Abstract: Methods of labeling, amplifying, and sequencing mitochondrial DNA are provided. The labeling methods typically include using polymerase-based extension of one or more unique molecular identifier (UMI) primers along a target mitochondrial DNA template, each UMI primer including a universal primer sequence, a unique molecular identifier sequence, and a mitochondrial DNA binding sequence. Amplification of the labeled-target mitochondrial DNA can include, for example, polymerase chain reaction (e.g., high-fidelity long-range). Methods of determining the sequence of labeled-target mitochondrial DNA can include use of a long-read sequencing platform. The methods may further including one or more of grouping sequences having the same UMI into one of more groups, determining the sequence of each labeled-target mitochondrial DNA by determining the consensus sequence of each group, and identifying polymorphisms in one or more of the labeled-target mitochondrial DNA.

Abstract: Methods for the rapid and accurate detection and characterization of a viral nucleic acid in a sample are provided. The method is a method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes. It can simultaneously detect SARS-CoV-2 and co-infecting respiratory viruses, and monitor mutations for up to 96 samples in real time. The method, termed NIRVANA for Nanopore sequencing of Isothermal Rapid Viral Amplification for Near real-time Analysis, showed high sensitivity and specificity for SARS-CoV-2 in 70 clinical samples. It also simultaneously detected other viral pathogens (e.g. influenza A) in clinical and municipal wastewater samples. It provides a rapid field-deployable solution of COVID-19 and co-infection detection and surveillance of the evolution of pandemic strains.

Abstract: Compositions and methods labeling individual nucleic acid (e.g., DNA) molecules with a unique molecular identifier (UMI), followed by amplification by PCR are provided. The PCR amplicons can be grouped by the UMI they contain and traced back to the original molecule. More specifically, the grouped reads with the same UMI represent one original nucleic acid (e.g., DNA) molecule, meaning they share the same nucleic acid sequence. Methods of sequencing the labeled nucleic acid are also provided. The methods can include determination of a consensus sequence, which thus eliminates errors that may be introduced in the amplification and sequencing process. Such methods can be used in, for example, the detection of rare genetic variants.

Abstract: Compositions and methods for enriching DNA from any loci of interest are provided. Endonuclease(s) such as RNA-guided nucleases Cpf1/Cas9 can cut the flanking regions of the gene of interest in, for example genomic DNA, followed by ligation of loop adapters. The loop adapters protect the gene of interest for subsequent digestion with one or more exonucleases. Multiple genes of interest can be digested and ligated with loop adapters at the same time. The undigested target sequence can be further purified by, for example, gel extraction using, for example, a commercial DNA purification kit. The enriched DNA can be used for sequencing.

Abstract: Methods of labeling, amplifying, and sequencing mitochondrial DNA are provided. The labeling methods typically include using polymerase-based extension of one or more unique molecular identifier (UMI) primers along a target mitochondrial DNA template, each UMI primer including a universal primer sequence, a unique molecular identifier sequence, and a mitochondrial DNA binding sequence. Amplification of the labeled-target mitochondrial DNA can include, for example, polymerase chain reaction (e.g., high-fidelity long-range). Methods of determining the sequence of labeled-target mitochondrial DNA can include use of a long-read sequencing platform. The methods may further including one or more of grouping sequences having the same UMI into one of more groups, determining the sequence of each labeled-target mitochondrial DNA by determining the consensus sequence of each group, and identifying polymorphisms in one or more of the labeled-target mitochondrial DNA.