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

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>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 (<jats:underline><jats:bold>s</jats:bold></jats:underline>equencing by <jats:underline><jats:bold>t</jats:bold></jats:underline>argeted <jats:underline><jats:bold>a</jats:bold></jats:underline>mplification of <jats:underline><jats:bold>m</jats:bold></jats:underline>ultiplex <jats:underline><jats:bold>p</jats:bold></jats:underline>robes) 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 PCR 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.</jats:p>

Accelerated expansion of pathogenic mitochondrial DNA heteroplasmies in Huntington’s disease

“…We developed a Python pipeline (the stamp toolkit) to process reads generated from STAMP (SI Appendix, Fig. S10) (41). In brief, paired-end reads were first demultiplexed into files for individual samples.…”

“…S15. mtDNA content was estimated by further using reads from five additional probe pairs targeting single-copy nuclear DNA regions (41). mtDNA content quantification and related quality control procedures (SI Appendix, Table S17), and comparisons with mtDNA content assessed by using a quantitative PCR-based method (SI Appendix, Table S12 and Fig.…”

“…mtDNA Variant Identification. mtDNA heteroplasmic variants (with VAF < 0.99) were determined by using the consensus reads that were aligned to mtDNA (mapping quality [MAPQ] ≥ 20; base alignment quality [BAQ] ≥ 30), and did not have an excess of nucleotide mismatches (>5 in the coding region and >8 in the D-loop region) compared to the individual's major mtDNA sequence (41). Variants were further filtered based on a list of criteria to reduce falsepositive calls, including 1) ≥100-fold (X) depth of coverage with ≥70% of the bases having BAQ ≥ 30; 2) not in low-complexity regions or low-quality sites of mtDNA; 3) ≥5 minor alleles detected; 4) a log likelihood quality score ≥5 computed with base quality scores (39); 5) comparable VAFs (Fisher's exact test, P ≥ 10 −4 , and fold change, <5) when estimated using consensus reads constructed with and without duplicate paired-end reads; for variants with VAF < 1%, VAF was at least 0.2% among consensus reads constructed with duplicate paired-end reads; 6) the detected number of minor alleles significantly larger than the expected number of errors, which was estimated at a rate of 0.02% in STAMP (SI Appendix, Fig.…”

“…Since HTT is universally expressed, and mitochondrial dysfunction has been repeatedly observed in peripheral tissues (11)(12)(13)(14)(15)(16), we surmised that HD-associated mtDNA changes can be detected in peripheral tissues and cell lines of HD patients, such as blood-derived lymphoblasts, which are readily available in large patient cohorts and thus can provide increased power for identifying mtDNA changes in HD. To test this hypothesis, we employed a sensitive mtDNA targeted sequencing approach, STAMP (sequencing by targeted amplification of multiplex probes) (41), to assess mtDNA heteroplasmies in lymphoblast and longitudinal blood samples from HD patients and healthy control individuals in the European Huntington's Disease Network's REGISTRY study (hereafter referred to as REGISTRY) (42). We achieved ultradeep sequencing coverage on mtDNA in these samples and revealed an accelerated expansion of pathogenic mitochondrial DNA heteroplasmies in HD, illustrating a molecular feature underlying HD biology.…”