Computational protocol: Burkitt lymphoma beyond MYC translocation: N-MYC and DNA methyltransferases dysregulation

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Protocol publication

[…] High-throughput RNA sequencing produced about 66 million of 75 bp paired ends reads (theoretical coverage calculated on Ref Seq transcriptome 84X). Chromosomal translocations were detected using a bioinformatic pipeline that combines results from three different fusion-detection tools (deFuse, Chimerascan and Tophat Fusion) [–] and filtered on non-tumor controls using previously sequenced control reactive lymph nodes. MYC gene expression was estimated in one MYC translocation-negative sample and in other 21 endemic Burkitt lymphomas using the transcripts parts per million (TPM) calculation method [].Single Nucleotide Variants (SNVs) and short insertions and deletions (Indels) were called using the Genome Analysis Toolkit (GATK) [] after mapping quality score recalibration and local realignment around indels. All of the mutations detected were filtered using tresholds based on quality, coverage and strand of the mapped reads and according to variants already present in public databases (Hapmap, dbSNP and 1000genome project) []. The Annovar tool [] was used for functional annotation of variants, including exonic functions and aminoacid changes. All the mutations found in the MYC gene, including variations in intergenic, intronic and UTR regions, were manually checked and explorated using the Integrative Genomic Viewer 2.03 (IGV) visualization tool []. [...] Quantitative RT-PCR was performed to validate results of both miRNA and gene expression profiling, and to assess relative expression of MYC in ten MYC translocation positive and ten MYC translocation negative cases. For validation of differentially expressed miRNAs identified by profiling, RNA samples were reverse transcribed using the Universal cDNA synthesis kit (Exiqon, Copenhagen, Denmark), according to the manufacturer’s instructions. RT-qPCR amplification was performed using microRNA LNA™ PCR primer sets (Exiqon, Copenhagen, Denmark) specific for hsa-miR-29a-b, hsa-miR-513a-5p, and hsa-miR-628-3p, and using hsa-Let-7c as a reference gene. Validation of genes potentially targeted by the differentially expressed miRNAs (DNA (cytosine-5)-methyltransferase 1 (DNMT1), 3 alpha (DNMT3A), 3 beta (DNMT3B) was also carried out by RT-qPCR using FluoCycle SYBR green (Euroclone, Celbio, Italy) in 10 MYC-translocation positive and 10 MYC-translocation negative cases according to manufacturer’s instructions. Non-neoplastic lymph nodes were meant as a negative control; HPRT was used as housekeeping gene. Primer sequences were designed using Primer-BLAST [] and are reported in Table . Differences in gene expression were calculated using the ΔΔCt method []. […]

Pipeline specifications

Software tools deFuse, chimerascan, TopHat-Fusion, GATK, ANNOVAR, IGV, Primer-BLAST
Databases dbSNP
Applications RNA-seq analysis, qPCR
Organisms Homo sapiens
Diseases Burkitt Lymphoma, Neoplasms, Lymphoma, B-Cell, Chronobiology Disorders