TY - JOUR
T1 - High-throughput method for the hybridisation-based targeted enrichment of long genomic fragments for PacBio third-generation sequencing
AU - Steiert, Tim Alexander
AU - Fuß, Janina
AU - Juzenas, Simonas
AU - Wittig, Michael
AU - Hoeppner, Marc Patrick
AU - Vollstedt, Melanie
AU - Varkalaite, Greta
AU - Elabd, Hesham
AU - Brockmann, Christian
AU - Görg, Siegfried
AU - Gassner, Christoph
AU - Forster, Michael
AU - Franke, Andre
N1 - Publisher Copyright:
© 2022 The Author(s). Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.
PY - 2022/9/1
Y1 - 2022/9/1
N2 - Hybridisation-based targeted enrichment is a widely used and well-established technique in high-throughput second-generation short-read sequencing. Despite the high potential to genetically resolve highly repetitive and variable genomic sequences by, for example PacBio third-generation sequencing, targeted enrichment for long fragments has not yet established the same high-throughput due to currently existing complex workflows and technological dependencies. We here describe a scalable targeted enrichment protocol for fragment sizes of >7 kb. For demonstration purposes we developed a custom blood group panel of challenging loci. Test results achieved > 65% on-target rate, good coverage (142.7×) and sufficient coverage evenness for both non-paralogous and paralogous targets, and sufficient non-duplicate read counts (83.5%) per sample for a highly multiplexed enrichment pool of 16 samples. We genotyped the blood groups of nine patients employing highly accurate phased assemblies at an allelic resolution that match reference blood group allele calls determined by SNP array and NGS genotyping. Seven Genome-in-a-Bottle reference samples achieved high recall (96%) and precision (99%) rates. Mendelian error rates were 0.04% and 0.13% for the included Ashkenazim and Han Chinese trios, respectively. In summary, we provide a protocol and first example for accurate targeted long-read sequencing that can be used in a high-throughput fashion.
AB - Hybridisation-based targeted enrichment is a widely used and well-established technique in high-throughput second-generation short-read sequencing. Despite the high potential to genetically resolve highly repetitive and variable genomic sequences by, for example PacBio third-generation sequencing, targeted enrichment for long fragments has not yet established the same high-throughput due to currently existing complex workflows and technological dependencies. We here describe a scalable targeted enrichment protocol for fragment sizes of >7 kb. For demonstration purposes we developed a custom blood group panel of challenging loci. Test results achieved > 65% on-target rate, good coverage (142.7×) and sufficient coverage evenness for both non-paralogous and paralogous targets, and sufficient non-duplicate read counts (83.5%) per sample for a highly multiplexed enrichment pool of 16 samples. We genotyped the blood groups of nine patients employing highly accurate phased assemblies at an allelic resolution that match reference blood group allele calls determined by SNP array and NGS genotyping. Seven Genome-in-a-Bottle reference samples achieved high recall (96%) and precision (99%) rates. Mendelian error rates were 0.04% and 0.13% for the included Ashkenazim and Han Chinese trios, respectively. In summary, we provide a protocol and first example for accurate targeted long-read sequencing that can be used in a high-throughput fashion.
UR - http://www.scopus.com/inward/record.url?scp=85134599848&partnerID=8YFLogxK
U2 - 10.1093/nargab/lqac051
DO - 10.1093/nargab/lqac051
M3 - Journal articles
AN - SCOPUS:85134599848
SN - 2631-9268
VL - 4
JO - NAR Genomics and Bioinformatics
JF - NAR Genomics and Bioinformatics
IS - 3
M1 - lqac051
ER -