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Donor-derived cell-free DNA (ddcfDNA) has been shown to be useful in monitoring lung graft health. Utilizing nanopore sequencing at relatively low coverage, single nucleotide variant (SNV) differences were identified between donor and recipient genomes in order to distinguish between donor and recipient derived plasma cfDNA. In this study, donor SNVs were called from ex vivo lung perfusion (EVLP) perfusate cfDNA using a Nanopore sequencer and validated by short-read whole genome sequencing (WGS) to verify its accuracy. cfDNA was extracted from 11 clinical EVLP perfusate samples and sequenced using a Nanopore sequencer. SNVs were identified by comparison to a reference genome. Nanopore SNVs were filtered as homozygous calls overlapping 1000Genomes SNP database. Matching short-read whole genome sequencing was performed on 4 matching samples to act as a gold standard. Furthermore, comparing Nanopore's accuracy in detecting ddcfDNA in plasma samples after transplant (Tx) to the WGS library were performed. Nanopore sequencing yielded genomic data with a median coverage of 4.88x using a single flow cell with a median run length of 72h. The median general error rate of the sequence was 5.55%, and the median number of SNP overlap with the 1000 Genomes SNP database was 2469207. When compared to the matching WGS database, the positive predictive value (PPV) of SNVs identified from 4X, 5X, 6X, 7X, and 8X regions were 92%, 95%, 97%, 98%, 98%. (Fig A-D). The nanopore results showed comparable results to the WGS library for ddcfDNA detection in post Tx plasma (Fig E). Despite the lower sequencing accuracy and depth obtained from nanopore sequencing, a high PPV can be achieved in a set of donor-specific SNVs when appropriately filtered by read depth and overlap with SNP databases. This demonstrates potential for the use of nanopore sequencing to generate personalized donor cfDNA maps for use in post-operative donor-derived plasma cfDNA identification. [ABSTRACT FROM AUTHOR]

Donor-derived cell-free DNA (cfDNA) has been shown to be useful to monitor the health of the lung graft. To distinguish donor from recipient cfDNA in the blood, single nucleotide variations (SNV) between the genome of the donor and recipient are generally used. However, this requires whole genome sequencing which is time-consuming and costly. A recently developed sequencing technology known as nanopore sequencing may prove to be a solution. Nanopore sequencing requires minimal preparation and sequences are read out in real-time. When combined with ex vivo lung perfusion (EVLP), we hypothesize that we can rapidly generate a donor-specific SNV map by nanopore sequencing of the perfusate cfDNA. In this study, we investigated the feasibility of using the nanopore sequencer to call donor SNVs from EVLP perfusate cfDNA (Fig A). EVLP perfusate samples from 4 clinical cases were collected at 4h of perfusion. DNA was extracted and library preparation performed using the Oxford Nanopore Rapid Sequencing Kit (Fig B). To correct for the low accuracy of nanopore sequencing, identified SNVs were compared to publicly accessible databases of common SNVs (dbSNP) in the population. We further compared the SNVs identified by nanopore sequencing with short-read sequencing of the same sample in 1 case. The mean and range of reads generated in the four cases were 69k and 6k-247k respectively. The average dbSNP overlap was 16,116, with a range of 907-57,558 (Fig C). In the case where we compared to short-read sequencing, we obtained 13,976 SNV overlaps. All sequencing was possible within 4 hours of preparation from perfusate samples. The nanopore sequencer allowed us to quickly search for donor SNVs from cfDNA in EVLP perfusate. In addition, comparison with short-read sequencing data demonstrated overlap and supports this approach. Further optimization of library preparation and validation with short-read sequencing is needed. [ABSTRACT FROM AUTHOR]