A method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome

Abstract

Methods to reconstruct the mitochondrial DNA (mtDNA) sequence using short-read sequencing come with an inherent bias due to amplification and mapping. They can fail to determine the phase of variants, to capture multiple deletions and to cover the mitochondrial genome evenly. Here we describe a method to target, multiplex and sequence at high coverage full-length human mitochondrial genomes as native single-molecules, utilizing the RNA-guided DNA endonuclease Cas9. Combining Cas9 induced breaks, that define the mtDNA beginning and end of the sequencing reads, as barcodes, we achieve high demultiplexing specificity and delineation of the full-length of the mtDNA, regardless of the structural variant pattern. The long-read sequencing data is analysed with a pipeline where our custom-developed software, baldur, efficiently detects single nucleotide heteroplasmy to below 1%, physically determines phase and can accurately disentangle complex deletions. Our workflow is a tool for studying mtDNA variation and will accelerate mitochondrial research.

Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging

“…For this test data, sequencing reads were simulated from template genomes in which deletion events were introduced beginning at bp 5547 and extending 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 kbp with read lengths and an error profile based on our sequencing data (representative aligned reads in Supplemental Figure 1). When test data was aligned with Minimap2 and deletions called based on the CIGAR sequence of the primary alignment as previously utilized to call mtDNA deletions in nanopore data [20,27], there was a rapid decline in detection with increasing deletion size (Figure 2B). Closer examination of aligned data demonstrated that many reads containing large deletions were aligned as chimeric alignments with a “primary” alignment and a non-overlapping “Supplemental” alignment.…”

“…Deletions were first identified through parsing of the CIGAR sequence from the primary alignment of each read as previously reported [20,27]. We identified a total of 10,712 deletion events greater than 100 bp across all 15 samples, ranging from 101 bp to 8131 bp.…”

Affordable de novo generation of fish mitogenomes using amplification‐free enrichment of mitochondrial DNA and deep sequencing of long fragments

“…We expect a few scattered errors in the homopolymeric regions in the mitogenomes generated herein as shown in De Vivo et al (2022) (sequencing error rates were ≥2% at the time of this study with Guppy5sup basecalling and R9.4 flow cells), but these are to be fixed as the base calling algorithms evolve or ultimately with the new sequencing kits and flow cells that increase yield and accuracy and allow for sequencing speed control (SQK‐LSK114 and flow cell R10.4) that were just released at the time of finalizing this manuscript, which were shown to reach >99% accuracy. These improvements will presumably allow single nucleotide variants and possible heteroplasmy detection (Keraite et al, 2022). On the other hand, the genomes generated in this study are likely to be more accurate than previous efforts since the methods overcomes the difficulty of sequencing the tandem repeats and long insertions, repetitions and rearrangements that can take place in vertebrate genomes (Formenti et al, 2021), provide read depth, favour long fragments of DNA and augment the proportion of the target.…”

“…At the time of these experiments a barcoding kit or protocol for mitosequencing of multiple samples was not available and it was more practical to carry out each mitogenome sequencing in a different run to guarantee good results and reuse the flow cell up to five times. Keraite et al (2022) ran multiple samples by using different guide RNAs for each sample of human origin. However, this is less attainable with de novo sequencing of mitogenomes of fishes given their diversity but could be useful for multiple samples of the same species.…”

“…The targeted mitosequencing approach can be further optimized by introducing some of the features used by Keraite et al (2022). They pre‐enriched by selecting the circular DNA using exonuclease V like we did here for the mitoenrichment approach with a 27%–49% of reads increase (useful for high integrity DNA only) and they also performed a digestion with proteinase K after the cleavage of the DNA to avoid the directional bias seen in our mitosequencing workflow from bound Cas9 enzymes to the DNA that increase the yield of full length reads two‐fold.…”