The mitochondrial genome and Epigenome of the Golden lion Tamarin from fecal DNA using Nanopore adaptive sequencing

Abstract

Background The golden lion tamarin (Leontopithecus rosalia) is an endangered Platyrrhine primate endemic to the Atlantic coastal forests of Brazil. Despite ongoing conservation efforts, genetic data on this species remains scarce. Complicating factors include limitations on sample collection and a lack of high-quality reference sequences. Here, we used nanopore adaptive sampling to resequence the L. rosalia mitogenome from feces, a sample which can be collected non-invasively. Results Adaptive sampling doubled the fraction of both host-derived and mitochondrial sequences compared to sequencing without enrichment. 258x coverage of the L. rosalia mitogenome was achieved in a single flow cell by targeting the unfinished genome of the distantly related emperor tamarin (Saguinus imperator) and the mitogenome of the closely related black lion tamarin (Leontopithecus chrysopygus). The L. rosalia mitogenome has a length of 16,597 bp, sharing 99.68% sequence identity with the L. chrysopygus mitogenome. A total of 38 SNPs between them were identified, with the majority being found in the non-coding D-loop region. DNA methylation and hydroxymethylation were directly detected using a neural network model applied to the raw signal from the MinION sequencer. In contrast to prior reports, DNA methylation was negligible in mitochondria in both CpG and non-CpG contexts. Surprisingly, a quarter of the 642 CpG sites exhibited DNA hydroxymethylation greater than 1% and 44 sites were above 5%, with concentration in the 3′ side of several coding regions. Conclusions Overall, we report a robust new mitogenome assembly for L. rosalia and direct detection of cytosine base modifications in all contexts.

Nanopore adaptive sampling for mitogenome sequencing and bloodmeal identification in hematophagous insects

“…Reads were filtered for mitogenome assembly using NanoFilt set to a minimum Q-score of 10 and read lengths between 1kb and 16kb (De Coster et al, 2018). Filtered reads for individual samples were de novo assembled using Flye v2.8.3 software following Wanner et al (2021).…”

“…Animal mitogenomes are traditionally circular in structure, relatively small in size (~16kb), and contain the same core set of 13 proteincoding genes (PCGs), 22 transfer RNAs (tRNAs), and 2 ribosomal RNAs (rRNAs) (Boore, 1999;Gissi et al, 2008). Furthermore, mitogenomes are present at many copies within each cell, making them convenient targets for molecular analyses using a variety of biological sample types (Boore, 1999;Cameron, 2014;Crampton-Platt et al, 2015;Crampton-Platt, Yu, Zhou, & Vogler, 2016;Romero, Weigand, & Pfenninger, 2016;Timmermans et al, 2010;Wanner, Larsen, McLain, & Faulk, 2021). Sequence data of numerous mitochondrial loci (e.g., cytochrome c oxidase subunit 1 (COI or cox1), cytochrome b (cyt-b), and D-Loop) are welldocumented for their phylogenetic and phylogeographic utility, having variable rates of evolution that can be leveraged to test taxonomic hypotheses and elucidate evolutionary histories (Beebe, 2018;Bradley & Baker, 2001;Creedy et al, 2021;Jinbo et al, 2011;Townzen et al, 2008).…”

“…Nanopore adaptive sampling (NAS) is a recently developed method that selectively sequences individual DNA, cDNA, or RNA molecules in real-time using ONT sequencing technology (Kipp et al, 2021;Payne et al, 2021;Wanner et al, 2021). NAS uses an advanced bioinformatic pipeline that compares nucleotides of individual molecules (~200-400 base pairs) against a reference file every ~0.4 sec, as sequencing is occurring.…”

“…Additionally, we show proof-of-concept for bloodmeal host identification using the NAS method (Fig 1). NAS, combined with the long-read sequencing capabilities of the ONT MinION, is ideally suited for the selective enrichment and assembly of complete mitogenomes (Wanner et al, 2021). Moreover, NAS mitogenome sequencing can effectively capture mitochondrial sequences having at least 75% identity with a given reference; thus, the method can be used for mitogenome assembly of a variety of taxa for which references are absent (Wanner et al, 2021).…”

“…NAS, combined with the long-read sequencing capabilities of the ONT MinION, is ideally suited for the selective enrichment and assembly of complete mitogenomes (Wanner et al, 2021). Moreover, NAS mitogenome sequencing can effectively capture mitochondrial sequences having at least 75% identity with a given reference; thus, the method can be used for mitogenome assembly of a variety of taxa for which references are absent (Wanner et al, 2021). We used the NAS mitogenome sequencing approach to molecularly characterize four mosquito species as well as a biting deer fly and its bloodmeal host from Minnesota.…”