Aim: The MinION Mk1B system from Oxford Nanopore Technologies (ONT) has allowed for the possibility of rapid NGS typing of deceased donors in a timely manner. This system also allows for re-use of the flow cells for subsequent HLA typings. One concern has been the possibility of residual DNA from a previous typing possibly contaminating a subsequent run.
Method: We undertook a series of HLA typings followed by a "blank run" of saline only on one MinION flow cells in a series of seven typings followed by seven blank runs. ONT, as well as the various HLA-specific vendors, have wash protocols to limit or prevent such DNA carryover. Our laboratory decided to see if such wash steps are sufficient to prevent carryover. We performed 7 typings by ONT MinION NGS, utilizing the GenDx NGS-Turbo® HLA typing system. After each typing, the appropriate cleaning of the MinION flow cell was performed according to the manufacturer’s instructions. After washing, a typing run was initiated with no DNA to see if residual HLA-specific DNA related to the previous sample typed could be detected.
Results: In six of the seven blank samples tested, there was HLA-specific DNA able to be detected. The average number of reads for the patient typing was 21,900 reads, while the blank run averaged 1,600 reads. Every blank sample that had runs detected, had a corresponding allele that was detected in the actual patient sample. Four of the seven samples had all loci from the sample detected in the blank; whereas two blanks detected only some of the loci and one blank run detected no previously typed HLA from the patient sample. Since the residual DNA only had about 7% of the reads found in the patient sample, it is not known if the residual DNA would affect a typing from a full sample, only that such DNA can be detected in a blank run.
Conclusion: It appears, based on our initial tests, that there is indeed DNA remaining after the flow cell wash that can be detected on a blank run afterwards. Since the residual DNA is only a fraction of the DNA that is added to a flow cell for an HLA typing, additional tests are being conducted to determine if carryover DNA from previous HLA typings is an issue that would prohibit HLA typing from previously used MinION flow cells, especially if the following typing contains homozygous loci.
