Nanopore Sequencing: An Enrichment-free Alternative to Mitochondrial DNA Sequencing

• NCJ Number: 254115
• Author(s): Roxanne R. Zascavage; Kelcie Thorson; John V. Planz
• Date Published: 2019
• Length: 9 pages
• Grant Number(s): 2015-DN-BX-K068
• Agencies: NIJ-Sponsored
• Publication Type: Research (Applied/Empirical); Report (Study/Research); Report (Grant Sponsored); Program/Project Description
• Award: Development of PCR-Free DNA Typing Strategies via Real-Time High Throughput Sequencing

Annotation

This article describes an efficient approach to sequencing directly from genomic DNA samples without prior enrichment or extensive library preparation steps.

Abstract

Mitochondrial DNA sequence data are often utilized in disease studies, conservation genetics and forensic identification. The current approaches for sequencing the full mtGenome typically require several rounds of PCR enrichment during Sanger or MPS protocols, followed by fairly tedious assembly and analysis. In the current project, a comparison is made between libraries sequenced directly from native DNA and the same samples sequenced from libraries generated with nine overlapping mtDNA amplicons on the Oxford Nanopore MinION device. The native and amplicon library preparation methods and alternative base calling strategies were assessed to establish error rates and identify trends of discordance between the two library preparation approaches. For the complete mtGenome, 16 569 nucleotides, an overall error rate of approximately 1.00 percent was observed. As expected with mtDNA, the majority of error was detected in homopolymeric regions. The use of a modified basecaller that corrects for ambiguous signal in homopolymeric stretches reduced the error rate for both library preparation methods to approximately 0.30 percent. This study indicates that direct mtDNA sequencing from native DNA on the MinION device provides comparable results to those obtained from common mtDNA sequencing methods and is a reliable alternative to approaches using PCR‐enriched libraries. (publisher abstract modified)