University of Chinese Academy of Sciences, Beijing 100049, PR China; CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, PR China.
Beijing Huayan Judicial Authentication Institute, Beijing 100192, PR China.
Forensic Sci Int Genet. 2020 Mar;45:102211. doi: 10.1016/j.fsigen.2019.102211. Epub 2019 Nov 29.
Whole genome amplification (WGA) allows for multiple genetic analyses with low template DNA, such as DNA derived from a single cell. WGA could increase the amount of input DNA from the pg to the μg level. However, there are no studies comparing the performance of forensic markers with DNA from a single cell after WGA evaluated on both capillary electrophoresis (CE) and massively parallel sequencing (MPS) platforms. In this study, cell lines consisting of female cultured B-lymphoblastoid cells and karyocytes from male venous blood were segregated into one, two, three and five cells. Including the references with the bulk cells, all samples were generated by WGA with the multiple displacement amplification (MDA) strategy in triplicate and genotyped on CE and MPS platforms. Allele balance, stutter ratio, accuracy, repeatability and concordance of short tandem repeat (STR) markers were used to evaluate the genotyping performance on both platforms. Additionally, the sequence coverage ratio (SCR) and SNP genotypes were evaluated for sequence information generated from the MPS. Heterozygous loci showed high allele balance, with an overall average allele balance ratio larger than 0.79 on the CE and 0.75 on the MPS platforms for the venous blood cell samples; the cultured B-lymphoblastoid cell samples had ratios of 0.62 and 0.70, respectively. The stutter ratio of every source and cell number from both cell line samples were very close, ranging from 5.3%-7.2% for autosomal STRs and approximately 10 % of Y chromosomal STRs on the CE platform. The average stutter, allele, and sequence-based and length-based noise ratios were 6.6 %, 88 %, 4.7 % and 0.7 %, respectively, in the single male cell sample. SNPs also showed high consistency and intralocus balance. Our study indicated that WGA with MDA strategy works relatively well of STR and SNP genotyping with low copy number samples on CE and MPS, even with one-cell sample.
全基因组扩增（WGA）允许使用低模板 DNA 进行多种基因分析，例如来自单个细胞的 DNA。WGA 可以将输入 DNA 的量从 pg 增加到 μg 水平。然而，目前尚无研究比较在毛细管电泳（CE）和大规模平行测序（MPS）平台上评估 WGA 后来自单个细胞的法医标记物的性能。在这项研究中，由女性培养的 B 淋巴细胞系和来自男性静脉血的核细胞组成的细胞系被分离成一个、两个、三个和五个细胞。包括与 bulk 细胞的参考文献，所有样品均通过 MDA 策略的 WGA 生成，一式三份，并在 CE 和 MPS 平台上进行基因分型。等位基因平衡、重复序列比、准确性、重复性和短串联重复（STR）标记的一致性用于评估两个平台上的基因分型性能。此外，还评估了 MPS 生成的序列信息的序列覆盖率比（SCR）和 SNP 基因型。杂合位点显示出高等位基因平衡，CE 平台上静脉血细胞样品的总体平均等位基因平衡比大于 0.79，MPS 平台上的比例为 0.75；培养的 B 淋巴细胞系样品的比例分别为 0.62 和 0.70。来自两个细胞系样品的每个来源和细胞数量的重复序列比非常接近，常染色体 STR 为 5.3%-7.2%，CE 平台上的 Y 染色体 STR 约为 10%。平均重复、等位基因、基于序列和基于长度的噪声比分别为 6.6%、88%、4.7%和 0.7%，在单个雄性细胞样品中。SNP 也表现出高度一致性和基因内平衡。我们的研究表明，即使使用单细胞样本，MDA 策略的 WGA 也能在 CE 和 MPS 上对低拷贝数样本进行相对较好的 STR 和 SNP 基因分型。
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