomal cfDNA using QIAamp; however, this could also be an artifact given the low concentrations. The Apostle mononucleosomal cfDNA yield was 70.0% higher than the yield for the QIAamp samples. （ 此结果显示对于核小体cfDNA， Apostle 试剂盒比 X 试剂盒产率高 70.0%。）

A summary provided in Table 2 shows the consistently higher yield achieved by the Apostle MiniMax High Efficiency cfDNA Isolation Kit as compared to the QIAamp® circulating nucleic acid kit. The average 40% improvement in yield underscores the importance of extraction kit selection in cfDNA assay development and highlights the potential impact of cfDNA isolation efficiency in ctDNA detection. （ 表2中的数据总结显示 Apostle MiniMax 高效游离核酸分离试剂盒比 X 试剂盒展现出稳定的更高产率。平均40% 的产率提高，提示在cfDNA 试剂研发中选择分离试剂盒的重要性，并提示在游离肿瘤DNA检测中游离核酸分离效率的潜在影响。）

cfDNA Extraction Efficiency Affects NGS Data.  Han Wei, Ph.D., Beckman Coulter Life Sciences. Indianapolis, IN. Technical Note 2020.

作者单位：美国贝克曼库尔特公司

Figure 1. cfDNA extraction method affects allele frequency (AF%). EGFR L858R standards with AF% of 0.3%, 1.0%, and 3.0% were spiked into plasma and isolated using Product A (red) or Apostle MiniMax cfDNA isolation kit (blue and green). The eluates were analyzed by NGS (performed by the Institute B) following standard protocol and AF% calculated using their established workflow. cfDNA isolated by Apostle MiniMax cfDNA isolation kit shows higher concordance between detected AF% and expected AF%. （使用 Apostle MiniMax cfDNA 分离试剂盒得到的cfDNA展现出更高的AF% 期待值与AF%实测值的吻合度）

In order to understand how cfDNA extraction efficiency affects NGS data, we present Institute B titration data. EGFR L858R standards with an allele frequency (AF%) of 0.3%, 1.0%, and 3.0% were spiked into plasma and isolated using either Apostle MiniMaxTM High Efficiency cfDNA Isolation or Product A. The cfDNA was analyzed via NGS and AF% was calculated by Institute B. cfDNA isolated by Apostle MiniMax cfDNA isolation kit shows concordance between detected AF% and expected AF%.

For cancer samples, the allele frequency represents the percentage of sequence reads carrying a mutant allele of an individual patient’s cancer. Researchers often set thresholds for defining the variant allele frequency as mutation; therefore, low cfDNA recovery efficiency can decrease sensitivity and lead to an increase in false negatives for mutation calling.

cfDNA Extraction from Plasma for Liquid Biopsy: Apostle MiniMaxTM High Efficiency cfDNA Isolation Kit. Beckman Coulter Life Sciences, Data Sheet. 2019. 

作者单位：美国贝克曼库尔特公司

(Figure 1) "For each tube the Apostle MiniMax extracted higher total yield of DNA. "

（图1）“对于任意一种采血管，相比较其他分离方法，Apostle MiniMax 技术的 （游离）DNA 回收值均更高。”

Apostle MiniMax High Efficiency cfDNA Isolation Kit, Apostle MiniMax is a cell–free DNA (cfDNA) isolation reagent kit, built on magnetic bead–based technology. Apostle MiniMax has been demonstrated to purify cfDNA from human plasma in both manual and automated workflows. 

• Data representative of results of cfDNA extracted from 1–5mL of plasma 

• Demonstrated compatibility with a variety of collection tubes 

• cfDNA purity shown to be suitable for downstream PCR based assays