# Absence of evidence is not evidence of absence: Nanopore sequencing and complete assembly of the European lobster (Homarus gammarus) mitogenome uncovers the missing nad2 and a new major gene cluster duplication

Han Ming Gan, Frédéric Grandjean, Tom Jenkins, Christopher Mervyn Austin

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Han Ming Gan, Frédéric Grandjean, Tom Jenkins, Christopher Mervyn Austin. Absence of evidence is not evidence of absence: Nanopore sequencing and complete assembly of the European lobster (Homarus gammarus) mitogenome uncovers the missing nad2 and a new major gene cluster duplication. BMC Genomics, 2019, 20 (1), pp.335. 10.1186/s12864-019-5704-3. hal-02295475

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# Absence of evidence is not evidence of absence: Nanopore sequencing and complete assembly of the European lobster (*Homarus gammarus*) mitogenome uncovers the missing *nad2* and a new major gene cluster duplication

Han Ming Gan¹,²,³,⁴* ID, Frederic Grandjean⁵, Tom L. Jenkins⁶ and Christopher Mervyn Austin¹,²,³,⁴

## Abstract

**Background:** The recently published complete mitogenome of the European lobster (*Homarus gammarus*) that was generated using long-range PCR exhibits unusual gene composition (missing *nad2*) and gene rearrangements among decapod crustaceans with strong implications in crustacean phylogenetics. Such atypical mitochondrial features will benefit greatly from validation with emerging long read sequencing technologies such as Oxford Nanopore that can more accurately identify structural variation.

**Results:** We re-sequenced the *H. gammarus* mitogenome on an Oxford Nanopore Minion flowcell and performed a long-read only assembly, generating a complete mitogenome assembly for *H. gammarus*. In contrast to previous reporting, we found an intact mitochondrial *nad2* gene in the *H. gammarus* mitogenome and showed that its gene organization is broadly similar to that of the American lobster (*H. americanus*) except for the presence of a large tandemly duplicated region with evidence of pseudogenization in one of each duplicated protein-coding genes.

**Conclusions:** Using the European lobster as an example, we demonstrate the value of Oxford Nanopore long read technology in resolving problematic mitogenome assemblies. The increasing accessibility of Oxford Nanopore technology will make it an attractive and useful tool for evolutionary biologists to verify new and existing unusual mitochondrial gene rearrangements recovered using first and second generation sequencing technologies, particularly those used to make phylogenetic inferences of evolutionary scenarios.

**Keywords:** *Homarus gammarus*, Mitogenome, Nanopore sequencing, Lobster, Tandem duplication, PCR bias

## Background

The European lobster (*Homarus gammarus* (Linnaeus, 1758)) is an economically important crustacean species with a broad geographic range across the eastern Atlantic Ocean, extending from the Arctic Circle to Morocco [1]. The only other species placed in the genus *Homarus*

is the closely related American lobster (*Homarus americanus* (Fabricius, 1775)) with natural range on the opposite side of the North Atlantic [2]. Despite its socio-economic importance, early population genetics of the clawed lobsters employing various genetic markers such as mtDNA, microsatellites and random amplified polymorphic DNA focused on the American lobster. It is only in the last two decades that the genetic diversity and population structure of *H. gammarus* have been actively investigated using molecular genetic techniques [3–6]. One of the most comprehensive genetic

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BMC

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

<footnote>* Correspondence: han.gan@deakin.edu.au
¹Deakin Genomics Centre, Deakin University, Geelong, VIC 3220, Australia
²Centre for Integrative Ecology, School of Life and Environmental Sciences, Deakin University, Geelong, VIC 3220, Australia
Full list of author information is available at the end of the article</footnote>
investigations of *Homarus gammarus* was by Triantafyl-
lidis and co-workers who sampled 3283 individuals from
44 populations across its native geographical distribution
and analyzed the diversity and distribution of restriction
fragment length polymorphism of a 3-kb mitochondrial
DNA amplicon [7].

The complete mitogenome of *H. americanus* has been
sequenced, assembled and annotated and is consistent
with the pancrustacean gene order [8]. However, in stark
contradistinction, the recently published *H. gammarus*
mitogenome [9] deviates substantially as it was reported
to have not only a large inversion but to be also missing
the *nad2* genes and a few tRNA genes [10]. While minor
and major departures from the pancrustacean gene
order have been observed in multiple lineages of decapod
crustaceans notably among scampi and Southern Hemi-
sphere freshwater crayfish species [11–16], all 13 mitochon-
drial protein-coding genes are always present in the
assembled mitogenome occasionally with some species hav-
ing duplicated tRNA genes and occasionally the control
region [14]. The finding of mitogenome structural varia-
tions is of interest as molecular synapomorphies and for
providing insights in mitogenome evolution and it is quite
rare to find such significant mitogenome rearrangements
and missing genes between closely related species. A neces-
sary caveat, however, is the assumption that mitogenome
studies reporting rearrangements and missing genes have
used appropriate wet lab and bioinformatics protocols that
are robust to potential errors or misinterpretations. It is
thus always beneficial that such atypical findings be verified
by other laboratories.

Traditionally, complete mitogenomes have been recov-
ered using long-range PCR followed by multiple Sanger
sequencing reactions (primer walking) using overlapping
fragments to produce a complete assembly. One of the
assumptions of this approach is that the mitogenome of
interest has a gene order that is identical to, or at least
highly similar to the reference sequence. In other words,
species with mitogenomes with substantial gene rearrange-
ment relative to an appropriate reference mitogenome (usu-
ally a related species) will present a significant challenge to
the complete recovery of mitogenomes using PCR-based ap-
proach as this will generally lead to failed PCR reactions,
amplicon of unanticipated lengths and multiple amplicons as
well as the potential amplification of nuclear-derived
mtDNAs [17–19]. The advent of high-throughput short-read
sequencing technologies has greatly enhanced the accuracy
and efficiency of mitogenome recovery by allowing mitogen-
omes to be sequenced without prior sequence knowledge or
gene order [20, 21]. By sequencing multiple small segments
of the DNA (~ 100–250 bp) in parallel, such an approach es-
sentially removes the assumptions and limitations of
long-range PCR and also allows the recovery of mitogen-
omes from degraded DNA samples including those from

museum specimens [22, 23]. Nevertheless, a major pitfall of
this technology is the inability of short reads to span highly
repetitive regions that exist in some mitogenomes particu-
larly in the control region. This means that assemblies may
give a fragmented mitogenome that precludes the accurate
inference of gene order organization, although gap closing
using custom design primers and Sanger sequencing can be
successful in resolving such incomplete assemblies [24].

Three common approaches are used for the assembly
of mitogenome from short read data e.g. whole mitogen-
ome reference mapping, bait-and-extend from a single
gene and de novo assembly; yet, these approaches can
lead to different outcomes when applied to the same
dataset [25]. Whole mitogenome reference mapping usu-
ally works well when the sequences are obtained from
individuals from the same or a closely related species.
However, some caution is advised in assembling mito-
genomes in this way as even congeneric species can
show variations in mitochondrial gene order as recently
reported in the Southern Hemisphere burrowing fresh-
water crayfish, *Engaeus* spp. [14]. Inferring novel mito-
chondrial gene organization is generally more robust
using either de novo assembly or single gene baiting ap-
proach as they do not make assumptions on a prior gene
order information. Failure to recover a complete circular
mitogenome is usually associated with anomalies in the
sequence composition ranging from an overly repetitive
control region to large-scale tandem duplications that will
require gap-closing via PCR which is not always successful.

The recent improvements in Oxford Nanopore tech-
nology most notably in its read accuracy, flow cell stabil-
ity, and sequencing algorithm have truly democratized
long-read sequencing, providing individual researchers
with sequencing capacity that is comparable to those previ-
ously found only in large sequencing centers [26]. To date,
several studies have explored combining accurate Illumina
reads with long (> 1 kb) but more error-prone Nanopore
reads to improve whole genome assemblies [27–29]. More
recently, this approach has been used to assemble the
complete mitogenome (~ 16 kb) of the economically import-
ant green-lipped mussel [30]. However, the mitogenome ex-
hibits the typical 13 protein-coding genes, 22 tRNAs, 2
rRNAs and a control region, suggesting that Illumina short
read data alone can also accomplish the same objective as re-
ported in various mussel mitogenomic studies [31–34]. Un-
like the green-lipped mussel mitogenome, the *Homarus*
*gammarus* mitogenome represents an interesting case due to
its atypical gene organization that has been partially verified
using PCR across a few individuals in a follow-up study [10].
Furthermore, the high genetic variation observed in the 3 kb
mitochondrial region that excludes the control region is un-
expected [7] but can be explained by the presence of a large
tandem duplication event followed by pseudogenization
which warrants further investigation. In this study, we
generated the first Illumina dataset for *Homarus gammarus* providing strong evidence for structural incongruence with the published *H. gammarus* mitogenome. By fully exploiting the ability of Nanopore long-read to span large repeats, we were able to circularize the mitogenome leading to the discovery of the large tandemly duplicated mitogenome region with strong evidence of pseudogenization and the presence of the purportedly missing *nad2* gene.

# Methods

## Sample collection and gDNA extraction

A wild-caught European lobster from the Atlantic Ocean was purchased from a commercial fisherman. Approximately 500 mg of tail muscle tissue was dissected and preserved in 95% ethanol. DNA extraction was performed using an SDS-based lysis method followed by chloroform purification as previously described [35]. To confirm mitogenome features, notably the presence of *nad2* gene, additional European lobsters were collected from five different and distant European regions (Fig. 2c) as previously described [36].

## Illumina sequencing

One hundred ng of gDNA was sonicated to a fragment size of 500 bp using a Covaris ultrasonicator and processed using NEB Ultra DNA library prep kit according to the manufacturer's instructions. The constructed library was quantified and size-estimated using Qubit and Tapestation2100, respectively. Sequencing of the library was performed on an Illumina MiSeq located at the Monash University Malaysia Genomics Facility using the run configuration 2 × 250 bp.

## Nanopore sequencing

Two µg of gDNA was repaired using NEB FFPE Repair Mix and subsequently processed using the Nanopore LSK108 kit according to the manufacturer's instructions. The library (premixed with loading beads) was pipetted into a previously used and washed FLO-MIN106 flow cell. The flow cell was fixed to a MINION device and sequencing was performed for 16 h (overnight) on a Linux Ubuntu desktop through the MinKnow software version 2.0.

## Mitogenome assembly and annotation

Illumina-only mitogenome assembly was performed using two reference-based assemblers (MITObim and NOVOplasty) and a de novo assembler (Megahit) [37–39]. For MITObim, 3 separate assemblies were performed using different mitochondrial gene baits (*nad2*, 16S rRNA, and *cytb*) from the American Lobster, *H. americanus* (GenBank accession number: NC_015607). Completeness and the presence of anomalies in the assembled mitogenome were determined by conducting BLASTX search of the contig against the 13 mitochondrial proteins from *H. americanus*. For long-read

only assembly, Nanopore long-reads were first aligned to the mitogenomes of *H. americanus* and *H. gammarus*. The aligned reads were error-corrected, trimmed and assembled using CANU v1.7 [40]. The graphical fragment assembly of contig flagged as circular by CANU was visualized on Bandage v0.8.1 [41]. A self-against-self nucleotide similarity search of the assembled contig was performed and visualized with EasyFig v2.2.2 [42] to confirm the presence of highly similar flanking regions in the contig. Polishing of the CANU assembly used Unicycler_Polish module in Unicycler v0.4.4 that mapped Illumina reads to the raw assembly using Bowtie2 (–very-sensitive-local) and piped the alignment file into Pilon v1.22 to specifically correct for small indel and SNP [43–45]. Circularization of the polished mitogenome and re-orientation of the contig 5' end to *cox1* gene used Circles.py python script that is part of the MITObim v1.9 software [39]. The circularized mitogenome was polished again to fix any remaining assembly errors in sequence previously originating from the flanking regions of the uncircularized contig. These errors may have been missed in the first polishing step due to the repetitiveness and lower read coverage in the flanking regions. The polished mitogenome was annotated using the MITOS webserver [46] and each protein-coding gene was manually inspected with their start and stop position adjusted when necessary using the *H. americanus* mitogenome as the reference. The polished *H. gammarus* mitogenome was subsequently used as the reference to reconstruct the mitogenomes of the 5 additional *H. gammarus* individuals through MITObim v1.9 “—quick” option.

## Visualization of mitochondrial gene organization and alignment

Nad2 proteins were aligned and visualized with MAFFT v7.123b and ESPrift v3.0, respectively [47, 48]. Maximum likelihood tree based on the amino alignment was inferred using IQ-TREE v1.65 with 1000 ultra-fast bootstrap replicates [49]. Circular comparisons between the newly assembled *H. gammarus* mitogenomes used BRIG v0.95 [50] while linear comparison of gene organization between *Homarus* spp. used EasyFig v2.2.2 [42]. Alignment of Illumina and Nanopore reads to the complete mitogenome was performed using bowtie2 v2.3.0 and minimap2 v2.10-r761, respectively and visualized in IGV v2.4.5 [51, 52]. GC plot of the mitogenome (windowsize = 500 bp) was constructed in Artemis v17.0.1 [53]. Nucleotide and amino acid alignment of pseudogenes used MACSE v2.01 (gc_code =5) followed by alignment visualization in Jalview [54, 55].

# Results

## The mitochondrial NADH dehydrogenase subunit 2 (*nad2*) gene has not been lost in *Homarus gammarus*

A total of 6,029,826 paired-end reads (~ 1.5 gigabases, SRA accession:SRP157954) were generated from MiSeq
sequencing. Such a sequencing output is typically sufficient for the reconstruction of mitogenome via reference mapping or de novo assembly approach. Contrary to the previously reported loss of *nad2* in *H. gammarus* [9] (Fig. 2a), mitogenome reconstruction using *H. americanus nad2* as the bait generated a contig which spans the entire *nad2* gene of *H. gammarus* in addition to other bona fide mitochondrial genes in its vicinity with. Most contig extensions occurring at the 3' end of the bait (Table 1), suggesting a difficult-to-assemble region possibly in the form of long repeats upstream of the *nad2* gene. The translated ORF of the assembled *nad2* shows high pairwise amino acid identity (96.7%) to that of *H. americanus* with only 11 amino acid mismatches out of 333 aligned protein length (Fig. 1a). Protein domains associated with NADH dehydrogenase subunit 2 are present in the translated ORF (Fig. 1b), suggesting a similar function. Maximum likelihood tree construction based on amino acid alignment provides phylogenetic support for its annotation as *nad2* given its well-supported monophyletic clustering (88% ultrafast bootstrap support) with the *nad2* protein of *H. americanus* (Fig. 1c).

## Illumina-based genome skimming approach fails to generate the complete *Homarus gammarus* mitogenome assembly

Both reference mapping (single-gene bait) and de novo assembly based on Illumina-only short reads failed to generate contigs that are longer than 15 kb or contain flanking identical region typically required to infer mitogenome circularity. The longest MITObim assembly was achieved using the *H. americanus cob* gene as the initial read bait with a final assembly length of 14 kb covering all 13 PCGs (Table 1). On the other hand, BlastX search against the de novo-assembled contigs identified multiple small mitochondrial contigs, some of which exhibit significant homology to one another. For example, ctg154136 (5 kb) and ctg252123 (4 kb) from the MEGA-HIT assembly harbor a nearly similar set of PCGs

although the some of the PCGs in the latter contig are likely pseudogenes.

## Nanopore long-read assembly confirms mitogenome circularity

An overnight run of a used nanopore flow cell generated 40,687 reads with an accumulated length of 100 megabases and N50 of 2811 bp. The basecalled fastq files were archived in Zenodo (https://doi.org/10.5281/zenodo.1345356). Approximately 0.19% of the sequencing reads were mapped to the *H. americanus* and *H. gammarus* mitogenomes (255 kb, N50 of 4639 bp). De novo assembly of the mapped long-reads generated a 26 kb contig suggested to be “circular” by CANU. This was subsequently verified by the circular representation of the assembly graph in Bandage (Additional file 1: Figure S1A). Self-to-self nucleotide alignment of the contig indicates that up to 6 kb of its flanking regions exhibit strikingly high similarity, thus providing additional evidence for mitogenome circularity (Additional file 1: Figure S1B). Four rounds of Pilon polishing identified and corrected 402 indels/SNPs in the initial CANU-assembled contig. Subsequent polishing of the circularized and re-oriented contig corrected an additional 5 indels/SNPs, generating a final, complete and circular mitogenome with a total length of 20,303 bp and 31.68% GC-content. MITOS annotation confirmed the presence of the typical 13 PCG but also identified 6 duplicated PCGs some of which contained internal stop codons and/or frameshift mutation. The final polished and annotated mitogenome assembly has been deposited in GenBank under the accession number MH747083.

## Consistent recovery of *nad2* in *H. gammarus* individuals from different populations

Linear comparison of the recently published *H. gammarus* complete mitogenome (GenBank: NC_020020) with our new assembly revealed three substantial structural variations namely a tandem duplication involving

<table><caption>**Table 1** Performance of different assemblers in the recovery of *Homarus gammarus* mitogenome</caption><thead><tr><th>Assembler</th><th>Contig Length (bait/contigID)</th><th>Protein coding gene (number of intact genes)</th></tr></thead><tbody><tr><td rowspan="3">MITObim v. 1.9</td><td>14,885 bp (rrnL)</td><td>nad1, cytb, nad6, nad4L, nad4, nad5, nad3, cox3, atp6, atp8, cox2, cox1, nad2 (13)</td></tr><tr><td>13,143 bp (cytb)</td><td>nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad5, nad4, nad4L, nad6, cytb, nad1 (13)</td></tr><tr><td>6984 bp (nad2)</td><td>nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad5, cox2 (9)</td></tr><tr><td rowspan="3">Megahit v1.1.2</td><td>5162 bp (ctg154136)</td><td>nad2, cox1, cox2, atp8, atp6, cox3 (6)</td></tr><tr><td>4045 bp (ctg252123)</td><td>cox3<sup>a</sup>, atp6, atp8, cox2<sup>a</sup>, nad5<sup>b</sup>, nad3, cox3 (4)</td></tr><tr><td>10,025 bp (ctg253367)</td><td>nad1, cytb, nad6, nad4L, nad4, nad5, nad3, cox3 (8)</td></tr><tr><td>NOVOplasty v2.7.1</td><td>22,032 bp</td><td>nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad5, cox2<sup>a</sup>, atp8, atp6, cox3<sup>a</sup>, nad3, nad5, cox2<sup>a</sup>, atp8, atp6, cox3<sup>a</sup>, nad3, nad5, cox3<sup>a</sup>, atp6, cox3<sup>a</sup>, nad3 (8)</td></tr></tbody></table>

<footnote><sup>a</sup>Internal stop codon <sup>b</sup> Fragmented</footnote>
<figure><img src="image_4.png" /></figure>

the *cox2-atp8-atp6-nad3* protein coding genes, a large inversion and as previously highlighted, the absence of *nad2* and a few tRNAs (Fig. 2a). On average 868 Mb (670–1128 Mb) of Illumina sequencing data were generated for each of the *H. gammarus* individuals and deposited in the SRA database (Bioproject ID: PRJNA486050). Reference-based mapping assembly successfully reconstructed the near-complete mitogenome except for samples She17_01 and Jer01 that contain minor assembly gaps in the ribosomal genes and non-coding control region (Fig. 2b and Additional file 2: Data 1). Notably, significant nucleotide alignment to the *H. gammarus* mitochondrial *nad2* gene with high pairwise identity was observed across all five individuals (Fig. 2c).

## Different lines of evidence indicate partial tandem duplication in the *Homarus gammarus* mitogenome

A gene region consisting of *cox2*, *atp8*, *atp6*, *cox3*, *nad3*, and *nad5* as well as their neighboring tRNAs were present in two copies and in tandem in the

newly assembled mitogenome. One of the *nad5* genes was substantially shorter with its start codon and a majority of 5' end truncated (Fig. 3a). The observed duplication was not due to sequencing or assembly error as multiple Nanopore reads spanned across both duplicated gene regions (Fig. 3b). In addition, de novo assembly using Illumina-only reads also recovered two separate contigs corresponding to each of the duplicated region suggesting that sufficient nucleotide similarity exists between the repeats to prevent collapsing of contigs by the assembler into a single contig (Fig. 3c). Substantial and similar coverage of Illumina reads to both regions lends additional support to the veracity of the nanopore sequences. Interestingly, unlike the Nanopore read alignment, Illumina reads show an uneven coverage across the mitogenome that negatively correlates with the genome AT-content (Fig. 3d and e). Notably, low or near-zero read depth was observed in regions with less than 22% GC that correspond to the location of the 12S and 16S ribosomal RNA genes.
<figure><img src="image_5.png" /><figcaption>**Fig. 2** Presence of *nad2* can be detected in at least five *H. gammarus* individuals collected from various European regions. a Linear gene organization comparison of the previous reported *H. gammarus* mitogenome with that reported in this study confirming the absence of *nad2* in the original report in addition to substantial structural variation. The direction of arrow indicates transcription orientation. Orange, teal, purple and red arrows correspond to protein-coding genes, putative pseudogenes, rRNAs and tRNAs, respectively. b Circular comparison of the reconstructed mitogenomes showing significant nucleotide alignment across the entire newly assembled *H. gammarus* mitogenome with minor assembly gaps in the high-AT ribosomal rRNA regions (See Fig. 3e for details). c Sampling map of the five *H. gammarus* individuals with their exact location indicated by a star symbol</figcaption></figure>

## Pseudogenization among tandemly duplicated mitochondrial genes

Duplicated genes that were disconnected from their original transcriptional track e.g. *cox2*, *atp8*, *atp6*, *cox3*, and *nad3* in the second duplicated region and *nad5* in the first duplicated region, appear to show signs of pseudogenization. For example, an internal stop codon was located in the duplicated *cox2* gene (Fig. 4a) while frameshift mutations were found in the *cox3*, *nad3*, and *atp8* genes (Fig. 4b). DNA mutations leading to pseudogenization were supported by multiple Illumina reads, precluding the possibility of assembly or sequencing errors. Although both duplicated *atp6* genes are still intact

and can be translated into protein sequence of the same length, protein similarity search using the ATPase6 of *H. americanus* as the query against both ATPase6 proteins indicates that the ATPase6 encoded by the gene located in the first duplicated region is likely the functional one as evidenced by its strikingly high protein identity to that of *H. americanus* (99.1%) compared to its duplicated partner (94.6%). Furthermore, the pseudo ATPase6 exhibits a substantially longer branch length compared to its homologs in the maximum likelihood tree (Fig. 4c), suggesting higher substitution rate presumably due to the lack of evolutionary constraints. It is also interesting to note that the pseudo ATPase6 clustered with the
<figure><img src="image_6.png" /></figure>

previously published *Homarus gammarus* ATPase6 that
also shows a similar branch length pattern.

**Discussion**

The veracity of the current European lobster reference
mitogenome in the NCBI database (Accession Number:
NC_020020.1) is questioned given its unusual features
e.g. missing *nad2*, large mitogenome inversion and
translocated tRNAs [9, 10, 24] that were not observed in
its close relative, the American lobster, *Homarus ameri-
canus* [8]. Although subsequent PCR and Sanger se-
quencing has been carried out on additional individuals
to validate the observed abnormalities [10], a secondary
validation by a separate group using different sequencing
technology is highly desirable to provide confirmation of
this atypical crustacean mitogenome sequence. We,
therefore, sequenced another individual of *Homarus*
*gammarus* using Illumina technology, which removes
the need for the laborious and presumably error-prone
steps required in traditional mitogenome sequencing
such as long-range PCR and primer walking that may
lead to the unintentional misassembly of mitogenome.

The absence of a repetitive region and/or duplicated
gene region in the previously reported *H. gammarus*
mitogenome [9] (Fig. 2a) suggests that it should be pos-
sible to recover a complete mitogenome using Illumina
short reads as demonstrated in various crustacean mito-
genome reports [16, 21, 22, 56, 57]. However, none of
our attempted Illumina-only assemblies recovered a cir-
cular mitogenome, indicating the potential presence of a
problematic region in the mitogenome that could not be
resolved, presumably due to the limitation of short reads
in recovering regions with long repeats. Only by lever-
aging on the availability of Oxford Nanopore long-read
technology, were we able to confidently close this mito-
genome. In doing so, we uncovered the presence of an
unforeseen duplicated region in the *H. gammarus* mito-
genome in addition to the presence of the *nad2* gene
which was previously reported as missing [9]. The iden-
tification of multiple Nanopore reads spanning the entire
duplicated region lends support to the authenticity of the
duplication observed in the mitogenome assembly. Fur-
thermore, by employing the Nanopore PCR-free approach
to sequencing e.g. by directly sequencing the native DNA,
<figure><img src="image_7.png" /></figure>

we can safely disregard misassembly due to chimeric sequences from PCR-amplified products commonly associated with long-range PCR [58, 59]. It is also fortuitous that the duplicated regions have accumulated sufficient

mutation resulting in the assembly of both regions as separate contigs using Illumina-only reads which provides additional and complementary support for the presence of tandem duplication in the *H. gammarus* mitogenome recovered from the Nanopore reads. The length of the observed large inversion between Shen et al [9] *H. gammarus* assembly (GenBank: NC_020020) and ours (GenBank: MH747083) (Fig. 2a) is consistent with the estimated amplicon length generated by the primers used for long-range PCR raising the possibility that misassembly could occur during the scaffolding of the assembled amplicons.

The clustering of the published *H. gammarus* AT6ase with the pseudo-ATP6ase recovered in this study coupled with its notably longer branch length compared to the bona fide *H. gammarus*, *H. americanus*, and *Nephrops norvegicus* ATP6ases strongly suggests that this *atp6* gene is a chimera of the bona fide and pseudogenes. The use of long-range PCR can result in the amplification of both duplicated region that will be considered as a single PCR product given their near-identical amplicon size. Subsequent Sanger sequencing of the “mixed” amplicons will result in the presence of non-random mixed peaks in the sequencing chromatogram that will likely be haphazardly regarded as heteroplasmy. Depending on the peak intensity, alleles originating from the other duplicated region may be called leading to the assembly of a chimeric sequence.

Based on an initial analysis of 100 *H. gammarus* individuals, Triantafyllidis et al. used restriction fragment length polymorphism (RFLP) profiling to show that a 3 kb mitochondrial region (amplified using the Hom3F and Hom3R primers) containing *cox2*, *cox3*, *atp6*, *atp8*, *nad3* and several tRNAs is the most variable region and hence selected that region for a population genetic study of 4018 individuals collected throughout the geographical range of the species [7]. This is quite surprising given that the 3 kb region does not contain the control region which is known to be the most polymorphic region in mitochondrial DNA [60], suggesting an unknown source of nucleotide variation in addition to population-specific genetic variation. Similarity search of Hom3F and Hom3R primers against the newly assembled *Homarus gammarus* mitogenome indicates that primer binding sites are present in both of the duplicated regions which will lead to the amplification of two amplicons with near exact length (~ 2.8 kb) (Additional file 3: Table S1), and may well have confounded the results of Triantafyllidis et al. [7].

Mapping of Illumina reads to the mitogenome revealed a GC-dependent coverage variation that was not observed in the Nanopore read alignment. Since the standard Nanopore library preparation does not involve any PCR step, the uneven mapping coverage is most definitely caused by PCR amplification bias during Illumina library preparation which is known to under-represent AT-rich fragments [61, 62]. AT-rich regions are prevalent in the
control region and ribosomal RNA genes. Such mitogenome regions will be harder to assemble due to low sequencing depth, leading to gaps in the mitogenome assembly and the incorrect inference of mitogenome linearity. Although our study highlights the value of Nanopore long-read technology in resolving a difficult-to-assemble mitogenome, we are conservative in advocating its use in routine mitogenome sequencing due to its stringent requirement for high-quality and intact gDNA. Having said that, the obvious advantage that Nanopore long reads offer in spanning repetitive sequencing make it a powerful new tool for elucidating or confirming duplicated mitochondrial genes and difficult-to-assemble mitogenomes in animals. Another third generations sequencing approach that can be used to improve a mitogenome assembly is PacBio technology albeit its reliance on polymerase chain reaction to generate sequencing data may reduce its efficiency in the sequencing of problematic region, as encountered in Illumina sequencing in this study.

Lastly, we emphasize as reported in other scientific domains, that the absence of evidence (for a mitochondrial gene) is not necessary evidence of absence. A gene may fail to be recovered due to limitation in a wet lab protocol or bioinformatics pipeline as we demonstrated for the *nad2* gene in *H. gammarus* in this study and others have demonstrated for the *atp8* gene in bivalve mollusks [63]. Thus, researchers need to be skeptical of assertions that a mitochondrial gene is missing and make efforts to verify that there really is a genuine evidence of absence.

## Conclusions

We demonstrate the utility of Oxford Nanopore long-read in resolving problematic mitogenome assemblies by re-sequencing the mitogenome of the European lobster that was previously reported to exhibit several atypical features. In addition to showing substantial deviation from the previously published mitogenome, we identified new gene features that were missed by previous studies including the complete recovery of the *nad2* gene and a large tandem duplication consisting of six protein-coding genes with evidence of pseudogenization. The power of long reads in resolving repetitive regions and accurately identifying tandemly duplicated mitochondrial gene regions highlights the value of new sequencing tools for evolutionary biologists to confidently infer novel mitochondrial gene organization. Furthermore, the veracity of unusual mitochondrial gene rearrangements recovered using first and second sequencing technologies, particularly those used to make phylogenetic inferences of evolutionary scenarios, can now be effectively tested using Oxford Nanopore long reads, marking another step forward in mitogenomics.

## Additional files

**Additional file 1: Figure S1.** Long-read only assembly enables the complete assembly of *H. gammarus* mitogenome. (A) Bandage visualization of the CANU assembly graph corresponding to the mitogenome contig. (B) Self-against-self comparison of the mitogenome contig in EasyFig with up to 6 kb of flanking regions exhibiting high nucleotide similarity (~ 98%) (TIF 76 kb)

**Additional file 2: Data 1.** Mitogenome assemblies in fasta format of 5 additional *Homarus gammarus* sampled from distant location in Europe (See Fig. 2c for details) (TXT 99 kb)

**Additional file 3: Table S1.** Binding sites of *I_Hom3F* and *I_Hom3R* primers on the newly assembled *Homarus gammarus* mitogenome and the estimated PCR product sizes. (PDF 16 kb)

### Abbreviations

PCG: Protein Coding Gene; PCR: Polymerase Chain Reaction; SDS: Sodium Dodecyl Sulfate; SNP: Single Nucleotide Polymorphism

### Acknowledgements

Not applicable.

### Funding

Funding for this project was provided by the Monash University Malaysia Tropical Medicine and Biology Platform. The funder had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

### Availability of data and materials

Illumina and Nanopore sequencing data are available through the NCBI SRA accession SRP157954 and Zenodo repository (https://doi.org/10.5281/zenodo.1345356), respectively.

### Authors' contributions

HMG, CMA, TLJ and FG designed and coordinated the research. FG and TLJ collected the sample. HMG performed sequencing and bioinformatics analysis. HMG, CMA and FG analyzed the data and drafted the manuscript. All authors read and approved the final manuscript.

### Ethics approval and consent to participate

Not applicable. Samples (deceased) were either purchased from or given by commercial fisherman.

### Consent for publication

Not applicable.

### Competing interests

The authors declare that they have no competing interests.

### Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

### Author details

<footnote n="1">¹Deakin Genomics Centre, Deakin University, Geelong, VIC 3220, Australia.</footnote>

<footnote n="2">²Centre for Integrative Ecology, School of Life and Environmental Sciences, Deakin University, Geelong, VIC 3220, Australia. ³School of Science, Monash University Malaysia, Bandar Sunway, 47500 Petaling Jaya, Malaysia. ⁴Monash University Malaysia Genomics Facility, Monash University, Bandar Sunway, 47500 Petaling Jaya, Malaysia. ⁵Laboratoire Ecologie et Biologie des Interactions, UMR CNRS 7267 Equipe Ecologie Evolution Symbiose 5 rue Albert Turpin, 86073 Poitiers, Cedex, France. ⁶Department of Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, UK.</footnote>
Received: 26 August 2018 Accepted: 16 April 2019

Published online: 03 May 2019

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