High throughput whole mitochondrial genome sequencing by two platforms of massively parallel sequencing

Seung Bum Seo,Bobby Larue,Antti Sajantila,Jonathan King,Mourad Assidi,Bruce Budowle,Xiangpei Zeng

doi:10.1186/1471-2164-15-s2-p7

Abstract

BackgroundMitochondrial DNA (mtDNA) is a valuable genetic bio-marker that has been implicated as a prognostic/diagnosticindicator for a number of diseases [1-3] as well as forhuman identification where forensic biologic evidencecontains too little or no nuclear DNA, such as a hair shaftwithout root or a fingernail, or where a sample from a dis-tant maternal relative is the only possibility for comparison[4-6]. Sanger sequencing has been the gold standardmethod for mtDNA typing, but the methodology has lim-itations with throughput, scalability, speed, and resolution[7]. Massively parallel sequencing technology (MPS) pro-vides platforms for more comprehensive coverage of thegenome per sample analyzed than currently is possiblewith Sanger sequencing [8,9]. Moreover, a number of dif-ferent samples, which can be distinguished by barcoding,may be sequenced simultaneously. Two of the availablepersonal genome sequencers are the Ion Torrent PersonalGenome Machine (PGM™) (LifeTechnologies, San Fran-cisco, CA) and the MiSeq

Highlights

Mitochondrial DNA is a valuable genetic biomarker that has been implicated as a prognostic/diagnostic indicator for a number of diseases [1,2,3] as well as for human identification where forensic biologic evidence contains too little or no nuclear DNA, such as a hair shaft without root or a fingernail, or where a sample from a distant maternal relative is the only possibility for comparison [4,5,6]
The protocol for whole mitochondrial genome sequencing on the PGM is described on ion community
Ion XpressTM Plus gDNA Fragment Library Kit, OneTouchTM 200 Template Kit v2 and Ion PGMTM 200 Sequencing Kit were mainly used for the library preparation, template preparation and sequencing reactions, respectively

Summary

Introduction

Mitochondrial DNA (mtDNA) is a valuable genetic biomarker that has been implicated as a prognostic/diagnostic indicator for a number of diseases [1,2,3] as well as for human identification where forensic biologic evidence contains too little or no nuclear DNA, such as a hair shaft without root or a fingernail, or where a sample from a distant maternal relative is the only possibility for comparison [4,5,6]. Sanger sequencing has been the gold standard method for mtDNA typing, but the methodology has limitations with throughput, scalability, speed, and resolution [7]. A number of different samples, which can be distinguished by barcoding, may be sequenced simultaneously. The PGM exploits non-optical sequencing on CMOS integrated circuits by detecting small changes in pH, due to release of H+ during addition of a nucleotide to the growing strand within a 2 hour run time. The MiSeq uses fluorescently tagged terminator chemistry and requires 39 hours for paired-end sequencing but has higher throughput and an associated simpler, less labor intensive library preparation methodology than the PGM

Methods

Results

Conclusion