Low-pass genome sequencing of LOUCY cell line

Massive parallel sequencing (MPS) techniques are rapidly evolving and continuously improving in accuracy, speed, and cost efficiency. An important factor determining the success of a sequencing run, is the whole genome amplification (WGA) of the DNA that has to be sequenced. The different currently available WGA methods lead to amplification bias resulting in over- and under-represented regions in the genome. Nevertheless, current WGA methods, such as SurePlex (Bluegnome) and subsequent aCGH analysis, make it possible to detect copy number variations (CNVs) at a 10 Mb resolution. A more uniform WGA combined with MPS instead of aCGH, however, could make it possible to screen at a higher resolution. Recently, a new Multiple Annealing and Looping Based Amplification Cycles (MALBAC) WGA method has been published, claiming unparalleled performance. The goal of this study was to compare the well-established SurePlex and MALBAC WGA for their ability to detect CNVs. Furthermore, we compared PCR-free MPS library preparation with the standard enrichment PCR library preparation. Samples consisting of either 1, 3 or 5 cells, in triplicate, were collected from the LOUCY lymphoblastoid cell line using micromanipulation and were amplified using both WGA methods. Illumina library preparation and sequencing was performed on the WGA products.

大规模平行测序（Massive parallel sequencing, MPS）技术正快速发展，在准确性、测序速度与成本效益层面持续优化。决定测序实验成败的关键因素之一，是待测序DNA的全基因组扩增（whole genome amplification, WGA）。目前已有的各类WGA方法均会引入扩增偏倚，导致基因组区域出现覆盖度偏高或偏低的情况。尽管如此，当前主流的WGA方法（如SurePlex（Bluegnome公司产品）及后续的阵列比较基因组杂交（array Comparative Genomic Hybridization, aCGH）分析），仍可实现10 Mb分辨率的拷贝数变异（copy number variations, CNVs）检测。然而，若采用更均一的WGA方法结合MPS测序替代aCGH分析，则有望实现更高分辨率的检测。近期，一种基于多重退火与环化扩增循环（Multiple Annealing and Looping Based Amplification Cycles, MALBAC）的新型WGA方法被报道，其宣称具备优异的性能表现。本研究旨在对比当前应用广泛的SurePlex与MALBAC两种WGA方法在CNV检测方面的性能。此外，本研究还对比了无PCR的MPS文库制备方案与标准富集PCR文库制备方案的效果。本研究通过显微操作技术从LOUCY淋巴母细胞系中分别获取1、3或5个细胞的样本，每组设置3次生物学重复，并分别采用两种WGA方法进行扩增。随后对扩增产物进行Illumina文库构建与测序。