Computational protocol: Genomic complexity and dynamics of clonal evolution in childhood acute myeloid leukemia studied with whole-exome sequencing

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Protocol publication

[…] Total DNA was extracted from BM leukemia/mononucleated cells of the 4 AML patients by QIAamp DNA Mini kit (Qiagen) and exome library preparation was performed by Nextera Rapid Capture Enrichement kit (Illumina, San Diego, CA) according to the manufacturer's recommendations. Bridge amplification was conducted through cBot cluster amplification system/TruSeq PE Cluster Kit v3-cBot-HS (Illumina). Sequencing by synthesis was performed on HiScanSQ sequencer (Illumina) at 100 bp in paired-end mode. After adapter and quality trimming, implemented by AdapterRemoval algorithm [], reads were aligned with Burrows-Wheeler Aligner [] to the reference human genome hg19/GRCh37. Genome Analysis Toolkit (GATK) (Haplotype Caller) and MuTect packages were used to detect Ins/Dels and SNVs respectively from exome-seq data [] []. Variants present in dbSNP database (http://www.ncbi.nlm.nih.gov/project/SNP), 138 edition, and in 1000 Genomes database (http://wwww.1000genomes.org), February 2012 edition with frequency greater than 1% were excluded. Synonym SNVs were excluded, thus resulting in evaluating only non-synonymous SNVs, nonsense SNVs, SNVs at splicing sites and Ins/Dels, both frameshift and non-frameshift. In further analysis, only high-quality somatic mutations were considered, according to their absence in the control sample obtained at time of complete remission from the corresponding patient (for detailed criteria refer to Supplementary Information). The effect of mutations at the protein level was predicted with the computational tools SIFT and PROVEAN []. Copy number alterations were detected by Control-FREEC Software [] on WES data, comparing primary and relapse samples to remission sample of corresponding patient. […]

Pipeline specifications

Software tools AdapterRemoval, BWA, GATK, MuTect, PROVEAN, Control-FREEC
Databases dbSNP
Application WES analysis
Organisms Homo sapiens
Diseases Leukemia, Leukemia, Lymphoid, Leukemia, Myeloid, Acute