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Article: Genome Skimming with Nanopore Sequencing Precisely Determines Global and Transposon DNA Methylation in Vertebrates.

Faulk, Christopher

bioRxiv : the preprint server for biology

2023

Abstract: Genome skimming is defined as low-pass sequencing below 0.05X coverage and is typically used for mitochondrial genome recovery and species identification. Long read nanopore sequencers enable simultaneous reading of both DNA sequence and methylation and ... ...

Abstract	Genome skimming is defined as low-pass sequencing below 0.05X coverage and is typically used for mitochondrial genome recovery and species identification. Long read nanopore sequencers enable simultaneous reading of both DNA sequence and methylation and can multiplex samples for low-cost genome skimming. Here I present nanopore sequencing as a highly precise platform for global DNA methylation and transposon assessment. At coverage of just 0.001X, or 30 Mb of reads, accuracy is sub-1%. Biological and technical replicates validate high precision. Skimming 40 vertebrate species reveals conserved patterns of global methylation consistent with whole genome bisulfite sequencing and an average mapping rate above 97%. Genome size directly correlates to global DNA methylation, explaining 44% of its variance. Accurate SINE and LINE transposon methylation in both mouse and primates can be obtained with just 0.0001X coverage, or 3 Mb of reads. Sample multiplexing, field portability, and the low price of this instrument combine to make genome skimming for DNA methylation an accessible method for epigenetic assessment from ecology to epidemiology, and by low resource groups.
Language	English
Publishing date	2023-01-25
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.01.25.525540
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Article ; Online: Genome skimming with nanopore sequencing precisely determines global and transposon DNA methylation in vertebrates.

Faulk, Christopher

Genome research

2023 Volume 33, Issue 6, Page(s) 948–956

Abstract: Genome skimming is defined as low-pass sequencing below 0.05× coverage and is typically used for mitochondrial genome recovery and species identification. Long-read nanopore sequencers enable simultaneous reading of both DNA sequence and methylation and ... ...

Abstract	Genome skimming is defined as low-pass sequencing below 0.05× coverage and is typically used for mitochondrial genome recovery and species identification. Long-read nanopore sequencers enable simultaneous reading of both DNA sequence and methylation and can multiplex samples for low-cost genome skimming. Here I present nanopore sequencing as a highly precise platform for global DNA methylation and transposon assessment. At coverage of just 0.001×, or 30 Mb of reads, accuracy is sub-1%. Biological and technical replicates validate high precision. Skimming 40 vertebrate species reveals conserved patterns of global methylation consistent with whole-genome bisulfite sequencing and an average mapping rate >97%. Genome size directly correlates to global DNA methylation, explaining 39% of its variance. Accurate SINE and LINE transposon methylation in both the mouse and primates can be obtained with just 0.0001× coverage, or 3 Mb of reads. Sample multiplexing, field portability, and the low price of this instrument combine to make genome skimming for DNA methylation an accessible method for epigenetic assessment from ecology to epidemiology and for low-resource groups.
MeSH term(s)	Animals ; Mice ; DNA Methylation ; Nanopore Sequencing ; Sequence Analysis, DNA/methods ; Genome ; Vertebrates/genetics ; High-Throughput Nucleotide Sequencing/methods
Language	English
Publishing date	2023-07-13
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1284872-4
ISSN	1549-5469 ; 1088-9051 ; 1054-9803
ISSN (online)	1549-5469
ISSN	1088-9051 ; 1054-9803
DOI	10.1101/gr.277743.123
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Article ; Online: De novo sequencing, diploid assembly, and annotation of the black carpenter ant, Camponotus pennsylvanicus, and its symbionts by one person for $1000, using nanopore sequencing.

Faulk, Christopher

Nucleic acids research

2022 Volume 51, Issue 1, Page(s) 17–28

Abstract: The black carpenter ant (Camponotus pennsylvanicus) is a pest species found widely throughout North America. From a single individual I used long-read nanopore sequencing to assemble a phased diploid genome of 306 Mb and 60X coverage, with quality ... ...

Abstract	The black carpenter ant (Camponotus pennsylvanicus) is a pest species found widely throughout North America. From a single individual I used long-read nanopore sequencing to assemble a phased diploid genome of 306 Mb and 60X coverage, with quality assessed by a 97.0% BUSCO score, improving upon other ant assemblies. The mitochondrial genome reveals minor rearrangements from other ants. The reads also allowed assembly of parasitic and symbiont genomes. I include a complete Wolbachia bacterial assembly with a size of 1.2 Mb, as well as a commensal symbiont Blochmannia pennsylvanicus, at 791 kb. DNA methylation and hydroxymethylation were measured at base-pair resolution level from the same reads and confirmed extremely low levels seen in the Formicidae family. There was moderate heterozygosity, with 0.16% of bases being biallelic from the parental haplotypes. Protein prediction yielded 14 415 amino acid sequences with 95.8% BUSCO score and 86% matching to previously known proteins. All assemblies were derived from a single MinION flow cell generating 20 Gb of sequence for a cost of $1047 including consumable reagents. Adding fixed costs for equipment brings the total for an ant-sized genome to less than $5000. All analyses were performed in 1 week on a single desktop computer.
MeSH term(s)	Animals ; Humans ; Ants/genetics ; Ants/microbiology ; Diploidy ; Genome, Mitochondrial ; High-Throughput Nucleotide Sequencing/economics ; High-Throughput Nucleotide Sequencing/methods ; Nanopore Sequencing ; Sequence Analysis, DNA/economics ; Sequence Analysis, DNA/methods ; Symbiosis ; Wolbachia/genetics ; Wolbachia/physiology ; Enterobacteriaceae/classification ; Enterobacteriaceae/genetics ; Enterobacteriaceae/physiology ; Genome, Insect
Language	English
Publishing date	2022-06-21
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkac510
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Article ; Online: De novo sequencing, diploid assembly, and annotation of the black carpenter ant, Camponotus pennsylvanicus, and its symbionts by one person for $1000, using nanopore sequencing

Faulk, Christopher

Nucleic Acids Research. 2022 June

2022

Abstract	The black carpenter ant (Camponotus pennsylvanicus) is a pest species found widely throughout North America. From a single individual I used long-read nanopore sequencing to assemble a phased diploid genome of 306 Mb and 60X coverage, with quality assessed by a 97.0% BUSCO score, improving upon other ant assemblies. The mitochondrial genome reveals minor rearrangements from other ants. The reads also allowed assembly of parasitic and symbiont genomes. I include a complete Wolbachia bacterial assembly with a size of 1.2 Mb, as well as a commensal symbiont Blochmannia pennsylvanicus, at 791 kb. DNA methylation and hydroxymethylation were measured at base-pair resolution level from the same reads and confirmed extremely low levels seen in the Formicidae family. There was moderate heterozygosity, with 0.16% of bases being biallelic from the parental haplotypes. Protein prediction yielded 14 415 amino acid sequences with 95.8% BUSCO score and 86% matching to previously known proteins. All assemblies were derived from a single MinION flow cell generating 20 Gb of sequence for a cost of $1047 including consumable reagents. Adding fixed costs for equipment brings the total for an ant-sized genome to less than $5000. All analyses were performed in 1 week on a single desktop computer.
Keywords	Camponotus pennsylvanicus ; DNA methylation ; Wolbachia ; amino acids ; computers ; diploidy ; equipment ; haplotypes ; heterozygosity ; mitochondrial genome ; nanopores ; pests ; prediction ; research ; symbionts ; North America
Language	English
Dates of publication	2022-0621
Publishing place	Oxford University Press (OUP)
Document type	Article ; Online
Note	Resource is Open Access ; CHORUS License Information
ZDB-ID	186809-3
ISSN	0301-5610 ; 0305-1048
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkac510
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Article ; Online: Suggested Absence of Horizontal Transfer of Retrotransposons between Humans and Domestic Mammal Species.

Wanner, Nicole M / Faulk, Christopher

Genes

2021 Volume 12, Issue 8

Abstract: Transposable element sequences are usually vertically inherited but have also spread across taxa via horizontal transfer. Previous investigations of ancient horizontal transfer of transposons have compared consensus sequences, but this method resists ... ...

Abstract	Transposable element sequences are usually vertically inherited but have also spread across taxa via horizontal transfer. Previous investigations of ancient horizontal transfer of transposons have compared consensus sequences, but this method resists detection of recent single or low copy number transfer events. The relationship between humans and domesticated animals represents an opportunity for potential horizontal transfer due to the consistent shared proximity and exposure to parasitic insects, which have been identified as plausible transfer vectors. The relatively short period of extended human-animal contact (tens of thousands of years or less) makes horizontal transfer of transposons between them unlikely. However, the availability of high-quality reference genomes allows individual element comparisons to detect low copy number events. Using pairwise all-versus-all megablast searches of the complete suite of retrotransposons of thirteen domestic animals against human, we searched a total of 27,949,823 individual TEs. Based on manual comparisons of stringently filtered BLAST search results for evidence of vertical inheritance, no plausible instances of HTT were identified. These results indicate that significant recent HTT between humans and domesticated animals has not occurred despite the close proximity, either due to the short timescale, inhospitable recipient genomes, a failure of vector activity, or other factors.
MeSH term(s)	Animals ; Gene Transfer, Horizontal ; Humans ; Mammals/genetics ; Retroelements
Chemical Substances	Retroelements
Language	English
Publishing date	2021-08-08
Publishing country	Switzerland
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2527218-4
ISSN	2073-4425 ; 2073-4425
ISSN (online)	2073-4425
ISSN	2073-4425
DOI	10.3390/genes12081223
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Article ; Online: Intergenerational arsenic exposure on the mouse epigenome and metabolic physiology.

Colwell, Mathia L / Flack, Nicole / Rezabek, Amanda / Faulk, Christopher

Environmental and molecular mutagenesis

2023 Volume 64, Issue 2, Page(s) 72–87

Abstract: Inorganic arsenic (iAs) is one of the largest toxic exposures to impact humanity worldwide. Exposure to iAs during pregnancy may disrupt the proper remodeling of the epigenome of F1 developing offspring and potentially their F2 grand-offspring via ... ...

Abstract	Inorganic arsenic (iAs) is one of the largest toxic exposures to impact humanity worldwide. Exposure to iAs during pregnancy may disrupt the proper remodeling of the epigenome of F1 developing offspring and potentially their F2 grand-offspring via disruption of fetal primordial germ cells (PGCs). There is a limited understanding between the correlation of disease phenotype and methylation profile within offspring of both generations and whether it persists to adulthood. Our study aims to understand the intergenerational effects of in utero iAs exposure on the epigenetic profile and onset of disease phenotypes within F1 and F2 adult offspring, despite the lifelong absence of direct arsenic exposure within these generations. We exposed F0 female mice (C57BL6/J) to the following doses of iAs in drinking water 2 weeks before pregnancy until the birth of the F1 offspring: 1, 10, 245, and 2300 ppb. We found sex- and dose-specific changes in weight and body composition that persist from early time to adulthood within both generations. Fasting blood glucose challenge suggests iAs exposure causes dysregulation of glucose metabolism, revealing generational, exposure, and sex-specific differences. Toward understanding the mechanism, genome-wide DNA methylation data highlights exposure-specific patterns in liver, finding dysregulation within genes associated with cancer, T2D, and obesity. We also identified regions containing persistently differentially methylated CpG sites between F1 and F2 generations. Our results indicate the F1 developing embryos and their PGCs, which will result in F2 progeny, retain epigenetic damage established during the prenatal period and are associated with adult metabolic dysfunction.
MeSH term(s)	Pregnancy ; Male ; Mice ; Animals ; Female ; Humans ; Arsenic ; Epigenesis, Genetic ; Epigenome ; DNA Methylation ; Germ Cells/metabolism ; Prenatal Exposure Delayed Effects/genetics
Chemical Substances	Arsenic (N712M78A8G)
Language	English
Publishing date	2023-01-23
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	639145-x
ISSN	1098-2280 ; 0893-6692
ISSN (online)	1098-2280
ISSN	0893-6692
DOI	10.1002/em.22526
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Article ; Online: Chromosome-level, nanopore-only genome and allele-specific DNA methylation of Pallas's cat,

Flack, Nicole / Drown, Melissa / Walls, Carrie / Pratte, Jay / McLain, Adam / Faulk, Christopher

NAR genomics and bioinformatics

2023 Volume 5, Issue 2, Page(s) lqad033

Abstract: Pallas's cat, or the manul cat ( ...

Abstract	Pallas's cat, or the manul cat (
Language	English
Publishing date	2023-04-04
Publishing country	England
Document type	Journal Article
ISSN	2631-9268
ISSN (online)	2631-9268
DOI	10.1093/nargab/lqad033
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Article ; Online: Metagenomic surveillance for bacterial tick-borne pathogens using nanopore adaptive sampling.

Kipp, Evan J / Lindsey, Laramie L / Khoo, Benedict / Faulk, Christopher / Oliver, Jonathan D / Larsen, Peter A

Scientific reports

2023 Volume 13, Issue 1, Page(s) 10991

Abstract: Technological and computational advancements in the fields of genomics and bioinformatics are providing exciting new opportunities for pathogen discovery and genomic surveillance. In particular, single-molecule nucleotide sequence data originating from ... ...

Abstract	Technological and computational advancements in the fields of genomics and bioinformatics are providing exciting new opportunities for pathogen discovery and genomic surveillance. In particular, single-molecule nucleotide sequence data originating from Oxford Nanopore Technologies (ONT) sequencing platforms can be bioinformatically leveraged, in real-time, for enhanced biosurveillance of a vast array of zoonoses. The recently released nanopore adaptive sampling (NAS) strategy facilitates immediate mapping of individual nucleotide molecules to a given reference as each molecule is being sequenced. User-defined thresholds then allow for the retention or rejection of specific molecules, informed by the real-time reference mapping results, as they are physically passing through a given sequencing nanopore. Here, we show how NAS can be used to selectively sequence DNA of multiple bacterial tick-borne pathogens circulating in wild populations of the blacklegged tick vector, Ixodes scapularis.
MeSH term(s)	Animals ; Nanopores ; Bacteria/genetics ; Ixodes/genetics ; Ixodes/microbiology ; Zoonoses
Language	English
Publishing date	2023-07-07
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-023-37134-9
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Article ; Online: Epigenetics, DNA damage, and aging.

Soto-Palma, Carolina / Niedernhofer, Laura J / Faulk, Christopher D / Dong, Xiao

The Journal of clinical investigation

2022 Volume 132, Issue 16

Abstract: Over the course of a human lifespan, genome integrity erodes, leading to an increased abundance of several types of chromatin changes. The abundance of DNA lesions (chemical perturbations to nucleotides) increases with age, as does the number of genomic ... ...

Abstract	Over the course of a human lifespan, genome integrity erodes, leading to an increased abundance of several types of chromatin changes. The abundance of DNA lesions (chemical perturbations to nucleotides) increases with age, as does the number of genomic mutations and transcriptional disruptions caused by replication or transcription of those lesions, respectively. At the epigenetic level, precise DNA methylation patterns degrade, likely causing increasingly stochastic variations in gene expression. Similarly, the tight regulation of histone modifications begins to unravel. The genomic instability caused by these mechanisms allows transposon element reactivation and remobilization, further mutations, gene dysregulation, and cytoplasmic chromatin fragments. This cumulative genomic instability promotes cell signaling events that drive cell fate decisions and extracellular communications known to disrupt tissue homeostasis and regeneration. In this Review, we focus on age-related epigenetic changes and their interactions with age-related genomic changes that instigate these events.
MeSH term(s)	Aging/genetics ; Aging/metabolism ; Chromatin/chemistry ; Chromatin/genetics ; Chromatin/metabolism ; DNA Damage ; DNA Methylation/physiology ; Epigenesis, Genetic ; Genomic Instability ; Histones/metabolism ; Humans ; Mutation ; Signal Transduction/genetics ; Signal Transduction/physiology ; Stochastic Processes
Chemical Substances	Chromatin ; Histones
Language	English
Publishing date	2022-08-23
Publishing country	United States
Document type	Journal Article ; Review ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	3067-3
ISSN	1558-8238 ; 0021-9738
ISSN (online)	1558-8238
ISSN	0021-9738
DOI	10.1172/JCI158446
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Article: Suggested Absence of Horizontal Transfer of Retrotransposons between Humans and Domestic Mammal Species

Wanner, Nicole M. / Faulk, Christopher

Genes. 2021 Aug. 08, v. 12, no. 8

2021

Abstract	Transposable element sequences are usually vertically inherited but have also spread across taxa via horizontal transfer. Previous investigations of ancient horizontal transfer of transposons have compared consensus sequences, but this method resists detection of recent single or low copy number transfer events. The relationship between humans and domesticated animals represents an opportunity for potential horizontal transfer due to the consistent shared proximity and exposure to parasitic insects, which have been identified as plausible transfer vectors. The relatively short period of extended human–animal contact (tens of thousands of years or less) makes horizontal transfer of transposons between them unlikely. However, the availability of high-quality reference genomes allows individual element comparisons to detect low copy number events. Using pairwise all-versus-all megablast searches of the complete suite of retrotransposons of thirteen domestic animals against human, we searched a total of 27,949,823 individual TEs. Based on manual comparisons of stringently filtered BLAST search results for evidence of vertical inheritance, no plausible instances of HTT were identified. These results indicate that significant recent HTT between humans and domesticated animals has not occurred despite the close proximity, either due to the short timescale, inhospitable recipient genomes, a failure of vector activity, or other factors.
Keywords	humans ; inheritance (genetics) ; retrotransposons ; transposons
Language	English
Dates of publication	2021-0808
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2527218-4
ISSN	2073-4425
ISSN	2073-4425
DOI	10.3390/genes12081223
Database	NAL-Catalogue (AGRICOLA)

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