Abstract


OBJECTIVE: The mitochondrial genome (mtDNA) is 16,571bp in length and there are multiple copies per cell. It contains mutations linked to diseases such as cancer, diabetes and deafness. These attributes make mtDNA an ideal model to evaluate performance metrics of whole genome amplification (WGA) technologies. Additionally, recent data suggests that mtDNA genome load may impact implantation potential of euploid embryos. The selection of embryos for IVF transfer using the additional information from mitochondria requires an accurate and high coverage WGA methodology. view more

OBJECTIVE: The mitochondrial genome (mtDNA) is 16,571bp in length and there are multiple copies per cell. It contains mutations linked to diseases such as cancer, diabetes and deafness. These attributes make mtDNA an ideal model to evaluate performance metrics of whole genome amplification (WGA) technologies. Additionally, recent data suggests that mtDNA genome load may impact implantation potential of euploid embryos. The selection of embryos for IVF transfer using the additional information from mitochondria requires an accurate and high coverage WGA methodology.
DESIGN: This study aimed to compare two different commercially available WGA kits using short and mid-range read length NGS; evaluating overall mtDNA genome coverage along with coverage of 23 common mitochondrial mutations.
MATERIALS AND METHODS: Single cells sorted from an aneuploid cell line (Coriell Institute for Medical Research) were subjected to WGA using DOPlify™ (n=2) and PicoPlex (n=2) according to manufacturer’s instructions. Nextera libraries (Illumina) were prepared with a total of 23 samples subsequently multiplexed and sequenced on a NextSeq according to standard 2x150bp protocol (Illumina). Then trophectoderm biopsies were subjected to WGA using DOPlify™ (n=3) and SurePlex (n=20) according to manufacturer’s instructions. Library generation and NGS was performed according to the standard 36bp VeriSeq protocol. The sequencing data was bioinformatically aligned to hg19 then analysed to determine mitochondrial genome coverage.
RESULTS: Overall coverage of the mtDNA genome following NextSeq NGS for the single cells analysed was on average 33x and 1763x for PicoPlex and DOPlify, respectively. NGS reads for the two DOPlifysamples covered 100% of the mtDNA genome; with minimum depth of 53 and maximum depth of 3229. The PicoPlex sequenced reads covered on average 79% of the mtDNA genome. Evaluation of DOPlifyamplified cells confirmed coverage at all 23 common mitochondrial mutation sites; with a minimum read depth of 120 and an average depth of 678 reads. For the two PicoPlex amplified cells, reads mapped to 16/23 and 11/23 of the common mutations, however for only 6 and 2 mutations the read depth was more than 50 reads. The differential coverage of the 2 different WGA technologies is normalised when the NGS read length is reduced to 36bp. A reduced number of mutations were sequenced; 9/23 and 8/23 for DOPlify and SurePlex respectively. The average maximum read depth for the detected mutations was slightly higher for DOPlify amplified samples; 21 compared 12 reads for SurePlex amplified samples.
CONCLUSIONS: While DOPlifywhole genome amplification resulted in significantly greater breadth and depth of coverage over the mitochondrial genome and replicated all 23 common mitochondrial genome mutation sites from single cells, the ability to accurately characterise mitochondrial genomes appears to be significantly limited by the use of short sequencing read length.

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