Computational protocol: Genomic and transcriptional landscape of P2RY8-CRLF2-positive childhood acute lymphoblastic leukemia

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Protocol publication

[…] WES was performed as described previously. Samples were prepared using Nextera Rapid Capture Exome Kit (Illumina, San Diego, CA, USA). Paired-end sequencing of 100 bp reads was performed on a HiSeq 2000 (Illumina) to obtain at least a 50 × coverage. More detailed coverage metrics computed by Picard (https://github.com/broadinstitute/picard) are provided in the , additional file 1. Mutations were validated with Sanger sequencing (). [...] Reads were aligned with BWA-MEM and mutations called with MuTect. SnpEff, dbNSFP and custom Perl scripts were used for variant annotation and filtering. WES-based CNA detection was performed with exomeCopy. [...] Total RNA was extracted from frozen primary leukemic samples (>90% blasts) upon Ficoll gradient separation using Quick-RNA MiniPrep kit (Zymo Research, Irvine, CA, USA). Selection of cases was based on the availability of material. Sequencing libraries were prepared using the NEBNext Poly(A) mRNA Magnetic Isolation Module and the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) and sequenced on a HiSeq 2000 (Illumina) at the Genomics Core Facility, EMBL, Heidelberg. RNA-Seq data analysis was performed with a custom pipeline implemented in Anduril using GSNAP for read alignment, htseq-count for read counting and DESeq2 for differential gene expression analysis. Gene set enrichment analysis was performed with GSEA. […]

Pipeline specifications

Software tools Picard, BWA, MuTect, SnpEff, exomeCopy
Databases dbNSFP
Application WES analysis
Organisms Homo sapiens
Diseases Leukemia, Lymphoma, Precursor Cell Lymphoblastic Leukemia-Lymphoma