The genome of the crustacean Parhyale hawaiensis, a model for animal development, regeneration, immunity and lignocellulose digestion
- University of Oxford, United Kingdom
- Janelia Farm Research Campus, United States
- Imperial College London, United Kingdom
- Centre National de la Recherche Scientifique (CNRS) and É cole Normale Supé rieure de Lyon, France
- University of California, United States
- Howard Hughes Medical Institute, University of California, United States
- Harvard University, United States
- Smithsonian National Museum of Natural History, United States
- Institut fur Biologie,Humboldt-Universitat zu Berlin, Germany
Figures
Figure 1
Introduction.
(A) Phylogenetic relationship of Arthropods showing the Chelicerata as an outgroup to Mandibulata and the Pancrustacea clade which includes crustaceans and insects. Species listed for each clade …
Figure 2
Parhyale karyotype.
(A) Frequency of the number of chromosomes observed in 42 mitotic spreads. Forty-six chromosomes were observed in more than half of all preparations. (B) Representative image of Hoechst-stained …
Figure 3
Parhyale genome assembly metrics.
(A) K-mer frequency spectra of all reads for k-lengths ranging from 20 to 50. (B) K-mer branching analysis showing the frequency of k-mer branches classified as variants compared to Homo sapiens …
Figure 4 with 1 supplement
Workflows of assembly, annotation, and proteome generation.
(A) Flowchart of the genome assembly. Two shotgun libraries and four mate-pair libraries with the indicated average sizes were prepared from a single male animal and sequenced to a predicted depth …
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Figure 4—source data 1
Catalog of repeat elements in Parhyale genome assembly.
Description of repeat content in the Parhyale genome.
- https://doi.org/10.7554/eLife.20062.008
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Figure 4—source data 2
Software and Data.
List of programs and bioinformatic tools and publicly available sequence data used in this study.
- https://doi.org/10.7554/eLife.20062.009
Figure 4—figure supplement 1
CEGMA assessment of Parhyale transcriptome and genome.
(A) CEGMA genes present in the transcriptome assembly scored by BLAST identity (y axis) and proportion of coverage (relative length, x axis) (B) CEGMA genes present in the genome assembly scored by …
Figure 5 with 1 supplement
Parhyale genome comparisons.
(A) Box plots comparing gene sizes between Parhyale and humans (H. sapiens), water fleas (D. pulex), flies (D. melanogaster) and nematodes (C. elegans). Ratios were calculated by dividing the size …
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Figure 5—source data 1
List of proteins currently unique to Parhyale.
List of proteins in Parhyale without identity to other species.
- https://doi.org/10.7554/eLife.20062.013
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Figure 5—source data 2
List of genes likely to be specific to the Malacostraca
List of genes likely to be specific to the Malacostraca.
- https://doi.org/10.7554/eLife.20062.014
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Figure 5—source data 3
Orthofinder analysis.
Orthofinder analysis using the Parhyale predicted proteome.
- https://doi.org/10.7554/eLife.20062.015
Figure 5—figure supplement 1
Expanded gene families in Parhyale.
Histograms showing number of paralogs in each listed species for (A) sidestep, (B) lachesin, (C) neurotrimin/DPR, (D) APN and (E) cathepsin genes for gene families over represented in Parhyale.
Figure 6 with 1 supplement
Variation analyses of predicted genes.
(A) A read coverage histogram of predicted genes. Reads were first mapped to the genome, then coverage was calculated for transcribed regions of each defined locus. (B) A coverage distribution plot …
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Figure 6—source data 1
Polymorphism in Parhyale devlopmental genes.
Description of polymorphism in previously identfied Parhyale developmental genes.
- https://doi.org/10.7554/eLife.20062.018
Figure 6—figure supplement 1
Confirmation of polymorphisms in the wider laboratory population of Parhyale.
(A) An example of laboratory population polymorphism in exon 1 of the gene aristalless. As well as heterozygoisty in the single Chicago-F male sequenced (pink and purple bases) there is additional …
Figure 7
Variation observed in contiguous BAC sequences.
(A) Schematic diagram of the contiguous BAC clones tiling across the HOX cluster and their% sequence identities. 'Overlap length' refers to the lengths (bp) of the overlapping regions between two …
Figure 8 with 2 supplements
Comparison of Wnt family members across Metazoa.
Comparison of Wnt genes across Metazoa. Tree on the left illustrates the phylogenetic relationships of species used. Dotted lines in the phylogenetic tree illustrate the alternative hypothesis of …
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Figure 8—source data 1
List of Parhyale transcription factors by family.
List of Parhyale transcript IDs for all transcription factors in the proteome, grouped by transcription factor family.
- https://doi.org/10.7554/eLife.20062.023
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Figure 8—source data 2
Wnt, TGFβ and FGF signaling pathways .
Parhyale transcript IDs for Wnt, Wnt ligand, FGF, FGFR and TGFβ pathway genes.
- https://doi.org/10.7554/eLife.20062.024
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Figure 8—source data 3
Homeobox transcription factors.
Annotation of homeobox transcription factor genes in Parhyale.
- https://doi.org/10.7554/eLife.20062.025
Figure 8—figure supplement 1
Phylogenetic tree of FGF and FGR molecules
(A) Phylogenetic tree of arthropod and vertebrate FGFs, including two FGFs from Parhyale (B) Phylogenetic tree of arthropod and vertebrate FGFRs, including a single FGFR in Parhyale.
Figure 8—figure supplement 2
Phylogenetic tree of CERS homeobox family genes.
A phylogenetic tree highlighting an expansion of CERS homeobox family genes in Parhyale.
Figure 9
Homeodomain protein family tree.
The overview of homeodomain radiation and phylogenetic relationships among homeodomain proteins from Arthropoda (P. hawaiensis, D. melanogaster and A. mellifera), Chordata (H. sapiens and B. floridae…
Figure 10
Evidence for an intact Hox cluster in Parhyale.
(A–F’’) Double fluorescent in situ hybridizations (FISH) for nascent transcripts of genes. (A–A’’) Deformed (Dfd) and Sex combs reduced (Scr), (B-B’’) engrailed 1 (en1) and Ultrabithorax (Ubx), …
Figure 11
Lignocellulose digestion overview.
(A) Simplified drawing of lignocellulose structure. The main component of lignocellulose is cellulose, which is a-1,4-linked chain of glucose monosaccharides. Cellulose and lignin are organized in …
Figure 12 with 1 supplement
Phylogenetic analysis of GH7 and GH9 family proteins.
(A) Phylogenetic tree showing the relationship between GH7 family proteins of Parhyale, other crustaceans (Malacostraca, Branchiopoda, Copepoda), fungi and symbiotic protists (root). UniProt and …
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Figure 12—source data 1.
Catalog of GH family genes in Parhyale.
IDs of all Parhyale GH genes and analyis of GH family membership across available malacostracan data sets.
- https://doi.org/10.7554/eLife.20062.032
Figure 12—figure supplement 1
Alignment of GH7 family genes.
Alignment of GH7 family genes in Parhyale with those from Chelura terebans and Limnoria quadripunctata.
Figure 13 with 1 supplement
Comparison of innate immunity genes.
(A) Phylogenetic tree of peptidoglycan recognition proteins (PGRPs). With the exception of Remipedes, PGRPs were not found in Crustaceans. PGRPs have been found in Arthropods, including insects, …
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Figure 13—source data 1
Catalog of innate immunity related genes in Parhyale.
Parhyale IDs and numbers of immune related genes in comparison to other species.
- https://doi.org/10.7554/eLife.20062.035
Figure 13—figure supplement 1
Overview of Parhyale Dscam structure and hypervariable regions
(A) Overview of domain structure of Parhyale Dscam protein and position of primers used to assess use of exons in 3 hypervariable regions. (B) Sequence alignments of cloned hypervariable regions in …
Figure 14 with 2 supplements
Evolution of miRNA families in Eumetazoans.
Phylogenetic tree showing the gains (in green) and losses (in red) of miRNA families at various taxonomic levels of the Eumetazoan tree leading to Parhyale. miRNAs marked with plain characters were …
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Figure 14—source data 1
RFAM based annotation of the Parhyale genome.
RFAM annotation of the Parhyale genome.
- https://doi.org/10.7554/eLife.20062.039
Figure 14—figure supplement 1
Phylogenetic trees of Dicer and PIWI/AGO genes.
(A) Phylogenetic tree of Dicer family genes, including two Dicer genes from Parhyale. (B) Phylogenetic tree of PIWI/AGO genes, including several Parhyale genes.
Figure 14—figure supplement 2
Examples of miRNAs in the Parhyale genome.
(A) Parhyale mir-100 and let-7 and clustered together in the intron of a putative lncRNA (B) A Parhyale mir-71/mir-2 family cluster (C) Parhyale mir-10 is in a conserved position in the genome …
Figure 15
Analysis of Parhyale genome methylation.
(A) Phylogenetic tree showing the families and numbers of DNA methyltransferases (DNMTs) present in the genomes of indicated species. Parhyale has one copy from each DNMT family. (B) Amounts of …
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Figure 15—source data 1
Genes involved with epigenetic modification.
Catalog of Parhyale genes involved in DNA methylation and histone modifications.
- https://doi.org/10.7554/eLife.20062.043
Figure 16 with 1 supplement
CRISPR/Cas9-based genome editing in Parhyale.
(A) Wild-type morphology. (B) Mutant Parhyale with truncated limbs after CRISPR-mediated knock-out (DllKO) of the limb patterning gene Distal-less (PhDll-e). Panels show ventral views of juveniles …
Figure 16—figure supplement 1
CRISPR experiments targeting the Distalless locus.
CRSIPR/Cas-based targeted genome editing in Parhyale. (A) Summary of gene knock-out experiments. (B) Illustration of the targeted PhDll-e (Dll) cDNA showing the 5’ and 3’ untranslated regions …
Tables
Table 1
Experimental resources. Available experimental resources in Parhyale and corresponding references.
| Experimental Resources | References |
|---|---|
| Embryological manipulations Cell microinjection, isolation, ablation | (Gerberding et al., 2002; Extavour, 2005; Price et al., 2010; Alwes et al., 2011; Hannibal et al., 2012; Rehm et al., 2009; Rehm et al., 2009; Kontarakis and Pavlopoulos, 2014; Nast and Extavour, 2014) |
| Gene expression studies In situ hybridization, antibody staining | (Rehm et al., 2009; Rehm et al., 2009) |
| Gene knock-down RNA interference, morpholinos | (Liubicich et al., 2009; Ozhan-Kizil et al., 2009) |
| Transgenesis Transposon-based, integrase-based | (Pavlopoulos and Averof, 2005; Kontarakis et al., 2011; Kontarakis and Pavlopoulos, 2014) |
| Gene trapping Exon/enhancer trapping, iTRAC (trap conversion) | (Kontarakis et al., 2011) |
| Gene misexpressionHeat-inducible | (Pavlopoulos et al., 2009) |
| Gene knock-outCRISPR/Cas | (Martin et al., 2015) |
| Gene knock-in CRISPR/Cas homology-dependent or homology-independent | (Serano et al., 2015) |
| Live imaging Bright-field, confocal, light-sheet microscopy | (Alwes et al., 2011; Hannibal et al., 2012; Chaw and Patel, 2012; Alwes et al., 2016) |
Table 2
Assembly statistics. Length metrics of assembled scaffolds and contigs.
| # sequences | N90 | N50 | N10 | Sum length | Max length | # Ns | |
|---|---|---|---|---|---|---|---|
| scaffolds | 133,035 | 14,799 | 81,190 | 289,705 | 3.63 GB | 1,285,385 | 1.10 GB |
| unplaced contigs | 259,343 | 304 | 627 | 1779 | 146 MB | 40,222 | 23,431 |
| hetero. contigs | 584,392 | 265 | 402 | 1038 | 240 MB | 24,461 | 627 |
| genic scaffolds | 15,160 | 52952 | 161,819 | 433836 | 1.49 GB | 1,285,385 | 323 MB |
Table 3
BAC variant statistics. Level of heterozygosity of each BAC sequence determined by mapping genomic reads to each BAC individually. Population variance rate represents additional alleles found (i.e. m…
| BAC ID | Length | Heterozygosity | Pop.Variance |
|---|---|---|---|
| PA81-D11 | 140,264 | 1.654 | 0.568 |
| PA40-O15 | 129,957 | 2.446 | 0.647 |
| PA76-H18 | 141,844 | 1.824 | 0.199 |
| PA120-H17 | 126,766 | 2.673 | 1.120 |
| PA222-D11 | 128,542 | 1.344 | 1.404 |
| PA31-H15 | 140,143 | 2.793 | 0.051 |
| PA284-I07 | 141,390 | 2.046 | 0.450 |
| PA221-A05 | 148,703 | 1.862 | 1.427 |
| PA93-L04 | 139,955 | 2.177 | 0.742 |
| PA272-M04 | 134,744 | 1.925 | 0.982 |
| PA179-K23 | 137,239 | 2.671 | 0.990 |
| PA92-D22 | 126,848 | 2.650 | 0.802 |
| PA268-E13 | 135,334 | 1.678 | 1.322 |
| PA264-B19 | 108,571 | 1.575 | 0.157 |
| PA24-C06 | 141,446 | 1.946 | 1.488 |
Table 4
Small RNA processing pathway members. The Parhyale orthologs of small RNA processing pathway members.
| Gene | Counts | Gen ID |
|---|---|---|
| Armitage | 2 | phaw_30_tra_m.006391 phaw_30_tra_m.007425 |
| Spindle_E | 3 | phaw_30_tra_m.000091 phaw_30_tra_m.020806 phaw_30_tra_m.018110 |
| rm62 | 7 | phaw_30_tra_m.014329 phaw_30_tra_m.012297 phaw_30_tra_m.004444 phaw_30_tra_m.012605 phaw_30_tra_m.001849 phaw_30_tra_m.006468 phaw_30_tra_m.023485 |
| Piwi/aubergine | 2 | phaw_30_tra_m.011247 phaw_30_tra_m.016012 |
| Dicer 1 | 1 | phaw_30_tra_m.001257 |
| Dicer 2 | 1 | phaw_30_tra_m.021619 |
| argonaute 1 | 1 | phaw_30_tra_m.006642 |
| arogonaute 2 | 3 | phaw_30_tra_m.021514 phaw_30_tra_m.018276 phaw_30_tra_m.012367 |
| Loquacious | 2 | phaw_30_tra_m.006389 phaw_30_tra_m.000074 |
| Drosha | 1 | phaw_30_tra_m.015433 |
Additional files
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Source code 1
iPython Notebook for Parhyale genome assembly.
Includes bioinformatic processsing of raw read data, k-mer analysis, contig assembly, scaffolding and CEGMA cased representation analyis.
- https://doi.org/10.7554/eLife.20062.046
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Source code 2
iPython Notebook for repeat analysis.
Includes repeat analysis of the Parhyale genome using Repeat Modeller and Repeat Masker.
- https://doi.org/10.7554/eLife.20062.047
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Source code 3
iPython Notebook for transcriptome and annotation.
Parhyale transcriptome assembly, genome annotation and generation of canonical proteome dataset.
- https://doi.org/10.7554/eLife.20062.048
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Source code 4
iPython Notebook for variant analysis.
Analysis of polymorphism in Parhyale using genome reads, transcriptome data and sanger sequenced BACs.
- https://doi.org/10.7554/eLife.20062.049
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Source code 5
iPython Notebook of orthology analysis.
Protein orthology analysis between Parhyale and other species
- https://doi.org/10.7554/eLife.20062.050
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Source code 6
iPython Notebook for RNA.
Analysis of microRNAs and putative lncRNAs in Parhyale.
- https://doi.org/10.7554/eLife.20062.051
