Rapid sequencing and identification for 18-STRs long amplicon panel using portable devices and nanopore sequencer.

Abstract

STRs are the most commonly used forensic genetic markers for human identification. Nanopore sequencing has shown the advantages of high portability and large data throughput. Previous studies indicate it has great potential for profiling STRs based on the ligation library preparation method. However, this method, which requires more library preparation time and operations, is unsuitable for rapid STR profiling, particularly for field forensic applications. The transposase-based rapid library preparation method offers the possibility to perform human identification using portable instruments. However, the amplicons of conventional STR panels are too small and would be cut into scraps with rapid methods, making them impractical for genotyping. In this study, we developed an 18-STRs multiplex amplification panel with amplicons of ∼1.4 Kbp. The PCR conditions were optimized to be finished within 2 h and 12 min, and the PCR products could undergo rapid methods that involved random fragmentation. We found that, on average, 29.16 % of reads from the long-amplicon panel and rapid library kit covered the whole STR region, sufficient for downstream STR profiling analysis. We conducted a small validation experiment on 24 samples using portable instruments powered by a 1.5 kW‧h portable power source. The entire process took 10.5 h and we obtained enough data from 24 samples to perform trustworthy pairwise identification analysis using the STR profiles. The overall accuracy of the analysis was 95.36 %. In sum, the study evaluated and demonstrated the viability and potential of nanopore sequencing for forensic application in the field.