The Mitochondrial Genome and Epigenome of the Golden Lion Tamarin from Fecal DNA using Nanopore Adaptive Sequencing

Wanner, Nicole; Larsen, Peter A.; McLain, Adam T.; Faulk, Christopher

doi:10.1101/2021.05.27.446055

Cited by 9 publications

(10 citation statements)

References 43 publications

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“…The enrichment of targets in human cells 30 – 34 or species in mock communities 31 , 35 or fecal samples of lions 36 via adaptive sampling was previously demonstrated. In the present study, we used clinical metagenomic samples obtained from vaginal swabs of pregnant women to evaluate the performance to deplete the high content of human DNA that heavily impairs downstream microbiome analyses.…”

Section: Discussionmentioning

confidence: 86%

Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore’s adaptive sequencing

Marquet

Zöllkau

Pastuschek

et al. 2022

Sci Rep

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Metagenomic sequencing is promising for clinical applications to study microbial composition concerning disease or patient outcomes. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA (> 90%), decreasing the overall microbial resolution. Nanopore's adaptive sampling method offers selective DNA depletion or target enrichment to directly reject or accept DNA molecules during sequencing without specialized sample preparation. Here, we demonstrate how selective ‘human host depletion’ resulted in a 1.70 fold (± 0.27 fold) increase in total sequencing depth, providing higher taxonomic profiling sensitivity. At the same time, the microbial composition remains consistent with the control experiments. The complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary. Adaptive sampling increased microbial sequencing yield in all 15 sequenced clinical routine vaginal samples, making it a valuable tool for clinical surveillance and medical-based research, which can be used in addition to other host depletion methods before sequencing.

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Section: Discussionmentioning

confidence: 86%

Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore’s adaptive sequencing

Marquet

Zöllkau

Pastuschek

et al. 2022

Sci Rep

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“…The layout was similar but not exactly convergent with other ant species. Previous nanopore sequencing has recovered mitogenomes from even highly degraded samples such as primate feces, and generated accurate assemblies and so was expected here (32).…”

Section: Mitochondrial Genomementioning

confidence: 59%

De novosequencing, diploid assembly, and annotation of the black carpenter ant,Camponotus pennsylvanicus, and its symbionts by one person for $1000, using nanopore sequencing

Faulk

2022

Preprint

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The black carpenter ant (Camponotus pennsylvanicus) is a pest species found widely throughout North America east of the Rocky Mountains. Its frequent infestation in human buildings justifies close genetic examination and its large size and small genome make it ideal for individual sequencing. From a single individual I used long-read nanopore sequencing to assemble a genome of 306 Mb, with an N50 of 565 Kb, and 60X coverage, with quality assessed by a 97.0% BUSCO score, improving upon related ant assemblies. The reads provided secondary information in the form of parasitic and symbiont genomes, as well as epigenetic information. I assembled a complete Wolbachia bacterial isolate with a genome size of 1.2 Mb and 76X coverage, as well as a commensal bacterium of the carpenter ant tribe, the species-specific symbiont Blochmannia pennsylvanicus, at 791 Kb, 2400X coverage, which matched to within 200 bp of its previously published reference. I also produced a complete mitochondrial genome with over 5000X coverage, revealing minor rearrangements and the first assembly for this species. DNA methylation and hydroxymethylation was measured simultaneously at whole genome, base-pair resolution level from the same nanopore reads and confirmed extremely low levels seen in the Formicidae family of Hymenoptera. A phased diploid assembly was built, revealing a moderate level of heterozygosity, with 0.16% of bases having biallelic SNPs from the two parental haplotypes. Protein prediction yielded 20,209 putative amino acid sequences and annotation identified 86% matched 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 all consumable reagents. Adding fixed costs for required equipment and batch costs for reagents brings the cost to assemble an ant-sized genome to less than $5000. Complete analysis was performed in under 1 week on a commodity computer desktop with 64 Gb memory.

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“…The enrichment of targets in human cells [29][30][31][32][33] or species in mock communities [30,34] or fecal samples of lions [35] via adaptive sampling was previously demonstrated. In the present study, we used clinical metagenomic samples obtained from vaginal swabs of pregnant women to evaluate the performance to deplete the high content of human DNA that heavily impairs downstream microbiome analyses.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore’s adaptive sequencing

Marquet

Zöllkau

Pastuschek

et al. 2021

Preprint

View full text Add to dashboard Cite

Metagenomic sequencing is a promising tool for clinical applications to study microbial composition in relation to disease or patient outcome. Alterations of the vaginal microbiome are associated with adverse pregnancy outcomes, like preterm premature rupture of membranes and preterm birth. Methodologically these samples often have to deal with low relative amounts of prokaryotic DNA and high amounts of host DNA, decreasing the overall microbial resolution. Nanopore's adaptive sampling method offers selective DNA depletion or target enrichment to reject unwanted DNA sequences directly during sequencing without specialized sample preparation. Here, we increase the relative amount of non-human DNA through either direct enrichment or depletion of human DNA. To assess which approach works better, we analyzed 15 clinical routine vaginal swabs with typically high relative amounts of human DNA (> 90%) through metagenomic sequencing. The selective host depletion approach resulted in a 1.70 fold (+/- 0.27 fold) increase in total sequencing depth. Rejected reads contained over 99.80% human reads. In contrast to host depletion, targeted bacterial enrichment resulted in higher human depletion, but the microbial composition was altered as bacterial reads not matching the targets were also depleted. On the other hand, host depletion is not changing the microbial composition, even if high amounts of human host DNA are present, making it a valuable tool for clinical surveillance and medical-based research. However, up to 3% (min: 0.26% max: 2.94%) of human reads were still detectable in the 'accepted' sequence fraction. Thus, the complete removal of all human host sequences is not yet possible and should be considered as an ethical approval statement might still be necessary.

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The Mitochondrial Genome and Epigenome of the Golden Lion Tamarin from Fecal DNA using Nanopore Adaptive Sequencing

Cited by 9 publications

References 43 publications

Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore’s adaptive sequencing

Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore’s adaptive sequencing

De novosequencing, diploid assembly, and annotation of the black carpenter ant,Camponotus pennsylvanicus, and its symbionts by one person for $1000, using nanopore sequencing

Evaluation of microbiome enrichment and host DNA depletion in human vaginal samples using Oxford Nanopore’s adaptive sequencing

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