Pacbio target sequencing of the entire genomic region encompassing CFH to CFHR5. CFH-CFHRs long read seq

Probes targeting CFH-CFHRs on the human genome reference hg19 (from chr1:196619000 to chr1:196979303) were designed using online Nimble Design Software (Roche Sequencing, Pleasanton, CA). Samples from 10 patients (new or rare SVs, n=6; heterozygous CFHR3-CFHR1 del, n=1; homozygous CFHR3-CFHR1 del, n=1; heterozygous CFHR1-CFHR4 del, n=1; CFHR3- CFHR1 del and CFHR1-CFHR4 del compound heterozygote, n=1) and 7 healthy controls (normal copy number, n=4; heterozygous CFHR3-CFHR1 del, n=3) were sequenced at the Norwegian Sequencing Centre.Libraries were prepared using the Pacific Biosciences (PacBio) protocol for Target Sequence Capture using SeqCap EZ Libraries with PacBio Barcoded Adapters. Following amplification, libraries were size selected by BluePippin with a 5 kb cut-off and then sequenced using Pacbio Sequel system. Data were obtained as multiplexed subreads and were demultiplexed with the PacBio read demultiplexer lima, retaining only those subreads with a barcode quality greater than 45.

针对人类参考基因组hg19（chr1:196619000至chr1:196979303区域）上的CFH-CFHRs基因位点，采用在线Nimble Design软件（罗氏测序，加州普莱森顿）设计了捕获探针。收集10例患者样本（含新发或罕见结构变异（Structural Variation, SV）6例、杂合型CFHR3-CFHR1缺失1例、纯合型CFHR3-CFHR1缺失1例、杂合型CFHR1-CFHR4缺失1例、复合杂合型CFHR3-CFHR1缺失合并CFHR1-CFHR4缺失1例）及7例健康对照样本（拷贝数正常4例、杂合型CFHR3-CFHR1缺失3例），于挪威测序中心完成测序。采用太平洋生物科学（Pacific Biosciences, PacBio）的靶序列捕获建库方案，使用SeqCap EZ文库（SeqCap EZ Libraries）搭配PacBio条形码接头完成文库构建。文库扩增完成后，通过BluePippin片段筛选仪以5kb为截选阈值进行片段大小分选，随后使用PacBio Sequel测序系统开展测序。所得原始数据为多路复用子读段，使用PacBio读段解多重工具lima进行解多重处理，仅保留条形码质量值大于45的子读段。