Cell free DNA Methylation Measurements: Invitation to Participate in an Interlaboratory Testing Program Evaluating Methylated Cell free DNA Test Materials

Summary

NIST invites participants to an Interlaboratory Testing Program to evaluate the suitability and utility of two sets of methylated cell free DNA (cfDNA) test materials to serve as reference materials or controls for the measurements of cfDNA methylation. The study is sponsored by the Early Detection Research Network (NCI) and organized as a collaborative project developed by NIST and LGC Clinical Diagnostics (formerly known as SeraCare Life Sciences, Inc).

GOALS OF THE INTERLAB STUDY

NIST will provide two sets of methylated cell free DNA (cfDNA) test materials free of charge to the participants for their evaluation using their own measurement methods. The laboratories will measure DNA methylation using the measurement techniques of their choice and NIST will coordinate the study and provide data analysis of the data provided by the participants. The results of the interlaboratory study will be published in a peer reviewed open-source scientific journal. The results of individual laboratories will be anonymized when published. The participants will have the opportunity to be authors on the publication. Depending upon the results of the study and feedback from participants, NIST may develop reference materials for cfDNA methylation measurements.

INTERLABORATORY STUDY PARTICIPATION

Please contact Hua-Jun He (hua-jun.he [at] nist.gov (hua-jun[dot]he[at]nist[dot]gov)) if you are interested in participating. By accepting and using these materials, you acknowledge that the materials are for research purposes only and not for commercial applications. Please provide contact information of an administrative officer who will be responsible for Material Transfer Agreement (MTA) with NIST Technology Partnerships Office.

Description

For this study, one set of materials was prepared by LGC Clinical Diagnostics and contains non-methylated cfDNA based on whole genome amplification, and methylated cfDNA by in vitro methylation. Both duplicate the size range of cfDNA. The other set of materials was prepared at NIST and was made with physically sheared genomic DNA from GM24385 cell line (Coriell Institute for Medical Research) and size selected at the size of cfDNA. The sheared and size selected DNA will serve as the native state cfDNA test material, and the in vitro methylated material as methylated cfDNA. Each NIST set consists of 5 components, which was formulated by mixing the in vitro methylated cfDNA at fractions of 0%, 5%, 25%, 50%, and 100% with the native state cfDNA.

NIST CHARACTERIZATION

The cfDNA concentration was determined using absorbance (260 nm), fluorescent dye binding, and digital PCR assays for reference genes (based on assays validated at NIST). The identity of the cell lines was confirmed by STR profiling. The cfDNA methylation level was confirmed by three digital PCR assays. The whole genome general coverage of these test materials is being evaluated by whole genome sequencing and the whole genome DNA methylation is being evaluated by whole genome bisulfite sequencing.

SAMPLES

The LGC test set will consist of two vials, one containing non-methylated cfDNA and the other in vitro methylated cfDNA. Each vial will contain approximately 10 µL with 20 ng/µL or total of 200 ng of cfDNA in 1 mmol/L Tris, 0.1 mmol/L EDTA, 10 mmol/L KCl, pH 8.0 aqueous buffer. The NIST test set will consist of 5 vials (each vial will contain a different fraction of in vitro methylated cfDNA as described above) containing approximately 10 µL with 20 ng/µL or total of 200 ng of cfDNA in 1x TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 8.0). All these samples were stored at -20 ⁰C and will be shipped on freeze packs or dry ice.

RESPONSIBILITIES FOR JOINING THE INTERLABORATORY STUDY

By joining this research study and accepting the samples, you agree to the following:

1. Return the results to the NIST contact organizers within 60 days after receiving the samples. If you require additional time, contact the NIST organizers and let them know how long you will require.
2. A results template will be provided in which you will provide the details of how the samples were used and the results. If the methods and details used in the measurements are proprietary you can respond with that option and not disclose those details.
3. You agree to provide NIST with feedback on the utility of the materials for your applications. It is extremely important to NIST to learn if the materials are useful and if can they be improved.
4. Store the materials protected from light at -20 ⁰C. Analyze the samples using your measurement method at least twice, triplicate measurements are better to determine reproducibility. If possible, replicates should be done on different days, operators, and instruments.
5. Complete the results template (Goggle form) providing as much information as possible and return to NIST.
6. NIST will unblind the samples and provide you with a summary of your institution results. NIST will not reveal the results of other participants, and any reports and publications of the results will be blinded to the identity of the participants.
7. Provide the shipping address, and contact information for the person receiving the samples and the contact for the contact person responsible for reporting the results.