Rapid Donor-Specific Single Nucleotide Variation Detection by Nanopore Sequencing of Ex Vivo Lung Perfusate

Abstract

<h3>Purpose</h3> 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 (<b>Fig A</b>). <h3>Methods</h3> 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 (<b>Fig B</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. <h3>Results</h3> 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 (<b>Fig C</b>). 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. <h3>Conclusion</h3> 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.