Candidatus Methanosphaera massiliense sp. nov., a methanogenic archaeal species found in a human fecal sample and prevalent in pigs and red kangaroos.

Methanosphaera stadtmanae was the sole Methanosphaera representative to be cultured and detected by molecular methods in the human gut microbiota, further associated with digestive and respiratory diseases, leaving unknown the actual diversity of human-associated Methanosphaera species. Here, a novel Methanosphaera species, Candidatus Methanosphaera massiliense (Ca. M. massiliense) sp. nov. was isolated by culture using a hydrogen- and carbon dioxide-free medium from one human feces sample. Ca. M. massiliense is a non-motile, 850 nm Gram-positive coccus autofluorescent at 420 nm. Whole-genome sequencing yielded a 29.7% GC content, gapless 1,785,773 bp genome sequence with an 84.5% coding ratio, encoding for alcohol and aldehyde dehydrogenases promoting the growth of Ca. M. massiliense without hydrogen. Screening additional mammal and human feces using a specific genome sequence-derived DNA-polymerase RT-PCR system yielded a prevalence of 22% in pigs, 12% in red kangaroos, and no detection in 149 other human samples. This study, extending the diversity of Methanosphaera in human microbiota, questions the zoonotic sources of Ca. M. massiliense and possible transfer between hosts.IMPORTANCEMethanogens are constant inhabitants in the human gut microbiota in which Methanosphaera stadtmanae was the only cultivated Methanosphaera representative. We grew Candidatus Methanosphaera massiliense sp. nov. from one human feces sample in a novel culture medium under a nitrogen atmosphere. Systematic research for methanogens in human and animal fecal samples detected Ca. M. massiliense in pig and red kangaroo feces, raising the possibility of its zoonotic acquisition. Host specificity, source of acquisition, and adaptation of methanogens should be further investigated.

Marsupials have monoallelic MEST expression with a conserved antisense lncRNA but MEST is not imprinted.

The imprinted isoform of the Mest gene in mice is involved in key mammalian traits such as placental and fetal growth, maternal care and mammary gland maturation. The imprinted isoform has a distinct differentially methylated region (DMR) at its promoter in eutherian mammals but in marsupials, there are no differentially methylated CpG islands between the parental alleles. Here, we examined similarities and differences in the MEST gene locus across mammals using a marsupial, the tammar wallaby, a monotreme, the platypus, and a eutherian, the mouse, to investigate how imprinting of this gene evolved in mammals. By confirming the presence of the short isoform in all mammalian groups (which is imprinted in eutherians), this study suggests that an alternative promoter for the short isoform evolved at the MEST gene locus in the common ancestor of mammals. In the tammar, the short isoform of MEST shared the putative promoter CpG island with an antisense lncRNA previously identified in humans and an isoform of a neighbouring gene CEP41. The antisense lncRNA was expressed in tammar sperm, as seen in humans. This suggested that the conserved lncRNA might be important in the establishment of MEST imprinting in therian mammals, but it was not imprinted in the tammar. In contrast to previous studies, this study shows that MEST is not imprinted in marsupials. MEST imprinting in eutherians, therefore must have occurred after the marsupial-eutherian split with the acquisition of a key epigenetic imprinting control region, the differentially methylated CpG islands between the parental alleles.

Characterization of the marsupial endogenous retrovirus walb with a focus on satellite DNA formation.

The walbRep megasatellite DNA found in the red-necked wallaby was formed from the walb endogenous retrovirus. Our previous PCR experiments suggested the presence of walb and absence of walbRep in the genome of the tammar wallaby, which diverged from the red-necked wallaby 2-3 Mya. The results failed to exclude the possibility that certain walbRep sequences might have remained undetected owing to variation in the primer-annealing regions; therefore, the aforementioned suggestion was not confirmed. To obtain conclusive evidence, we analyzed the structure of walb sequences drawn from the tammar wallaby genome database recently updated to a chromosome-level assembly. All walb copies existed as separate DNA segments, not constituting tandem repeats. We concluded that walbRep was formed in the red-necked wallaby lineage after its divergence from the tammar wallaby. We also confirm the presence of a walb copy with an anomalistic, complex structure and propose a simple model for its generation mechanism.

Kangaroo endogenous retrovirus (KERV) forms megasatellite DNA with a simple repetition pattern in which the provirus structure is retained.

The kangaroo endogenous retrovirus (KERV) was previously reported to have undergone a rapid copy number increase in the red-necked wallaby; however, the mode of amplification was left to be clarified. The present study revealed that the long terminal repeat (LTR) (0.6 kb) and internal region (2.0 kb) of a provirus are repeated alternately, forming megasatellite DNA which we named kervRep. This repetition pattern was the same as that observed for walbRep, megasatellite DNA originating from another endogenous retrovirus. Their formation process can be explained using a simple model: pairing slippage followed by homologous recombination. This model features that the initial step is triggered by the presence of two identical sequences within a short distance; the possession of LTRs by endogenous retroviruses fulfills this condition. The discovery of two cases suggests that formation of this type of satellite DNA is one of non-negligible effects of endogenous retroviruses on their host genomes.

Marsupial genome analysis suggests that satellite DNA formation from walb endogenous retrovirus is an event specific to the red-necked wallaby.

We recently identified walbRep, a satellite DNA residing in the genome of the red-necked wallaby Notamacropus rufogriseus. It originates from the walb endogenous retrovirus and is organized in a manner in which the provirus structure is retained. The walbRep repeat units feature an average pairwise nucleotide identity as high as 99.5%, raising the possibility of a recent origin. The tammar wallaby N. eugenii is a species estimated to have diverged from the red-necked wallaby 2-3 million years ago. In PCR analyses of these two and other related species, walbRep-specific fragment amplification was observed only in the red-necked wallaby. Sequence database searches for the tammar wallaby resulted in sequence alignment lists that were sufficiently powerful to exclude the possibility of walbRep existence. These results suggested that the walbRep formation occurred in the red-necked wallaby lineage after its divergence from the tammar wallaby lineage, thus in a time span of maximum 3 million years.

Evolution of the Short Form of DNMT3A, DNMT3A2, Occurred in the Common Ancestor of Mammals.

Genomic imprinting is found in marsupial and eutherian mammals, but not in monotremes. While the primary regulator of genomic imprinting in eutherians is differential DNA methylation between parental alleles, conserved imprinted genes in marsupials tend to lack DNA methylation at their promoters. DNA methylation at eutherian imprinted genes is mainly catalyzed by a DNA methyltransferase (DNMT) enzyme, DNMT3A. There are two isoforms of eutherian DNMT3A: DNMT3A and DNMT3A2. DNMT3A2 is the primary isoform for establishing DNA methylation at eutherian imprinted genes and is essential for eutherian genomic imprinting. In this study, we investigated whether DNMT3A2 is also present in the two other mammalian lineages, marsupials and monotremes. We identified DNMT3A2 in both marsupials and monotremes, although imprinting has not been identified in monotremes. By analyzing genomic sequences and transcriptome data across vertebrates, we concluded that the evolution of DNMT3A2 occurred in the common ancestor of mammals. In addition, DNMT3A/3A2 gene and protein expression during gametogenesis showed distinct sexual dimorphisms in a marsupial, the tammar wallaby, and this pattern coincided with the sex-specific DNA methylation reprogramming in this species as it does in mice. Our results show that DNMT3A2 is present in all mammalian groups and suggests that the basic DNMT3A/3A2-based DNA methylation mechanism is conserved at least in therian mammals.

Epigenetic clock and methylation studies in marsupials: opossums, Tasmanian devils, kangaroos, and wallabies.

The opossum (Monodelphis domestica), with its sequenced genome, ease of laboratory care and experimental manipulation, and unique biology, is the most used laboratory marsupial. Using the mammalian methylation array, we generated DNA methylation data from n = 100 opossum samples from the ear, liver, and tail. We contrasted postnatal development and later aging effects in the opossum methylome with those in mouse (Mus musculus, C57BL/6 J strain) and other marsupial species such as Tasmanian devil, kangaroos, and wallabies. While the opossum methylome is similar to that of mouse during postnatal development, it is distinct from that shared by other mammals when it comes to the age-related gain of methylation at target sites of polycomb repressive complex 2. Our immunohistochemical staining results provide additional support for the hypothesis that PRC2 activity increases with later aging in mouse tissues but remains constant in opossum tissues. We present several epigenetic clocks for opossums that are distinguished by their compatibility with tissue type (pan-tissue and blood clock) and species (opossum and human). Two dual-species human-opossum pan-tissue clocks accurately measure chronological age and relative age, respectively. The human-opossum epigenetic clocks are expected to provide a significant boost to the attractiveness of opossum as a biological model. Additional epigenetic clocks for Tasmanian devil, red kangaroos and other species of the genus Macropus may aid species conservation efforts.

Strategies for meiotic sex chromosome dynamics and telomeric elongation in Marsupials.

During meiotic prophase I, homologous chromosomes pair, synapse and recombine in a tightly regulated process that ensures the generation of genetically variable haploid gametes. Although the mechanisms underlying meiotic cell division have been well studied in model species, our understanding of the dynamics of meiotic prophase I in non-traditional model mammals remains in its infancy. Here, we reveal key meiotic features in previously uncharacterised marsupial species (the tammar wallaby and the fat-tailed dunnart), plus the fat-tailed mouse opossum, with a focus on sex chromosome pairing strategies, recombination and meiotic telomere homeostasis. We uncovered differences between phylogroups with important functional and evolutionary implications. First, sex chromosomes, which lack a pseudo-autosomal region in marsupials, had species specific pairing and silencing strategies, with implications for sex chromosome evolution. Second, we detected two waves of γH2AX accumulation during prophase I. The first wave was accompanied by low γH2AX levels on autosomes, which correlated with the low recombination rates that distinguish marsupials from eutherian mammals. In the second wave, γH2AX was restricted to sex chromosomes in all three species, which correlated with transcription from the X in tammar wallaby. This suggests non-canonical functions of γH2AX on meiotic sex chromosomes. Finally, we uncover evidence for telomere elongation in primary spermatocytes of the fat-tailed dunnart, a unique strategy within mammals. Our results provide new insights into meiotic progression and telomere homeostasis in marsupials, highlighting the importance of capturing the diversity of meiotic strategies within mammals.

An endogenous retrovirus presumed to have been endogenized or relocated recently in a marsupial, the red-necked wallaby.

An albino infant wallaby was born to a mother with wild-type body color. PCR and sequencing analyses of TYR (encoding tyrosinase, which is essential for melanin biosynthesis) of this albino wallaby revealed a 7.1-kb-long DNA fragment inserted in the first exon. Since the fragment carried long terminal repeats, we assumed it to be a copy of an endogenous retrovirus, which we named walb. We cloned other walb copies residing in the genomes of this species and of another wallaby species. The copies exhibited length variation, and the longest copy (>8.0 kb) contained open reading frames whose deduced amino acid sequences were well aligned with those of gag, pol, and env of retroviruses. It is unknown through which of the following likely processes the walb copy was inserted into TYR: endogenization (infection of a germline cell by an exogenous virus), reinfection (infection by a virus produced from a previously endogenized provirus), or retrotransposition (intracellular relocation of a provirus). In any case, the insertion into TYR is considered to have been a recent event on an evolutionary timescale because albino mutant alleles generally do not persist for long because of their deleterious effects in wild circumstances.

Limited Introgression between Rock-Wallabies with Extensive Chromosomal Rearrangements.

Chromosome rearrangements can result in the rapid evolution of hybrid incompatibilities. Robertsonian fusions, particularly those with monobrachial homology, can drive reproductive isolation amongst recently diverged taxa. The recent radiation of rock-wallabies (genus Petrogale) is an important model to explore the role of Robertsonian fusions in speciation. Here, we pursue that goal using an extensive sampling of populations and genomes of Petrogale from north-eastern Australia. In contrast to previous assessments using mitochondrial DNA or nuclear microsatellite loci, genomic data are able to separate the most closely related species and to resolve their divergence histories. Both phylogenetic and population genetic analyses indicate introgression between two species that differ by a single Robertsonian fusion. Based on the available data, there is also evidence for introgression between two species which share complex chromosomal rearrangements. However, the remaining results show no consistent signature of introgression amongst species pairs and where evident, indicate generally low introgression overall. X-linked loci have elevated divergence compared with autosomal loci indicating a potential role for genic evolution to produce reproductive isolation in concert with chromosome change. Our results highlight the value of genome scale data in evaluating the role of Robertsonian fusions and structural variation in divergence, speciation, and patterns of molecular evolution.

Male germline development in the tammar wallaby, Macropus eugenii.

Male germ cells undergo two consecutive processes - pre-spermatogenesis and spermatogenesis - to generate mature sperm. In eutherian mammals, epigenetic information such as DNA methylation is dynamically reprogrammed during pre-spermatogenesis, before and during mitotic arrest. In mice, by the time germ cells resume mitosis, the majority of DNA methylation is reprogrammed. The tammar wallaby has a similar pattern of germ cell global DNA methylation reprogramming to that of the mouse during early pre-spermatogenesis. However, early male germline development in the tammar or in any marsupial has not been described previously, so it is unknown whether this is a general feature regulating male germline development or a more recent phenomenon in mammalian evolutionary history. To answer this, we examined germ cell nuclear morphology and mitotic arrest during male germline development in the tammar wallaby (Macropus eugenii), a marsupial that diverged from mice and humans around 160 million years ago. Tammar pro-spermatogonia proliferated after birth and entered mitotic arrest after day 30 postpartum (pp). At this time, they began moving towards the periphery of the testis cords and their nuclear size increased. Germ cells increased in number after day 100 pp which is the time that DNA methylation is known to be re-established in the tammar. This is similar to the pattern observed in the mouse, suggesting that resumption of germ cell mitosis and the timing of DNA methylation reprogramming are correlated and conserved across mammals and over long evolutionary timescales.

Understanding Historical Demographic Processes to Inform Contemporary Conservation of an Arid zone Specialist: The Yellow-Footed Rock-Wallaby.

Little genetic research has been undertaken on mammals across the vast expanse of the arid biome in Australia, despite continuing species decline and need for conservation management. Here, we evaluate the contemporary and historical genetic connectivity of the yellow-footed rock-wallaby, Petrogalexanthopusxanthopus, a threatened macropodid which inhabits rocky outcrops across the disconnected mountain range systems of the southern arid biome. We use 17 microsatellite loci together with mitochondrial control region data to determine the genetic diversity of populations and the evolutionary processes shaping contemporary population dynamics on which to base conservation recommendations. Our results indicate the highly fragmented populations have reduced diversity and limited contemporary gene flow, with most populations having been through population bottlenecks. Despite limited contemporary gene flow, the phylogeographic relationships of the mitochondrial control region indicate a lack of structure and suggests greater historical connectivity. This is an emerging outcome for mammals across this arid region. On the basis of our results, we recommend augmentation of populations of P. x.xanthopus, mixing populations from disjunct mountain range systems to reduce the chance of continued diversity loss and inbreeding depression, and therefore maximize the potential for populations to adapt and survive into the future.

Effective machine-learning assembly for next-generation amplicon sequencing with very low coverage.

BACKGROUND: In short-read DNA sequencing experiments, the read coverage is a key parameter to successfully assemble the reads and reconstruct the sequence of the input DNA. When coverage is very low, the original sequence reconstruction from the reads can be difficult because of the occurrence of uncovered gaps. Reference guided assembly can then improve these assemblies. However, when the available reference is phylogenetically distant from the sequencing reads, the mapping rate of the reads can be extremely low. Some recent improvements in read mapping approaches aim at modifying the reference according to the reads dynamically. Such approaches can significantly improve the alignment rate of the reads onto distant references but the processing of insertions and deletions remains challenging. RESULTS: Here, we introduce a new algorithm to update the reference sequence according to previously aligned reads. Substitutions, insertions and deletions are performed in the reference sequence dynamically. We evaluate this approach to assemble a western-grey kangaroo mitochondrial amplicon. Our results show that more reads can be aligned and that this method produces assemblies of length comparable to the truth while limiting error rate when classic approaches fail to recover the correct length. Finally, we discuss how the core algorithm of this method could be improved and combined with other approaches to analyse larger genomic sequences. CONCLUSIONS: We introduced an algorithm to perform dynamic alignment of reads on a distant reference. We showed that such approach can improve the reconstruction of an amplicon compared to classically used bioinformatic pipelines. Although not portable to genomic scale in the current form, we suggested several improvements to be investigated to make this method more flexible and allow dynamic alignment to be used for large genome assemblies.

Reconstructing the Evolution of Giant Extinct Kangaroos: Comparing the Utility of DNA, Morphology, and Total Evidence.

Combined "total evidence" analysis of molecular and morphological data offers the opportunity to objectively merge fossils into the tree of life, and challenges the primacy of solely DNA based phylogenetic and dating inference, even among modern taxa. To investigate the relative utility of DNA, morphology, and total evidence for evolutionary inference, we sequenced the first near-complete mitochondrial genomes from extinct Australian megafauna: a 40-50 thousand year old giant short-faced kangaroo (Simosthenurus occidentalis) and giant wallaby (Protemnodon anak). We analyzed the ancient DNA and fossil data alongside comparable data from extant species to infer phylogeny, divergence times, and ancestral body mass among macropods (kangaroos and wallabies). Our results confirm a close relationship between Protemnodon and the iconic kangaroo genus complex "Macropus", and unite the giant Simothenurus with the hare-sized Lagostrophus fasciatus (banded hare-wallaby), suggesting that the latter is the closest living link to the once diverse sthenurine kangaroo radiation. We find that large body size evolved multiple times among kangaroos, coincident with expansion of open woodland habitats beginning in the Late Miocene. In addition, our results suggest that morphological data mislead macropod phylogeny reconstruction and in turn can distort total evidence estimation of divergence dates. However, a novel result with potentially broad application is that the accuracy and precision of reconstructing ancestral body mass was improved by tracing body mass on morphological branch lengths. This is likely due to positive allometric correlation between morphological and body size variation-a relationship that may be masked or even misleadingly inverted with the temporal or molecular branch lengths that typically underpin ancestral body size reconstruction. Our study supports complementary roles for DNA and morphology in evolutionary inference, and opens a new window into the evolution of Australia's unique marsupial fauna.

DNA methylation dynamics in the germline of the marsupial tammar wallaby, Macropus eugenii.

Parent specific-DNA methylation is the genomic imprint that induces mono-allelic gene expression dependent on parental origin. Resetting of DNA methylation in the germ line is mediated by a genome-wide re-methylation following demethylation known as epigenetic reprogramming. Most of our understanding of epigenetic reprogramming in germ cells is based on studies in mice, but little is known about this in marsupials. We examined genome-wide changes in DNA methylation levels by measuring 5-methylcytosine expression, and mRNA expression and protein localization of the key enzyme DNA methyltransferase 3 L (DNMT3L) during germ cell development of the marsupial tammar wallaby, Macropus eugenii. Our data clearly showed that the relative timing of genome-wide changes in DNA methylation was conserved between the tammar and mouse, but in the tammar it all occurred post-natally. In the female tammar, genome-wide demethylation occurred in two phases, I and II, suggesting that there is an unidentified demethylation mechanism in this species. Although the localization pattern of DNMT3L in male germ cells differed, the expression patterns of DNMT3L were broadly conserved between tammar, mouse and human. Thus, the basic mechanisms of DNA methylation-reprogramming must have been established before the marsupial-eutherian mammal divergence over 160 Mya.

Transcriptomic Analysis of MAP3K1 and MAP3K4 in the Developing Marsupial Gonad.

MAPKs affect gonadal differentiation in mice and humans, but whether this applies to all mammals is as yet unknown. Thus, we investigated MAPK expression during gonadal differentiation and after treatment with oestrogen in a distantly related mammal, the marsupial tammar wallaby, using our model of oestrogen-induced gonadal sex reversal. High-throughput RNA-sequencing was carried out on gonads collected from developing tammar 2 days before birth to 8 days after birth to characterise MAPK and key sexual differentiation markers. Day 25 foetal testes were cultured for 120 h in control medium or medium supplemented with exogenous oestrogen and processed for RNA-seq to identify changes in gene expression in response to oestrogen. MAPK pathway genes in the tammar were highly conserved at the sequence and amino acid level with those of mice and humans. Marsupial MAP3K1 and MAP3K4 clustered together in a separate branch from eutherian mammals. There was a marked decrease in the expression of male-determining genes SOX9 and AMH and increase in the female marker FOXL2 in oestrogen-treated male gonads. Only MAP3K1 expression increased in male gonads in response to oestrogen while other MAPK genes remained unaffected. This study suggests that MAP3K1 can be influenced by exogenous oestrogens during gonadal differentiation in this marsupial.

The genetic relatedness of a peri-urban population of eastern grey kangaroos.

OBJECTIVES: The genetic diversity of an eastern grey kangaroo (Macropus giganteus) population surrounded by landscape barriers was examined. DNA was extracted from tissue samples from 22 road-killed kangaroos, and blood samples from four live captured kangaroos. Amplified loci were used to determine relatedness between individual kangaroos. The level of relatedness and location of road-killed kangaroos were compared to evaluate spatial autocorrelation. RESULTS: The expected and observed heterozygosity confirmed the loci were polymorphic and highly informative for use in this population. One pair of kangaroos were identified to be full siblings, and a high proportion were identified as half siblings. Six positive parentage assignments were detected. No correlation between relatedness and crossing site was detected.

Hormone-responsive genes in the SHH and WNT/ß-catenin signaling pathways influence urethral closure and phallus growth.

Environmental endocrine disruptors (EEDs) that affect androgen or estrogen activity may disrupt gene regulation during phallus development to cause hypospadias or a masculinized clitoris. We treated developing male tammar wallabies with estrogen and females with androgen from day 20-40 postpartum (pp) during the androgen imprinting window of sensitivity. Estrogen inhibited phallus elongation but had no effect on urethral closure and did not significantly depress testicular androgen synthesis. Androgen treatment in females did not promote phallus elongation but initiated urethral closure. Phalluses were collected for transcriptome sequencing at day 50 pp when they first become sexually dimorphic to examine changes in two signaling pathways, sonic hedgehog (SHH) and wingless-type MMTV integration site family (WNT)/ß-catenin. SHH mRNA and ß-catenin were predominantly expressed in the urethral epithelium in the tammar phallus, as in eutherian mammals. Estrogen treatment and castration of males induced an upregulation of SHH, while androgen treatment downregulated SHH. These effects appear to be direct since we detected putative estrogen receptor α (ERα) and androgen receptor (AR) binding sites near SHH. WNT5A, like SHH, was downregulated by androgen, while WNT4 was upregulated in female phalluses after androgen treatment. After estrogen treatment, WIF1 and WNT7A were both downregulated in male phalluses. After castration, WNT9A was upregulated. These results suggest that SHH and WNT pathways are regulated by both estrogen and androgen to direct the proliferation and elongation of the phallus during differentiation. Their response to exogenous hormones makes these genes potential targets of EEDs in the etiology of abnormal phallus development including hypospadias.

Phylogenetic analysis of the tree-kangaroos (Dendrolagus) reveals multiple divergent lineages within New Guinea.

Amongst the Australasian kangaroos and wallabies (Macropodidae) one anomalous genus, the tree-kangaroos, Dendrolagus, has secondarily returned to arboreality. Modern tree-kangaroos are confined to the wet tropical forests of north Queensland, Australia (2 species) and New Guinea (8 species). Due to their behavior, distribution and habitat most species are poorly known and our understanding of the evolutionary history and systematics of the genus is limited and controversial. We obtained tissue samples from 36 individual Dendrolagus including representatives from 14 of the 17 currently recognised or proposed subspecies and generated DNA sequence data from three mitochondrial (3116 bp) and five nuclear (4097 bp) loci. Phylogenetic analysis of these multi-locus data resolved long-standing questions regarding inter-relationships within Dendrolagus. The presence of a paraphyletic ancestral long-footed and derived monophyletic short-footed group was confirmed. Six major lineages were identified: one in Australia (D. lumholtzi, D. bennettianus) and five in New Guinea (D. inustus, D. ursinus, a Goodfellow's group, D. mbaiso and a Doria's group). Two major episodes of diversification within Dendrolagus were identified: the first during the late Miocene/early Pliocene associated with orogenic processes in New Guinea and the second mostly during the early Pleistocene associated with the intensification of climatic cycling. All sampled subspecies showed high levels of genetic divergence and currently recognized species within both the Doria's and Goodfellow's groups were paraphyletic indicating that adjustments to current taxonomy are warranted.

Reassembling haplotypes in a mixture of pooled amplicons when the relative concentrations are known: A proof-of-concept study on the efficient design of next-generation sequencing strategies.

Next-generation sequencing can be costly and labour intensive. Usually, the sequencing cost per sample is reduced by pooling amplified DNA = amplicons) derived from different individuals on the same sequencing lane. Barcodes unique to each amplicon permit short-read sequences to be assigned appropriately. However, the cost of the library preparation increases with the number of barcodes used. We propose an alternative to barcoding: by using different known proportions of individually-derived amplicons in a pooled sample, each is characterised a priori by an expected depth of coverage. We have developed a Hidden Markov Model that uses these expected proportions to reconstruct the input sequences. We apply this method to pools of mitochondrial DNA amplicons extracted from kangaroo meat, genus Macropus. Our experiments indicate that the sequence coverage can be efficiently used to index the short-reads and that we can reassemble the input haplotypes when secondary factors impacting the coverage are controlled. We therefore demonstrate that, by combining our approach with standard barcoding, the cost of the library preparation is reduced to a third.