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Pipeline publication

[…] g, see for a list of included genes. Briefly, mononuclear cells (MNCs) were isolated from bone marrow aspirates by Lymphoprep® density gradient centrifugation. Genomic DNA was extracted from 1×106 CD34- cells or MNCs using Gene Elute genomic DNA extraction kit (Sigma-Aldrich, Stockholm, Sweden). HaloplexTM target enrichment kit G9901A/B was designed using SureDesigntm wizard available by Agilent (https://earray.chem.agilent.com/suredesign/) and we achieved 99.2% coverage of the 42 selected genes. All samples were individually barcoded during enrichment and sequenced using Illumina HiSeQ 2000 system at the Sci-Life lab, Stockholm, Sweden. Sequencing reads were mapped over Human genome 19 by Bowtie and the variants were called using SAMTOOLS [, ]. The minimum of variant reads to consider was 20 with a minimum allelic burden of 5%. Sequence variations were annotated and functionally classified using ANNOVAR []. Variants previously reported as germline polymorphisms in the 1000 genome and the ESP5400 databases were excluded [, ]. Finally, variants located in none coding regions as well as synonymous variants were filtered out. Variant allele ratio was calculated for each mutation identified as number of variant reads divided by total reads., DNA was extracted from bone marrow CD34+ cells or MNCs using the QIAamp DNA extraction kit (Qiagen, Limburg, Netherlands) according to the manufacturer's protocol. A targeted ‘24 gene’ myeloid gene panel was used for the analysis of presentation samples from MDS and AML patients (n=45). Target enrichment was achieved using an in-house True SeqCustom […]

Pipeline specifications

Software tools Bowtie, SAMtools, ANNOVAR