ena-STUDY-Mario Negri Institute-07-04-2021-11:19:52:313-846
Pacbio target sequencing of the entire genomic region encompassing CFH to CFHR5
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.
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.