Development and assessment of an optimized next-generation DNA sequencing approach for the mtgenome using the Illumina MiSeq

Abstract

The development of molecular tools to detect and report mitochondrial DNA (mtDNA) heteroplasmy will increase the discrimination potential of the testing method when applied to forensic cases. The inherent limitations of the current state-of-the-art, Sanger-based sequencing, including constrictions in speed, throughput, and resolution, have hindered progress in this area. With the advent of next-generation sequencing (NGS) approaches, it is now possible to clearly identify heteroplasmic variants, and at a much lower level than previously possible. However, in order to bring these approaches into forensic laboratories and subsequently as accepted scientific information in a court of law, validated methods will be required to produce and analyze NGS data. We report here on the development of an optimized approach to NGS analysis for the mtDNA genome (mtgenome) using the Illumina MiSeq instrument. This optimized protocol allows for the production of more than 5 gigabases of mtDNA sequence per run, sufficient for detection and reliable reporting of minor heteroplasmic variants down to approximately 0.5–1.0% when multiplexing twelve samples. Depending on sample throughput needs, sequence coverage rates can be set at various levels, but were optimized here for at least 5,000 reads. In addition, analysis parameters are provided for a commercially available software package that identify the highest quality sequencing reads and effectively filter out sequencing-based noise. With this method it will be possible to measure the rates of low-level heteroplasmy across the mtgenome, evaluate the transmission of heteroplasmy between the generations of maternal lineages, and assess the drift of variant sequences between different tissue types within an individual.

Maternal age effect and severe germ-line bottleneck in the inheritance of human mitochondrial DNA

“…Before MiSeq sequening, mtDNA was amplified in two ∼9-kb amplicons (16) that were mixed at an equimolar ratio and spiked with 5% (wt/wt) of pUC18 or PhiX174 DNA, or with no spike-in. Sequencing libraries were prepared according to the customized Nextera XT protocol (57).…”

Effect of Seasonal Temperature on the Performance and on the Microbial Community of a Novel AWFR for Decentralized Domestic Wastewater Pretreatment

“…Nevertheless, most of the studies still applied the techniques of terminal restriction fragment length polymorphism (T-RFLP) [27], fluorescence in situ hybridization (FISH) [19,28], denaturing gradient gel electrophoresis (DGGE) [29][30][31] and quantitative polymerase chain reaction (q-PCR) [32]. Compared to the conventional ones, the high-throughput sequencing, especially Illumina MiSeq sequencing, is becoming one of the most popular molecular tools for microbial community analysis due to low-cost, fast turnaround time producing several gigabases of sequence and greater coverage [33]. Nevertheless, there is no study that focused on the microbial communities in the AWFR system.…”

Whole mitochondrial genome genetic diversity in an Estonian population sample

“…STS. With the utility of bench-top sequencers like Illumina MiSeq and Ion Torrent PGM (Personal Genome Machine), generation of whole mtGenome data is feasible for the application-orientated laboratory [6][7][8][9][10].…”