Application Guide - QIAxcel Advanced

14 QIAxcel Advanced Application Guide 10/2016 Identifying rare mutations in Diamond-Blackfan anemia using target enrichment and next-generation sequencing G. Gerrard Imperial Molecular Pathology Center for Hematology, Faculty of Medicine, Imperial College of London, UK Introduction Diamond-Blackfan anemia (DBA) is a rare congenital stem cell disorder associated with monoallelic inactivating mutations in the ribosomal protein (RP) genes. It leads to bone marrow failure syndrome by causing defects in erythroid progenitor and precursor cell development (1). Loss of function mutations in 10 of the c. 80 RP genes have been definitively associated with DBA. RPS19 is mutated in up to 25% of DBA cases, and 13 other RP genes are mutated in a further 25–35%. The molecular basis of the remaining 40–50% of cases is unknown. Since such cases may harbor mutations in one or more of the remaining RP genes and such mutations may occur at very low frequencies, genetic screening using conventional Sanger sequencing on a per- exon/per-gene basis is challenging. Therefore, we developed a methodology based on custom target enrichment technology combined with high-throughput sequencing. To ensure high quality libraries, rapid quality control using the QIAxcel® Advanced System was used at several steps of the library preparation process. We chose the QIAxcel for capillary electrophoresis because it uses ready- to-run gel cartridges and has very short, automated runs that are suitable for high- throughput analysis. Next-generation sequencing was performed using MiSeq® , an Illumina® platform, to screen all 80 RP genes. This proved to be a powerful approach for finding rare mutations in a large set of genes. Materials and methods Target enrichment SureSelect® XP (Agilent® , US) was used for the target enrichment, which employed a custom designed RNA bait hybridization solution to capture the target genes, including the intronic regions and 500 bp of the flanking untranslated region. The regions of interest were collated from the Ribosomal Protein Gene Database (http://ribosome.med. miyazaki-u.ac.jp) and uploaded to the Agilent eArray design facility. Library preparation and sequencing Library preparation is a complex process with multiple steps. Therefore it is important to assess the sample quality after several steps to ensure the appropriate quality of the final libraries. DNA was purified from peripheral blood leukocytes using QIAamp® DNA Mini Kit on the QIAcube® . A 3 μl sample of gDNA was sheared using a Covaris® e220 sonication platform, and the fragment size was determined via capillary electrophoresis using the QIAxcel Advanced System. Samples were diluted 1 μl to 10 μl with DNA dilution buffer and run on the instrument with the appropriate screening kit. The AM320 method was used in combination with the 15 bp/5 kb alignment marker and a 100 bp – 2.5 kb DNA size marker.

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