Environmental transcriptomics under heat stress: Can environmental RNA reveal changes in gene expression of aquatic organisms?

To safeguard biodiversity in a changing climate, taxonomic information about species turnover and insights into the health of organisms are required. Environmental DNA approaches are increasingly used for species identification, but cannot provide functional insights. Transcriptomic methods reveal the physiological states of macroorganisms, but are currently species-specific and require tissue sampling or animal sacrifice, making community-wide assessments challenging. Here, we test whether broad functional information (expression level of the transcribed genes) can be harnessed from environmental RNA (eRNA), which includes extra-organismal RNA from macroorganisms along with whole microorganisms. We exposed Daphnia pulex as well as phytoplankton prey and microorganism colonizers to control (20°C) and heat stress (28°C) conditions for 7 days. We sequenced eRNA from tank water (after complete removal of Daphnia) as well as RNA from Daphnia tissue, enabling comparisons of extra-organismal and organismal RNA-based gene expression profiles. Both RNA types detected similar heat stress responses of Daphnia. Using eRNA, we identified 32 Daphnia genes to be differentially expressed following heat stress. Of these, 17 were also differentially expressed and exhibited similar levels of relative expression in organismal RNA. In addition to the extra-organismal Daphnia response, eRNA detected community-wide heat stress responses consisting of distinct functional profiles and 121 differentially expressed genes across eight taxa. Our study demonstrates that environmental transcriptomics based on extra-organismal eRNA can noninvasively reveal gene expression responses of macroorganisms following environmental changes, with broad potential implications for the biomonitoring of health across the trophic chain.

Origin matters: Using a local reference genome improves measures in population genomics.

Genome sequencing enables answering fundamental questions about the genetic basis of adaptation, population structure and epigenetic mechanisms. Yet, we usually need a suitable reference genome for mapping population-level resequencing data. In some model systems, multiple reference genomes are available, giving the challenging task of determining which reference genome best suits the data. Here, we compared the use of two different reference genomes for the three-spined stickleback (Gasterosteus aculeatus), one novel genome derived from a European gynogenetic individual and the published reference genome of a North American individual. Specifically, we investigated the impact of using a local reference versus one generated from a distinct lineage on several common population genomics analyses. Through mapping genome resequencing data of 60 sticklebacks from across Europe and North America, we demonstrate that genetic distance among samples and the reference genomes impacts downstream analyses. Using a local reference genome increased mapping efficiency and genotyping accuracy, effectively retaining more and better data. Despite comparable distributions of the metrics generated across the genome using SNP data (i.e. π, Tajima's D and FST ), window-based statistics using different references resulted in different outlier genes and enriched gene functions. A marker-based analysis of DNA methylation distributions had a comparably high overlap in outlier genes and functions, yet with distinct differences depending on the reference genome. Overall, our results highlight how using a local reference genome decreases reference bias to increase confidence in downstream analyses of the data. Such results have significant implications in all reference-genome-based population genomic analyses.

Very Low Rates of Spontaneous Gene Deletions and Gene Duplications in Dictyostelium discoideum.

The study of spontaneous mutation rates has revealed a wide range of heritable point mutation rates across species, but there are comparatively few estimates for large-scale deletion and duplication rates. The handful of studies that have directly calculated spontaneous rates of deletion and duplication using mutation accumulation lines have estimated that genes are duplicated and deleted at orders of magnitude greater rates than the spontaneous point mutation rate. In our study, we tested whether spontaneous gene deletion and gene duplication rates are also high in Dictyostelium discoideum, a eukaryote with among the lowest point mutation rates (2.5 × 10-11 per site per generation) and an AT-rich genome (GC content of 22%). We calculated mutation rates of gene deletions and duplications using whole-genome sequencing data originating from a mutation accumulation experiment and determined the association between the copy number mutations and GC content. Overall, we estimated an average of 3.93 × 10-8 gene deletions and 1.18 × 10-8 gene duplications per gene per generation. While orders of magnitude greater than their point mutation rate, these rates are much lower compared to gene deletion and duplication rates estimated from mutation accumulation lines in other organisms (that are on the order of ~ 10-6 per gene/generation). The deletions and duplications were enriched in regions that were AT-rich even compared to the genomic background, in contrast to our expectations if low GC content was contributing to low mutation rates. The low deletion and duplication mutation rates in D. discoideum compared to other eukaryotes mirror their low point mutation rates, supporting previous work suggesting that this organism has high replication fidelity and effective molecular machinery to avoid the accumulation of mutations in their genome.

Diversity in Expression Biases of Lineage-Specific Genes During Development and Anhydrobiosis Among Tardigrade Species.

Lineage-specific genes can contribute to the emergence and evolution of novel traits and adaptations. Tardigrades are animals that have adapted to tolerate extreme conditions by undergoing a form of cryptobiosis called anhydrobiosis, a physical transformation to an inactive desiccated state. While studies to understand the genetics underlying the interspecies diversity in anhydrobiotic transitions have identified tardigrade-specific genes and family expansions involved in this process, the contributions of species-specific genes to the variation in tardigrade development and cryptobiosis are less clear. We used previously published transcriptomes throughout development and anhydrobiosis (5 embryonic stages, 7 juvenile stages, active adults, and tun adults) to assess the transcriptional biases of different classes of genes between 2 tardigrade species, Hypsibius exemplaris and Ramazzottius varieornatus. We also used the transcriptomes of 2 other tardigrades, Echiniscoides sigismundi and Richtersius coronifer, and data from 3 non-tardigrade species (Adenita vaga, Drosophila melanogaster, and Caenorhabditis elegans) to help identify lineage-specific genes. We found that lineage-specific genes have generally low and narrow expression but are enriched among biased genes in different stages of development depending on the species. Biased genes tend to be specific to early and late development, but there is little overlap in functional enrichment of biased genes between species. Gene expansions in the 2 tardigrades also involve families with different functions despite homologous genes being expressed during anhydrobiosis in both species. Our results demonstrate the interspecific variation in transcriptional contributions and biases of lineage-specific genes during development and anhydrobiosis in 2 tardigrades.

Lineage-Specific Genes and Family Expansions in Dictyostelid Genomes Display Expression Bias and Evolutionary Diversification during Development.

Gene duplications generate new genes that can contribute to expression changes and the evolution of new functions. Genomes often consist of gene families that undergo expansions, some of which occur in specific lineages that reflect recent adaptive diversification. In this study, lineage-specific genes and gene family expansions were studied across five dictyostelid species to determine when and how they are expressed during multicellular development. Lineage-specific genes were found to be enriched among genes with biased expression (predominant expression in one developmental stage) in each species and at most developmental time points, suggesting independent functional innovations of new genes throughout the phylogeny. Biased duplicate genes had greater expression divergence than their orthologs and paralogs, consistent with subfunctionalization or neofunctionalization. Lineage-specific expansions in particular had biased genes with both molecular signals of positive selection and high expression, suggesting adaptive genetic and transcriptional diversification following duplication. Our results present insights into the potential contributions of lineage-specific genes and families in generating species-specific phenotypes during multicellular development in dictyostelids.

Concatenation of paired-end reads improves taxonomic classification of amplicons for profiling microbial communities.

BACKGROUND: Taxonomic classification of genetic markers for microbiome analysis is affected by the numerous choices made from sample preparation to bioinformatics analysis. Paired-end read merging is routinely used to capture the entire amplicon sequence when the read ends overlap. However, the exclusion of unmerged reads from further analysis can result in underestimating the diversity in the sequenced microbial community and is influenced by bioinformatic processes such as read trimming and the choice of reference database. A potential solution to overcome this is to concatenate (join) reads that do not overlap and keep them for taxonomic classification. The use of concatenated reads can outperform taxonomic recovery from single-end reads, but it remains unclear how their performance compares to merged reads. Using various sequenced mock communities with different amplicons, read length, read depth, taxonomic composition, and sequence quality, we tested how merging and concatenating reads performed for genus recall and precision in bioinformatic pipelines combining different parameters for read trimming and taxonomic classification using different reference databases. RESULTS: The addition of concatenated reads to merged reads always increased pipeline performance. The top two performing pipelines both included read concatenation, with variable strengths depending on the mock community. The pipeline that combined merged and concatenated reads that were quality-trimmed performed best for mock communities with larger amplicons and higher average quality sequences. The pipeline that used length-trimmed concatenated reads outperformed quality trimming in mock communities with lower quality sequences but lost a significant amount of input sequences for taxonomic classification during processing. Genus level classification was more accurate using the SILVA reference database compared to Greengenes. CONCLUSIONS: Merged sequences with the addition of concatenated sequences that were unable to be merged increased performance of taxonomic classifications. This was especially beneficial in mock communities with larger amplicons. We have shown for the first time, using an in-depth comparison of pipelines containing merged vs concatenated reads combined with different trimming parameters and reference databases, the potential advantages of concatenating sequences in improving resolution in microbiome investigations.

BLAST from the Past: Impacts of Evolving Approaches on Studies of Evolution by Gene Duplication.

In 1970, Susumu Ohno hypothesized that gene duplication was a major reservoir of adaptive innovation. However, it was not until over two decades later that DNA sequencing studies uncovered the ubiquity of gene duplication across all domains of life, highlighting its global importance in the evolution of phenotypic complexity and species diversification. Today, it seems that there are no limits to the study of evolution by gene duplication, as it has rapidly coevolved with numerous experimental and computational advances in genomics. In this perspective, we examine word stem usage in PubMed abstracts to infer how evolving discoveries and technologies have shaped the landscape of studying evolution by gene duplication, leading to a more refined understanding of its role in the emergence of novel phenotypes.

Refining analyses of existing data sets is valuable for macrogenetics: a response to Paz-Vinas, Jensen et al., (2021).

Paz-Vinas, Jensen et al. (2021) comment on data and methodological limits of Millette, Fugère, Debyser et al. (2020)-some affect a small proportion of our data sets and analyses and others need to be tackled more generally. These points do not refute our main conclusion of no strong signal of human impacts on COI variation globally.

Copy number variations and young duplicate genes have high methylation levels in sticklebacks.

Gene duplication is an important driver of genomic diversity that can promote adaptive evolution. However, like most mutations, a newly duplicated gene is often deleterious and removed from the genome by drift or natural selection. The early molecular changes that occur soon after duplication therefore may influence the long-term survival of gene duplicates, but relatively little empirical data exist on the events near the onset of duplication before mutations have time to accumulate. In this study, we contrast gene expression and DNA methylation levels of duplicate genes in the threespine stickleback, Gasterosteus aculeatus, including recently emerged duplications that segregate as copy number variations (CNVs). We find that younger duplicate genes have higher levels of promoter methylation than older genes, and that gene CNVs have higher promoter methylation than non-CNVs. These results suggest preferential duplication of highly methylated genes or rapid methylation changes soon after duplication. We also find a negative association between methylation and expression, providing a putative role for methylation in suppressing transcription that compensates for increases in gene copy numbers and promoting paralog retention. We propose that methylation contributes to the longevity of young duplicate genes, extending the window of opportunity for functional divergence via mutation.

Freshwater zooplankton metapopulations and metacommunities respond differently to environmental and spatial variation.

Theory predicts that population genetic structure and metacommunity structure are linked by the common processes of drift and migration, but how population genetic structure and metacommunity structure are related in nature is still unknown. Deeper understanding of the processes influencing both genetic and community diversity is vital for better predicting how environmental change will impact biodiversity patterns. We examined how crustacean zooplankton and rotifer species' metapopulation genetic structure and metacommunities respond to environmental and spatial variation both within and across four regions of boreal Canada. Metapopulation and metacommunity variation partitioning results were compared within and across the four regions. Metapopulations and metacommunities responded differently to environmental variation and spatial structure both within and across regions, as metapopulations were influenced by different environmental variables compared to metacommunities. At larger spatial scales both metapopulations and metacommunities exhibited greater spatial and environmental structuring, again responding to a different subset of environmental variables. Our findings suggest that even though both genetic and species diversity are linked by the same processes, regional variation in environmental characteristics and spatial structure influence resulting biodiversity patterns differently. To date, no other empirical research has explored relationships between entire metapopulation and metacommunity assemblages at large regional spatial scales.

The Challenges of Microbial Control of Mosquito-Borne Diseases Due to the Gut Microbiome.

Mosquitoes are one of the deadliest animals on earth because of their ability to transmit a wide range of human pathogens. Traditional mosquito control methods use chemical insecticides, but with dwindling long-term effectiveness and negative effects on the environment, microbial forms of control have become common alternatives. The insecticide Bacillus thuringiensis subspecies israelensis (Bti) is the most popular of these alternatives, although it can also have direct effects on lowering environmental biodiversity and indirect effects on food-web relationships in the ecosystems where it is deployed. In addition, microbial control agents that impede pathogen development or transmission from mosquito to human are under investigation, including Wolbachia and Asaia, but unexpected interactions with mosquito gut bacteria can hinder their effectiveness. Improved characterization of mosquito gut bacterial communities is needed to determine the taxa that interfere with microbial controls and their effectiveness in wild populations. This mini-review briefly discusses relationships between mosquito gut bacteria and microbial forms of control, and the challenges in ensuring their success.

Evolution of novel genes in three-spined stickleback populations.

Eukaryotic genomes frequently acquire new protein-coding genes which may significantly impact an organism's fitness. Novel genes can be created, for example, by duplication of large genomic regions or de novo, from previously non-coding DNA. Either way, creation of a novel transcript is an essential early step during novel gene emergence. Most studies on the gain-and-loss dynamics of novel genes so far have compared genomes between species, constraining analyses to genes that have remained fixed over long time scales. However, the importance of novel genes for rapid adaptation among populations has recently been shown. Therefore, since little is known about the evolutionary dynamics of transcripts across natural populations, we here study transcriptomes from several tissues and nine geographically distinct populations of an ecological model species, the three-spined stickleback. Our findings suggest that novel genes typically start out as transcripts with low expression and high tissue specificity. Early expression regulation appears to be mediated by gene-body methylation. Although most new and narrowly expressed genes are rapidly lost, those that survive and subsequently spread through populations tend to gain broader and higher expression levels. The properties of the encoded proteins, such as disorder and aggregation propensity, hardly change. Correspondingly, young novel genes are not preferentially under positive selection but older novel genes more often overlap with FST outlier regions. Taken together, expression of the surviving novel genes is rapidly regulated, probably via epigenetic mechanisms, while structural properties of encoded proteins are non-debilitating and might only change much later.

Alternative splicing is highly variable among Daphnia pulex lineages in response to acute copper exposure.

BACKGROUND: Despite being one of the primary mechanisms of gene expression regulation in eukaryotes, alternative splicing is often overlooked in ecotoxicogenomic studies. The process of alternative splicing facilitates the production of multiple mRNA isoforms from a single gene thereby greatly increasing the diversity of the transcriptome and proteome. This process can be important in enabling the organism to cope with stressful conditions. Accurate identification of splice sites using RNA sequencing requires alignment to independent exonic positions within the genome, presenting bioinformatic challenges, particularly when using short read data. Although technological advances allow for the detection of splicing patterns on a genome-wide scale, very little is known about the extent of intraspecies variation in splicing patterns, particularly in response to environmental stressors. In this study, we used RNA-sequencing to study the molecular responses to acute copper exposure in three lineages of Daphnia pulex by focusing on the contribution of alternative splicing in addition to gene expression responses. RESULTS: By comparing the overall gene expression and splicing patterns among all 15 copper-exposed samples and 6 controls, we identified 588 differentially expressed (DE) genes and 16 differentially spliced (DS) genes. Most of the DS genes (13) were not found to be DE, suggesting unique transcriptional regulation in response to copper that went unnoticed with conventional DE analysis. To understand the influence of genetic background on gene expression and alternative splicing responses to Cu, each of the three lineages was analyzed separately. In contrast to the overall analysis, each lineage had a higher proportion of unique DS genes than DE genes suggesting that genetic background has a larger influence on DS than on DE. Gene Ontology analysis revealed that some pathways involved in stress response were jointly regulated by DS and DE genes while others were regulated by only transcription or only splicing. CONCLUSIONS: Our findings suggest an important role for alternative splicing in shaping transcriptome diversity in response to metal exposure in Daphnia, highlighting the importance of integrating splicing analyses with gene expression surveys to characterize molecular pathways in evolutionary and environmental studies.

No consistent effects of humans on animal genetic diversity worldwide.

Human impacts on genetic diversity are poorly understood yet critical to biodiversity conservation. We used 175 247 COI sequences collected between 1980 and 2016 to assess the global effects of land use and human density on the intraspecific genetic diversity of 17 082 species of birds, fishes, insects and mammals. Human impacts on mtDNA diversity were taxon and scale-dependent, and were generally weak or non-significant. Spatial analyses identified weak latitudinal diversity gradients as well as negative effects of human density on insect diversity, and negative effects of intensive land use on fish diversity. The observed effects were predominantly associated with species turnover. Time series analyses found nearly an equal number of positive and negative temporal trends in diversity, resulting in no net monotonic trend in diversity over this time period. Our analyses reveal critical data and theory gaps and call for increased efforts to monitor global genetic diversity.

Genome-Wide Genotype-Expression Relationships Reveal Both Copy Number and Single Nucleotide Differentiation Contribute to Differential Gene Expression between Stickleback Ecotypes.

Repeated and independent emergence of trait divergence that matches habitat differences is a sign of parallel evolution by natural selection. Yet, the molecular underpinnings that are targeted by adaptive evolution often remain elusive. We investigate this question by combining genome-wide analyses of copy number variants (CNVs), single nucleotide polymorphisms (SNPs), and gene expression across four pairs of lake and river populations of the three-spined stickleback (Gasterosteus aculeatus). We tested whether CNVs that span entire genes and SNPs occurring in putative cis-regulatory regions contribute to gene expression differences between sticklebacks from lake and river origins. We found 135 gene CNVs that showed a significant positive association between gene copy number and gene expression, suggesting that CNVs result in dosage effects that can fuel phenotypic variation and serve as substrates for habitat-specific selection. Copy number differentiation between lake and river sticklebacks also contributed to expression differences of two immune-related genes in immune tissues, cathepsin A and GIMAP7. In addition, we identified SNPs in cis-regulatory regions (eSNPs) associated with the expression of 1,865 genes, including one eSNP upstream of a carboxypeptidase gene where both the SNP alleles differentiated and the gene was differentially expressed between lake and river populations. Our study highlights two types of mutations as important sources of genetic variation involved in the evolution of gene expression and in potentially facilitating repeated adaptation to novel environments.

Variation in transcriptional responses to copper exposure across Daphnia pulex lineages.

Copper pollution is pervasive in aquatic habitats and is particularly harmful to invertebrates sensitive to environmental changes such as Daphnia pulex. Mechanisms of toxicity and tolerance to copper are not well understood. We used RNA-sequencing to investigate these mechanisms in three genetically distinct D. pulex clonal lineages with different histories of copper exposure. Upregulated genes after copper exposure were enriched with Gene Ontology (GO) categories involved in digestion, molting and growth, whereas downregulated genes after copper exposure were enriched in the metal-regulatory system, immune response and epigenetic modifications. The three D. pulex clones in our study show largely similar transcriptional patterns in response to copper, with only a total of twenty genes differentially expressed in a single clonal lineages. We also detected lower relative expression of some genes known to be important for copper tolerance, metallothionein and glutathione-S-transferase, in a sensitive lineage sampled from an uncontaminated habitat. Daphnia-specific genes (without orthologs outside the genus) and Daphnia-specific duplications (genes duplicated in the Daphnia lineage) were overrepresented in differentially expressed genes, highlighting an important role for newly emerged genes in tolerating environmental stressors. The results indicate that the D. pulex lineages tested in this study generally respond to copper stress using the same major pathways, but that the more resistant clone with previous copper exposure might be better able to regulate key genes. This finding highlights the important nuances in gene expression among clones, shaped by historical exposure and influencing copper tolerance.

Fitness and Genomic Consequences of Chronic Exposure to Low Levels of Copper and Nickel in Daphnia pulex Mutation Accumulation Lines.

In at least some unicellular organisms, mutation rates are temporarily raised upon exposure to environmental stress, potentially contributing to the evolutionary response to stress. Whether this is true for multicellular organisms, however, has received little attention. This study investigated the effects of chronic mild stress, in the form of low-level copper and nickel exposure, on mutational processes in Daphnia pulex using a combination of mutation accumulation, whole genome sequencing and life-history assays. After over 100 generations of mutation accumulation, we found no effects of metal exposure on the rates of single nucleotide mutations and of loss of heterozygosity events, the two mutation classes that occurred in sufficient numbers to allow statistical analysis. Similarly, rates of decline in fitness, as measured by intrinsic rate of population increase and of body size at first reproduction, were negligibly affected by metal exposure. We can reject the possibility that Daphnia were insufficiently stressed to invoke genetic responses as we have previously shown rates of large-scale deletions and duplications are elevated under metal exposure in this experiment. Overall, the mutation accumulation lines did not significantly depart from initial values for phenotypic traits measured, indicating the lineage used was broadly mutationally robust. Taken together, these results indicate that the mutagenic effects of chronic low-level exposure to these metals are restricted to certain mutation classes and that fitness consequences are likely minor and therefore unlikely to be relevant in determining the evolutionary responses of populations exposed to these stressors.

Accelerated rates of large-scale mutations in the presence of copper and nickel.

Mutation rate variation has been under intense investigation for decades. Despite these efforts, little is known about the extent to which environmental stressors accelerate mutation rates and influence the genetic load of populations. Moreover, most studies on stressors have focused on unicellular organisms and point mutations rather than large-scale deletions and duplications (copy number variations [CNVs]). We estimated mutation rates in Daphnia pulex exposed to low levels of environmental stressors as well as the effect of selection on de novo mutations. We conducted a mutation accumulation (MA) experiment in which selection was minimized, coupled with an experiment in which a population was propagated under competitive conditions in a benign environment. After an average of 103 generations of MA propagation, we sequenced 60 genomes and found significantly accelerated rates of deletions and duplications in MA lines exposed to ecologically relevant concentrations of metals. Whereas control lines had gene deletion and duplication rates comparable to other multicellular eukaryotes (1.8 × 10-6 per gene per generation), the presence of nickel and copper increased these rates fourfold. The realized mutation rate under selection was reduced to 0.4× that of control MA lines, providing evidence that CNVs contribute to mutational load. Our CNV breakpoint analysis revealed that nonhomologous recombination associated with regions of DNA fragility is the primary source of CNVs, plausibly linking metal-induced DNA strand breaks with higher CNV rates. Our findings suggest that environmental stress, in particular multiple stressors, can have profound effects on large-scale mutation rates and mutational load of multicellular organisms.

Metabarcoding using multiplexed markers increases species detection in complex zooplankton communities.

Metabarcoding combines DNA barcoding with high-throughput sequencing, often using one genetic marker to understand complex and taxonomically diverse samples. However, species-level identification depends heavily on the choice of marker and the selected primer pair, often with a trade-off between successful species amplification and taxonomic resolution. We present a versatile metabarcoding protocol for biomonitoring that involves the use of two barcode markers (COI and 18S) and four primer pairs in a single high-throughput sequencing run, via sample multiplexing. We validate the protocol using a series of 24 mock zooplanktonic communities incorporating various levels of genetic variation. With the use of a single marker and single primer pair, the highest species recovery was 77%. With all three COI fragments, we detected 62%-83% of species across the mock communities, while the use of the 18S fragment alone resulted in the detection of 73%-75% of species. The species detection level was significantly improved to 89%-93% when both markers were used. Furthermore, multiplexing did not have a negative impact on the proportion of reads assigned to each species and the total number of species detected was similar to when markers were sequenced alone. Overall, our metabarcoding approach utilizing two barcode markers and multiple primer pairs per barcode improved species detection rates over a single marker/primer pair by 14% to 35%, making it an attractive and relatively cost-effective method for biomonitoring natural zooplankton communities. We strongly recommend combining evolutionary independent markers and, when necessary, multiple primer pairs per marker to increase species detection (i.e., reduce false negatives) in metabarcoding studies.

Optimization and performance testing of a sequence processing pipeline applied to detection of nonindigenous species.

Genetic taxonomic assignment can be more sensitive than morphological taxonomic assignment, particularly for small, cryptic or rare species. Sequence processing is essential to taxonomic assignment, but can also produce errors because optimal parameters are not known a priori. Here, we explored how sequence processing parameters influence taxonomic assignment of 18S sequences from bulk zooplankton samples produced by 454 pyrosequencing. We optimized a sequence processing pipeline for two common research goals, estimation of species richness and early detection of aquatic invasive species (AIS), and then tested most optimal models' performances through simulations. We tested 1,050 parameter sets on 18S sequences from 20 AIS to determine optimal parameters for each research goal. We tested optimized pipelines' performances (detectability and sensitivity) by computationally inoculating sequences of 20 AIS into ten bulk zooplankton samples from ports across Canada. We found that optimal parameter selection generally depends on the research goal. However, regardless of research goal, we found that metazoan 18S sequences produced by 454 pyrosequencing should be trimmed to 375-400 bp and sequence quality filtering should be relaxed (1.5 ≤ maximum expected error ≤ 3.0, Phred score = 10). Clustering and denoising were only viable for estimating species richness, because these processing steps made some species undetectable at low sequence abundances which would not be useful for early detection of AIS. With parameter sets optimized for early detection of AIS, 90% of AIS were detected with fewer than 11 target sequences, regardless of whether clustering or denoising was used. Despite developments in next-generation sequencing, sequence processing remains an important issue owing to difficulties in balancing false-positive and false-negative errors in metabarcoding data.