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Comparison of SureSelect and Nextera Exome Capture Performance in Single-Cell Sequencing.
Human Heredity ( IF 1.8 ) Pub Date : 2019-01-22 , DOI: 10.1159/000490506
Wendy J Huss 1 , Qiang Hu 2 , Sean T Glenn 3, 4 , Kalyan J Gangavarapu 1 , Jianmin Wang 2 , Jesse D Luce 3 , Paul K Quinn 3 , Elizabeth A Brese 5 , Fenglin Zhan 6 , Jeffrey M Conroy 3 , Gyorgy Paragh 7, 8 , Barbara A Foster 1 , Carl D Morrison 3 , Song Liu 2 , Lei Wei 9
Affiliation  

BACKGROUND Advances in single-cell sequencing provide unprecedented opportunities for clinical examination of circulating tumor cells, cancer stem cells, and other rare cells responsible for disease progression and drug resistance. On the genomic level, single-cell whole exome sequencing (scWES) started to gain popularity with its unique potentials in characterizing mutational landscapes at a single-cell level. Currently, there is little known about the performance of different exome capture kits in scWES. Nextera rapid capture (NXT; Illumina, Inc.) has been the only exome capture kit recommended for scWES by Fluidigm C1, a widely accessed system in single-cell preparation. RESULTS In this study, we compared the performance of NXT following Fluidigm's protocol with Agilent SureSelectXT Target Enrichment System (AGL), another exome capture kit widely used for bulk sequencing. We created DNA libraries of 192 single cells isolated from spheres grown from a melanoma specimen using Fluidigm C1. Twelve high-yield cells were selected to perform dual-exome capture and sequencing using AGL and NXT in parallel. After mapping and coverage analysis, AGL outperformed NXT in coverage uniformity, mapping rates of reads, exome capture rates, and low PCR duplicate rates. For germline variant calling, AGL achieved better performance in overlap with known variants in dbSNP and transition-transversion ratios. Using calls from high coverage bulk sequencing from blood DNA as the golden standard, AGL-based scWES demonstrated high positive predictive values, and medium to high sensitivity. Lastly, we evaluated somatic mutation calling by comparing single-cell data with the matched blood sequence as control. On average, 300 mutations were identified in each cell. In 10 of 12 cells, higher numbers of mutations were identified using AGL than NXT, probably caused by coverage depth. When mutations are adequately covered in both AGL and NXT data, the two methods showed very high concordance (93-100% per cell). CONCLUSIONS Our results suggest that AGL can also be used for scWES when there is sufficient DNA, and it yields better data quality than the current Fluidigm's protocol using NXT.

中文翻译:

单细胞测序中 SureSelect 和 Nextera 外显子组捕获性能的比较。

背景单细胞测序的进步为循环肿瘤细胞、癌症干细胞和其他负责疾病进展和耐药性的稀有细胞的临床检查提供了前所未有的机会。在基因组水平上,单细胞全外显子组测序 (scWES) 凭借其在单细胞水平上表征突变景观的独特潜力开始受到欢迎。目前,关于不同外显子组捕获试剂盒在 scWES 中的性能知之甚少。Nextera 快速捕获 (NXT; Illumina, Inc.) 是 Fl​​uidigm C1 推荐用于 scWES 的唯一外显子组捕获试剂盒,Fluidigm C1 是一种广泛使用的单细胞制备系统。结果 在本研究中,我们比较了遵循 Fluidigm 方案的 NXT 与安捷伦 SureSelectXT 目标富集系统 (AGL) 的性能,另一种广泛用于批量测序的外显子组捕获试剂盒。我们创建了 192 个单细胞的 DNA 文库,这些单细胞从使用 Fluidigm C1 的黑色素瘤标本生长的球体中分离出来。选择了 12 个高产细胞,并行使用 AGL 和 NXT 进行双外显子组捕获和测序。在映射和覆盖分析之后,AGL 在覆盖均匀性、读数映射率、外显子组捕获率和低 PCR 重复率方面优于 NXT。对于种系变异调用,AGL 在与 dbSNP 和转换-转换比率中的已知变异重叠方面取得了更好的性能。使用来自血液 DNA 的高覆盖批量测序的调用作为黄金标准,基于 AGL 的 scWES 显示出高阳性预测值和中到高灵敏度。最后,我们通过将单细胞数据与匹配的血液序列作为对照来评估体细胞突变调用。平均而言,在每个细胞中鉴定出 300 个突变。在 12 个细胞中的 10 个中,使用 AGL 鉴定出的突变数量高于 NXT,这可能是由于覆盖深度所致。当 AGL 和 NXT 数据充分涵盖突变时,这两种方法显示出非常高的一致性(每个细胞 93-100%)。结论 我们的结果表明,当有足够的 DNA 时,AGL 也可用于 scWES,并且它产生的数据质量比当前使用 NXT 的 Fluidigm 协议更好。当 AGL 和 NXT 数据充分涵盖突变时,这两种方法显示出非常高的一致性(每个细胞 93-100%)。结论 我们的结果表明,当有足够的 DNA 时,AGL 也可用于 scWES,并且它产生的数据质量比当前使用 NXT 的 Fluidigm 协议更好。当 AGL 和 NXT 数据充分涵盖突变时,这两种方法显示出非常高的一致性(每个细胞 93-100%)。结论 我们的结果表明,当有足够的 DNA 时,AGL 也可用于 scWES,并且它产生的数据质量比当前使用 NXT 的 Fluidigm 协议更好。
更新日期:2019-11-01
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