From Forensics to Clinical Research: Expanding the Variant Calling Pipeline for the Precision ID mtDNA Whole Genome Panel

Despite a multitude of methods for the sample preparation, sequencing, and data analysis of mitochondrial DNA (mtDNA), the demand for innovation remains, particularly in comparison with nuclear DNA (nDNA) research. The Applied Biosystems™ Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific, USA) is an innovative library preparation kit suitable for degraded samples and low DNA input. However, its bioinformatic processing occurs in the enterprise Ion Torrent Suite™ Software (TSS), yielding BAM files aligned to an unorthodox version of the revised Cambridge Reference Sequence (rCRS), with a heteroplasmy threshold level of 10%. Here, we present an alternative customizable pipeline, the PrecisionCallerPipeline (PCP), for processing samples with the correct rCRS output after Ion Torrent sequencing with the Precision ID library kit. Using 18 samples (3 original samples and 15 mixtures) derived from the 1000 Genomes Project, we achieved overall improved performance metrics in comparison with the proprietary TSS, with optimal performance at a 2.5% heteroplasmy threshold. We further validated our findings with 50 samples from an ongoing independent cohort of stroke patients, with PCP finding 98.31% of TSS’s variants (TSS found 57.92% of PCP’s variants), with a significant correlation between the variant levels of variants found with both pipelines.     

Borrowing Nuclear DNA Helicases to Protect Mitochondrial DNA

In normal cells, mitochondria are the primary organelles that generate energy, which is critical for cellular metabolism. Mitochondrial dysfunction, caused by mitochondrial DNA (mtDNA) mutations or an abnormal mtDNA copy number, is linked to a range of human diseases, including Alzheimer’s disease, premature aging‎ and cancer. mtDNA resides in the mitochondrial lumen, and its duplication requires the mtDNA replicative helicase, Twinkle. In addition to Twinkle, many DNA helicases, which are encoded by the nuclear genome and are crucial for nuclear genome integrity, are transported into the mitochondrion to also function in mtDNA replication and repair. To date, these helicases include RecQ-like helicase 4 (RECQ4), petite integration frequency 1 (PIF1), DNA replication helicase/nuclease 2 (DNA2) and suppressor of var1 3-like protein 1 (SUV3). Although the nuclear functions of some of these DNA helicases have been extensively studied, the regulation of their mitochondrial transport and the mechanisms by which they contribute to mtDNA synthesis and maintenance remain largely unknown. In this review, we attempt to summarize recent research progress on the role of mammalian DNA helicases in mitochondrial genome maintenance and the effects on mitochondria-associated diseases.     

Fine-Tuning Phylogenetic Alignment and Haplogrouping of mtDNA Sequences

In this paper, we present a new algorithm for alignment and haplogroup estimation of mitochondrial DNA (mtDNA) sequences. Based on 26,011 vetted full mitogenome sequences, we refined the 5435 original haplogroup motifs of Phylotree Build 17 without changing the haplogroup nomenclature. We adapted 430 motifs (about 8%) and added 966 motifs for yet undetermined subclades. In summary, this led to an 18% increase of haplogroup defining motifs for full mitogenomes and a 30% increase for the mtDNA control region that is of interest for a variety of scientific disciplines, such as medical, population and forensic genetics. The new algorithm is implemented in the EMPOP mtDNA database and is freely accessible.     

Progress on the Physiological Function of Mitochondrial DNA and Its Specific Detection and Therapy

Mitochondrial DNA (mtDNA) is the genetic information of mitochondrion, and its structure is circular double-stranded. Despite the diminutive size of the mitochondrial genome, mtDNA mutations are an important cause of mitochondrial diseases which are characterized by defects in oxidative phosphorylation (OXPHOS). Mitochondrial diseases are involved in multiple systems, particularly in the organs that are highly dependent on aerobic metabolism. The diagnosis of mitochondrial disease is more complicated since mtDNA mutations can cause various clinical symptoms. To realize more accurate diagnosis and treatment of mitochondrial diseases, the detection of mtDNA and the design of drugs acting on it are extremely important. Over the past few years, many probes and therapeutic drugs targeting mtDNA have been developed, making significant contributions to fundamental research including elucidation of the mechanisms of mitochondrial diseases at the genetic level. In this review, we summarize the structure, function, and detection approaches for mtDNA. The most current topics in this field, such as mechanistic exploration and treatment of mtDNA mutation-related disorders, are also reviewed. Specific attention is given to discussing the design and development of these probes and drugs for mtDNA. We hope that this review will provide readers with a comprehensive understanding of the importance of mtDNA, and promote the development of effective molecules for theragnosis of mtDNA mutation-related diseases.     

In-Situ Configuration Studies on Segmented DNA Origami Nanotubes

One-dimensional nanotubes are of considerable interest in materials and biochemical sciences. A particular desire is to create DNA nanotubes with user-defined structural features and biological relevance, which will facilitate the application of these nanotubes in the controlled release of drugs, templating of other materials into linear arrays and the construction of artificial membrane channels. However, little is known about the structures of assembled DNA nanotubes in solution. Here we report an in situ exploration of segmented DNA nanotubes, composed of multiple units with set length distributions, by using synchrotron small-angle X-ray scattering (SAXS). Through joint experimental and theoretical studies, we show that the SAXS data are highly informative in the context of heterogeneous mixtures of DNA nanotubes. The structural parameters obtained by SAXS are in good agreement with those determined by atomic force microscopy (AFM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). In particular, the SAXS data revealed important structural information on these DNA nanotubes, such as the in-solution diameters (≈25 nm), which could be obtained only with difficulty by use of other methods. Our results establish SAXS as a reliable structural analysis method for long DNA nanotubes and could assist in the rational design of these structures.     

Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA

Genetic alphabet expansion technology, creating new replicable and functional DNA molecules with unnatural base pairs (UBPs), is the novel promising research area of xenobiology. Recently, this technology has rapidly advanced, resulting in the need for a sequencing method for DNA molecules containing UBPs. However, all of the conventional sequencing methods, such as Sanger methods, are for four-letter DNA molecules. Here, we present an improved Sanger sequencing method (Sanger gap sequencing) for DNAs containing our UBP, Ds-Px, which appears as gaps in the sequencing peak patterns. By improving the sequencing reaction for efficient Ds-Px pairing and using modified Px substrates, we have developed a sequencing method with increased processivity and clear gap patterns for multiple Ds-Px pairs in various sequence contexts. This method is useful for UBP applications such as high-affinity DNA aptamer generation and semisynthetic organism creation involving UBPs. In addition, through this research, we found that the side chains of UBs greatly affect the efficiency of UB pairings in replication, thus suggesting further development of UBPs.     

A Yeast-Based Repurposing Approach for the Treatment of Mitochondrial DNA Depletion Syndromes Led to the Identification of Molecules Able to Modulate the dNTP Pool

Mitochondrial DNA depletion syndromes (MDS) are clinically heterogenous and often severe diseases, characterized by a reduction of the number of copies of mitochondrial DNA (mtDNA) in affected tissues. In the context of MDS, yeast has proved to be both an excellent model for the study of the mechanisms underlying mitochondrial pathologies and for the discovery of new therapies via high-throughput assays. Among the several genes involved in MDS, it has been shown that recessive mutations in MPV17 cause a hepatocerebral form of MDS and Navajo neurohepatopathy. MPV17 encodes a non selective channel in the inner mitochondrial membrane, but its physiological role and the nature of its cargo remains elusive. In this study we identify ten drugs active against MPV17 disorder, modelled in yeast using the homologous gene SYM1. All ten of the identified molecules cause a concomitant increase of both the mitochondrial deoxyribonucleoside triphosphate (mtdNTP) pool and mtDNA stability, which suggests that the reduced availability of DNA synthesis precursors is the cause for the mtDNA deletion and depletion associated with Sym1 deficiency. We finally evaluated the effect of these molecules on mtDNA stability in two other MDS yeast models, extending the potential use of these drugs to a wider range of MDS patients.     

OCRDetector: Accurately Detecting Open Chromatin Regions via Plasma Cell-Free DNA Sequencing Data

Open chromatin regions (OCRs) are special regions of the human genome that can be accessed by DNA regulatory elements. Several studies have reported that a series of OCRs are associated with mechanisms involved in human diseases, such as cancers. Identifying OCRs using ATAC-seq or DNase-seq is often expensive. It has become popular to detect OCRs from plasma cell-free DNA (cfDNA) sequencing data, because both the fragmentation modes of cfDNA and the sequencing coverage in OCRs are significantly different from those in other regions. However, it is a challenging computational problem to accurately detect OCRs from plasma cfDNA-seq data, as multiple factors—e.g., sequencing and mapping bias, insufficient read depth, etc.—often mislead the computational model. In this paper, we propose a novel bioinformatics pipeline, OCRDetector, for detecting OCRs from whole-genome cfDNA sequencing data. The pipeline calculates the window protection score (WPS) waveform and the cfDNA sequencing coverage. To validate the proposed pipeline, we compared the percentage overlap of our OCRs with those obtained by other methods. The experimental results show that 81% of the TSS regions of housekeeping genes are detected, and our results have obvious tissue specificity. In addition, the overlap percentage between our OCRs and the high-confidence OCRs obtained by ATAC-seq or DNase-seq is greater than 70%.     

A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings

The reconstruction of individual haplotypes can facilitate the interpretation of disease risks; however, high costs and technical challenges still hinder their assessment in clinical settings. Second-generation sequencing is the gold standard for variant discovery but, due to the production of short reads covering small genomic regions, allows only indirect haplotyping based on statistical methods. In contrast, third-generation methods such as the nanopore sequencing platform developed by Oxford Nanopore Technologies (ONT) generate long reads that can be used for direct haplotyping, with fewer drawbacks. However, robust standards for variant phasing in ONT-based target resequencing efforts are not yet available. In this study, we presented a streamlined proof-of-concept workflow for variant calling and phasing based on ONT data in a clinically relevant 12-kb region of the APOE locus, a hotspot for variants and haplotypes associated with aging-related diseases and longevity. Starting with sequencing data from simple amplicons of the target locus, we demonstrated that ONT data allow for reliable single-nucleotide variant (SNV) calling and phasing from as little as 60 reads, although the recognition of indels is less efficient. Even so, we identified the best combination of ONT read sets (600) and software (BWA/Minimap2 and HapCUT2) that enables full haplotype reconstruction when both SNVs and indels have been identified previously using a highly-accurate sequencing platform. In conclusion, we established a rapid and inexpensive workflow for variant phasing based on ONT long reads. This allowed for the analysis of multiple samples in parallel and can easily be implemented in routine clinical practice, including diagnostic testing.     

Advancing DNA Steganography with Incorporation of Randomness

DNA has become a promising candidate as a future data storage medium; this makes DNA steganography indispensable in DNA data security. PCR primers are conventional secret keys in DNA steganography. Brute force testing of different primers will be extremely time consuming, and practically unaffordable when high-throughput sequencing is used. However, the encrypted information can be sequenced and read once the primers are intercepted. A new steganography approach is needed to make the DNA-encoded information safer, if not unhackable. Mixing information-carrying DNA with a partially degenerated DNA library containing single or multiple restriction sites, we have built an additional protective layer that can be removed by desired restriction enzymes as secondary secret keys. As PCR is inevitable for reading DNA-encrypted information, heating will cause reshuffling and generate endonuclease-resistant mismatched duplexes, especially for DNA with high sequence diversity. Consequently, with the incorporation of randomness, DNA steganography possesses both quantum key distribution (QKD)-like function for detecting PCR by an interceptor and a self-destructive property. It is noteworthy that the background noise generated through the protective layer is independent from any sequencing technology including Sanger and high-throughput sequencing. With a DNA ink incorporating the steganography, we have shown that the authenticity of a piece of writing can be confirmed only by authorized persons with knowledge of all embedded keys.     

Detection of HER2+ Breast Cancer Cells using Bioinspired DNA-Based Signal Amplification

Circulating tumor cells (CTC) are promising biomarkers for metastatic cancer detection and monitoring progression. However, detection of CTCs remains challenging due to their low frequency and heterogeneity. Herein, we report a bioinspired approach to detect individual cancer cells, based on a signal amplification cascade using a programmable DNA hybridization chain reaction (HCR) circuit. We applied this approach to detect HER2⁺ cancer cells using the anti-HER2 antibody (trastuzumab) coupled to initiator DNA eliciting a HCR cascade that leads to a fluorescent signal at the cell surface. At 4 °C, this HCR detection scheme resulted in highly efficient, specific and sensitive signal amplification of the DNA hairpins specifically on the membrane of the HER2⁺ cells in a background of HER2⁻ cells and peripheral blood leukocytes, which remained almost non-fluorescent. The results indicate that this system offers a new strategy that may be further developed toward an in vitro diagnostic platform for the sensitive and efficient detection of CTC.     

Leukocyte mitochondrial DNA alteration in systemic lupus erythematosus and its relevance to the susceptibility to lupus nephritis

The role of mitochondrial DNA (mtDNA) alterations in the pathophysiology of systemic lupus erythematosus (SLE) remains unclear. We investigated sequence variations in the D310 region and copy number change of mtDNA in 85 SLE patients and 45 normal subjects. Leukocyte DNA and RNA were extracted from leukocytes of the peripheral venous blood. The D310 sequence variations and copy number of mtDNA, and mRNA expression levels of mtDNA-encoded genes in leukocytes were determined by quantitative real-time polymerase chain reaction (Q-PCR) and PCR-based direct sequencing, respectively. We found that leukocyte mtDNA in SLE patients exhibited higher frequency of D310 heteroplasmy (69.4% vs. 48.9%, p = 0.022) and more D310 variants (2.2 vs. 1.7, p = 0.014) than those found in controls. Among normal controls and patients with low, medium or high SLE disease activity index (SLEDAI), an ever-increasing frequency of D310 heteroplasmy was observed (p = 0.021). Leukocyte mtDNA copy number tended to be low in patients of high SLEDAI group (p = 0.068), especially in those harboring mtDNA with D310 heteroplasmy (p = 0.020). Moreover, the mtDNA copy number was positively correlated with the mRNA level of mtDNA-encoded ND1 (NADH dehydrogenase subunit 1) (p = 0.041) and ATPase 6 (ATP synthase subunit 6) (p = 0.030) genes. Patients with more D310 variants were more susceptible to lupus nephritis (p = 0.035). Taken together, our findings suggest that decrease in the mtDNA copy number and increase in D310 heteroplasmy of mtDNA are related to the development and progression of SLE, and that the patients harboring more D310 variants of mtDNA are more susceptible to lupus nephritis.     

Identification of DNA Damage Repair-Associated Prognostic Biomarkers for Prostate Cancer Using Transcriptomic Data Analysis

In the recent decade, the importance of DNA damage repair (DDR) and its clinical application have been firmly recognized in prostate cancer (PC). For example, olaparib was just approved in May 2020 to treat metastatic castration-resistant PC with homologous recombination repair-mutated genes; however, not all patients can benefit from olaparib, and the treatment response depends on patient-specific mutations. This highlights the need to understand the detailed DDR biology further and develop DDR-based biomarkers. In this study, we establish a four-gene panel of which the expression is significantly associated with overall survival (OS) and progression-free survival (PFS) in PC patients from the TCGA-PRAD database. This panel includes DNTT, EXO1, NEIL3, and EME2 genes. Patients with higher expression of the four identified genes have significantly worse OS and PFS. This significance also exists in a multivariate Cox regression model adjusting for age, PSA, TNM stages, and Gleason scores. Moreover, the expression of the four-gene panel is highly correlated with aggressiveness based on well-known PAM50 and PCS subtyping classifiers. Using publicly available databases, we successfully validate the four-gene panel as having the potential to serve as a prognostic and predictive biomarker for PC specifically based on DDR biology.     

Genetic Variants and Tumor Immune Microenvironment: Clues for Targeted Therapies in Inflammatory Breast Cancer (IBC)

To better understand the etiology of inflammatory breast cancer (IBC) and identify potential therapies, we studied genomic alterations in IBC patients. Targeted, next-generation sequencing (NGS) was performed on cell-free DNA (cfDNA) (n = 33) and paired DNA from tumor tissues (n = 29) from 32 IBC patients. We confirmed complementarity between cfDNA and tumor tissue genetic profiles. We found a high incidence of germline variants in IBC patients that could be associated with an increased risk of developing the disease. Furthermore, 31% of IBC patients showed deficiencies in the homologous recombination repair (HRR) pathway (BRCA1, BRCA2, PALB2, RAD51C, ATM, BARD1) making them sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. We also characterized the tumor-infiltrating lymphocytes (TILs) in tumor tissue biopsies by studying several markers (CD4, CD8, FoxP3, CD20, PD-1, and PD-L1) through immunohistochemistry (IHC) staining. In 7 of 24 (29%) patients, tumor biopsies were positive for PD-L1 and PD-1 expression on TILs, making them sensitive to PD-1/PD-L1 blocking therapies. Our results provide a rationale for considering PARP inhibitors and PD-1/PDL1 blocking immunotherapy in qualifying IBC patients.     

Nanopore/electrode structures for single-molecule biosensing

Biological and solid-state nanopores have recently attracted much interest as ultrafast DNA fragment sizing and sequencing devices. Their potential however goes far beyond DNA sequencing. In particular, nanopores offer perspectives of single-molecule (bio)sensing at physiologically relevant concentrations, which is key for studying protein/protein or protein/DNA interactions. Integration of electrode structures into solid-state nanopore devices moreover enables control and fast switching of the pore properties, e.g. for active control of biopolymer transport through the nanopore. We present some of recent work in this area, namely the fabrication and characterization of nanopore/electrode architectures for single-(bio)molecule sensing. Specifically, we introduce a new technique to fabricate ultra-small metal nanopores with diameters smaller than 20 nm based on ion current feedback (ICF) controlled electrodeposition. It offers precise control of the pore conductance, is easily multiplexed, and can be extended to a wide range of different metals.     

Pervaporation of Organic Liquids from Binary Aqueous Mixtures using Poly(trifluoropropylmethylsiloxane) and Poly(dimethylsiloxane) Dense Membranes

In this work we have compared and contrasted the pervaporation behaviour (separation factor and flux) of fluorosilicone dense membranes based on poly(trifluoropropylmethylsiloxane) (PTFPMS) with poly(dimethylsiloxane) (PDMS) dense membranes. In particular, pervaporation experiments were carried out at 298 K using lab-made PTFPMS, lab-made PDMS and commercial PDMS membranes in order to remove three different organic liquids pyridine (PY), isopropanol (IPA) and methylethylketone (MEK) from dilute (<10 wt.%) binary aqueous mixtures. All of the silicone membranes studied were found to be successful for the desired separations. The permeation flux of pyridine–water liquid mixtures for the PTFPMS membranes was found to increase with the pyridine concentration in the feed mixtures. The separation factor for PDMS membranes for the removal of pyridine, IPA and MEK from aqueous binary mixtures (1 wt.%) was found to be higher than that of PTFPMS membranes while the normalized flux was higher for PTFPMS membranes under identical test conditions. The effect of crosslink density of the PTFPMS membranes on the separation of pyridine–water mixtures was also studied. For a 1 wt.% feed solution the total flux increased with the molar mass between crosslinks, whereas the separation factor for pyridine–water was highest for a molar mass between crosslinks of 15,320 g mol⁻¹.     

The Impact of Modern Technologies on Molecular Diagnostic Success Rates,with a Focus on Inherited Retinal Dystrophy and Hearing Loss

The identification of pathogenic variants in monogenic diseases has been of interest to researchers and clinicians for several decades. However, for inherited diseases with extremely high genetic heterogeneity, such as hearing loss and retinal dystrophies, establishing a molecular diagnosis requires an enormous effort. In this review, we use these two genetic conditions as examples to describe the initial molecular genetic identification approaches, as performed since the early 90s, and subsequent improvements and refinements introduced over the years. Next, the history of DNA sequencing from conventional Sanger sequencing to high-throughput massive parallel sequencing, a.k.a. next-generation sequencing, is outlined, including their advantages and limitations and their impact on identifying the remaining genetic defects. Moreover, the development of recent technologies, also coined “third-generation” sequencing, is reviewed, which holds the promise to overcome these limitations. Furthermore, we outline the importance and complexity of variant interpretation in clinical diagnostic settings concerning the massive number of different variants identified by these methods. Finally, we briefly mention the development of novel approaches such as optical mapping and multiomics, which can help to further identify genetic defects in the near future.     

N4-Allyldeoxycytidine: A New DNA Tag with Chemical Sequencing Power for Pinpointing Labelling Sites,Mapping Epigenetic Markers,and In Situ Imaging

DNA tagging with base analogues has found numerous applications. To precisely record the DNA labelling information, it would be highly beneficial to develop chemical sequencing tags that can be encoded into DNA as regular bases and decoded as mutant bases following a mild, efficient and bioorthogonal chemical treatment. Here we reported such a DNA tag, N⁴-allyldeoxycytidine (a⁴dC), for labeling and identifying DNA by in vitro assays. The iodination of a⁴dC led to fast and complete formation of 3 , N⁴-cyclized deoxycytidine, which induced base misincorporation during DNA replication and thus could be located at single base resolution. We explored the applications of a⁴dC in pinpointing DNA labelling sites at single base resolution, mapping epigenetic marker N⁴-methyldeoxycytidine, and imaging nucleic acids in situ. In addition, mammalian cellular DNA could be metabolically labelled with a⁴dC. Our study sheds light on the design of next generation DNA tags with chemical sequencing power.