STAMP: a multiplex sequencing method for simultaneous evaluation of mitochondrial DNA heteroplasmies and content

Abstract

Human mitochondrial genome (mtDNA) variations, such as mtDNA heteroplasmies (the co-existence of mutated and wild-type mtDNA), have received increasing attention in recent years for their clinical relevance to numerous diseases. But large-scale population studies of mtDNA heteroplasmies have been lagging due to the lack of a labor- and cost-effective method. Here, we present a novel human mtDNA sequencing method called STAMP (sequencing by targeted amplification of multiplex probes) for measuring mtDNA heteroplasmies and content in a streamlined workflow. We show that STAMP has high-mapping rates to mtDNA, deep coverage of unique reads and high tolerance to sequencing and polymerase chain reaction errors when applied to human samples. STAMP also has high sensitivity and low false positive rates in identifying artificial mtDNA variants at fractions as low as 0.5% in genomic DNA samples. We further extend STAMP, by including nuclear DNA-targeting probes, to enable assessment of relative mtDNA content in the same assay. The high cost-effectiveness of STAMP, along with the flexibility of using it for measuring various aspects of mtDNA variations, will accelerate the research of mtDNA heteroplasmies and content in large population cohorts, and in the context of human diseases and aging.

Association of mitochondrial DNA content, heteroplasmies and inter-generational transmission with autism

“…Paired-end reads obtained were analyzed using “stamp toolkit” 33 (Supplementary Table 22 ). We first sorted paired-end reads into clusters of capture products according to the probe arm sequences and sample barcodes identified.…”

“…We used a residual method to compute mtDNA content from the STAMP data 33 , 72 based on the number of consensus reads from mtDNA and the number of consensus reads from nuclear DNA (Supplementary Table 22 ). To avoid variation in capture efficiency due to mtDNA polymorphisms in probe-annealing regions 33 , 72 , consensus reads used were from six target regions of probes whose arm sequences did not overlap with mtDNA polymorphisms having the minor allele detected in ≥5 (frequency > 0.2%) BBC samples. Consensus reads representing nuclear DNA were from probe-target regions as previously described 33 , 72 .…”

“…Third, methodologically, we performed ultradeep sequencing of mtDNA and systematically evaluated mtDNA variations, encompassing low-fraction mtDNA heteroplasmies, mtDNA content variations, and mtDNA inter-generational transmission and mutation patterns in both the SSC and BBC. The sequencing method (STAMP) that we developed for evaluating mtDNA heteroplasmies and content is reproducible and time- and cost-effective 33 , thus scalable in research, clinical, and public health settings.

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A Genetic Bottleneck of Mitochondrial DNA During Human Lymphocyte Development

“…Furthermore, the mutation rate in our model is sensitive to the detection of low-frequency mutations. Ultradeep sequencing of the mitochondrial genome through other single-cell mtDNA sequencing methods ( Guo et al 2020 ) would aid in estimating the mutation rate accurately and would further facilitate the estimation of selection strength.…”

“Empowering” Cardiac Cells via Stem Cell Derived Mitochondrial Transplantation- Does Age Matter?

“…By applying screening methods for mitochondria to be used, possible underlying mtDNA defects might be prevented. In this context, recently a new method for mtDNA sequencing (STAMP) has been proposed which could aid in identifying the optimal mitochondria for transplantation [ 174 ].…”