Experimental Methods for Burkitt Lymphoma Genome Sequencing Project

On this page researchers can find detailed information describing how CGCI data was generated by genomic platform, including protocols for establishing high-quality nucleic acid samples.

CGCI Sample Naming 

CGCI samples are named using a coding system specific to OCG characterization programs. Please use the OCG Sample Codes document to properly discern the metadata reiterated within the sample name. OCG Sample Codes_finalized 05 2017.pdf

Nucleic Acid Sample Processing

CGCI project teams use high-quality RNA and DNA from case-matched tumor and normal tissues to generate comprehensive genomics data. Below is a table outlining how the project team generated those samples.

General Methodology Sample Preparation Protocols
DNA/RNA AllPrep Kit (Qiagen), mirVana miRNA Isolation Kit (Ambion), High Pure miRNA Kit (Roche), and QiaAmp blood midi kit (Qiagen) Burkitt Lymphoma Data Generation Protocol IconBL
Sample Preparation Protocols

The protocol herein describes the procedures used by Nationwide Children's Hospital to process normal and disease tissues for RNA and/or DNA subsequently used for characterization in the NCI’s CGCI initiative.

The experimental protocols for the Burkitt Lymphoma (BL) project were acquired from the following manuscript.

Grande BM, Gerhard DS, Jiang A, et al. Genome-wide discovery of somatic coding and non-coding mutations in pediatric endemic and sporadic Burkitt lymphoma. Blood. March 2019; 21;133(12):1313-1324. (PMID: 30617194)

Sample processing and nucleic acid extraction

Frozen specimens were shipped to and from Nationwide Children’s Hospital (Columbus, OH) using a cryoport that maintained an average temperature of less than -180°C (SOP #308). A top and bottom histologic section were cut from tumor and uninvolved tissue (if it was to be used for healthy tissue control) for pathologic quality control review. These were either stained with H&Eor Wright-Giemsa and imaged at 40X using an Aperio AT Turbo or Aperio AT2 scanner. Images were reviewed by a board-certified pathologist to confirm that the tumor specimen was histologically consistent with Burkitt lymphoma, and that uninvolved specimens contained no tumor cells. The tumor sections were required to contain a minimum of 50% tumor cell nuclei, and less than 50% necrosis for inclusion in the study. Nearly all samples had less than 20% necrosis. RNA and DNA were extracted from frozen (SOP #305) and FFPE tumor (SOP #315-316) and normal tissue specimens (mainly blood or granulocytes) using a modification of the DNA/RNA AllPrep kit (Qiagen). Frozen samples were homogenized and applied to a Qiagen DNA column, and FFPE samples were deparaffinized and applied to a Qiagen FFPE DNA column. The flow through from the Qiagen DNA column was processed using a mirVana miRNA Isolation Kit (Ambion) for frozen tissues, and a High Pure miRNA Kit (Roche) for FFPE tissues. This latter step generated RNA preparations that included RNA <200 nt suitable for miRNA analysis. DNA was extracted from blood using the QiaAmp blood midi kit (Qiagen; SOP #307). DNA was quantified by PicoGreen assay and was resolved by 1% agarose gel electrophoresis to confirm high molecular weight fragments. A custom Sequenom SNP panel or the AmpF/STR Identifiler (Applied Biosystems) was utilized to verify tumor DNA and germline DNA were derived from the same patient. One hundred nanograms of each tumor and normal DNA were sent in duplicate to Qiagen for REPLI-g whole genome amplification using a 100 μg reaction scale. RNA was quantified by measuring Abs260 with a UV spectrophotometer, and integrity was measured using the RNA6000 nano assay (Agilent) to determine the RNA Integrity Number (RIN) for frozen samples or DV200 for FFPE samples. For inclusion in the discovery set, a tumor needed to pass pathology consensus review (University of Nebraska Medical Center, Omaha, NE) and the specimen pathology quality control review (Nationwide Children’s Hospital, Columbus, OH). In addition, a primary tumor and a matched germline (blood, buccal, or uninvolved tissue) sample needed to pass the following metrics: a minimum of 0.7 μg of DNA from frozen or 0.25 μg of DNA from FFPE, and 3 μg RNA from frozen or 1 μg RNA from FFPE. The minimum RNA integrity metrics were a RIN above 7.0 or DV200 above 30. Cases that did not meet these metrics were included in the validation set if there was at least 0.7 μg of DNA from the primary tumor available for DNA sequencing. Tumor RNA sequencing was also performed for validation cases if there was sufficient RNA material.

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Last updated: November 01, 2019