No evidence for extensive horizontal gene transfer in the genome of the tardigrade <i>Hypsibius dujardini</i>

No evidence for extensive horizontal gene transfer in the genome of the tardigrade Hypsibius dujardini These files accompany the peer-reviewed version of http://dx.doi.org/10.1101/033464 A previous dataset https://zenodo.org/record/45162 accompanied the version of this manuscript at BioRxiv - biorxiv.org/content/early/2015/12/13/033464 This dataset includes all files from https://zenodo.org/record/45162 plus all the Supplemental files, and one additional file HGT_phylogenetic_files.tgz. All files are described in Hypsibius_dujardini_files_README.md Abstract Tardigrades are meiofaunal ecdysozoans that are key to understanding the origins of Arthropoda. Many species of Tardigrada can survive extreme conditions through cryptobiosis. In a recent paper (Boothby TC et al (2015) Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade. Proc Natl Acad Sci USA 112:15976-15981) the authors concluded that the tardigrade Hypsibius dujardini had an unprecedented proportion (17%) of genes originating through functional horizontal gene transfer (fHGT), and speculated that fHGT was likely formative in the evolution of cryptobiosis. We independently sequenced the genome of H. dujardini. As expected from whole-organism DNA sampling, our raw data contained reads from non-target genomes. Filtering using metagenomics approaches generated a draft H. dujardini genome assembly of 135 Mb with superior assembly metrics to the previously published assembly. Additional microbial contamination likely remains. We found no support for extensive fHGT. Among 23,021 gene predictions we identified 0.2% strong candidates for fHGT from bacteria, and 0.2% strong candidates for fHGT from non-metazoan eukaryotes. Cross-comparison of assemblies showed that the overwhelming majority of HGT candidates in the Boothby et al. genome derived from contaminants. We conclude that fHGT into H. dujardini accounts for at most 1-2% of genes and that the proposal that one sixth of tardigrade genes originate from functional HGT events is an artefact of undetected contamination

The role of FGF signalling in otic placode induction and specification

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Fgf3 and Fgf8 are required together for formation of the otic placode and vesicle

Fgf3 has long been implicated in otic placode induction and early development of the otocyst; however, the results of experiments in mouse and chick embryos to determine its function have proved to be conflicting. In this study, we determined fgf3 expression in relation to otic development in the zebrafish and used antisense morpholino oligonucleotides to inhibit Fgf3 translation. Successful knockdown of Fgf3 protein was demonstrated and this resulted in a reduction of otocyst size together with reduction in expression of early markers of the otic placode.fgf3 is co-expressed with fgf8 in the hindbrain prior to otic induction and, strikingly, when Fgf3 morpholinos were co-injected together with Fgf8 morpholinos, a significant number of embryos failed to form otocysts. These effects were made manifest at early stages of otic development by an absence of early placode markers (pax2.1 and dlx3) but were not accompanied by effects on cell division or death. The temporal requirement for Fgf signalling was established as being between 60% epiboly and tailbud stages using the Fgf receptor inhibitor SU5402. However, the earliest molecular event in induction of the otic territory, pax8 expression, did not require Fgf signalling, indicating an inductive event upstream of signalling by Fgf3 and Fgf8. We propose that Fgf3 and Fgf8 are required together for formation of the otic placode and act during the earliest stages of its induction

The genome of the tardigrade Hypsibius dujardini

<p>These data files accompany the bioRxiv preprint "The genome of the tardigrade Hypsibius dujardini"</p> <p>Edinburgh genome assembly and annotation<br> ========================================</p> <p>1. nHd.2.3.abv500.fna.gz - Edinburgh (EDI) genome assembly version 2.3. Reads were assembled as single-end with CLC to calculate the insert size distributions of the libraries and check for contaminants. Insert size distributions are calculated by mapping the reads back to the assembly with CLC. The MP library insert distribution wasn't normally distributed. The single-end assembly is checked for contamination using the blobtools software package which creates a TAGC plot. Inspection of the TAGC plot revealed multiple contaminations with distinct coverage and GC content that did not have a reference genome in public databases. The PE reads were normalised with one-pass khmer and were assembled with Velvet using a k-mer size of 55. Contaminants in the Velvet assembly were identified based on the coverage and GC of the scaffolds. The non-normalised reads were mapped to the assembly using CLC and reads were removed if either pair mapped to a contig identified as contaminant. The process was repeated two more times since newly assembled contaminants could be identified. Gaps were filled in the final assembly using GapFiller. Finally the MP library was used to scaffold the gap-filled assembly with SSPACE, accepting only the information from reads mapping 2 kb from the ends of the scaffolds. The final assembly spans 140 megabases (Mb) with median coverage of 86X.</p> <p>2. nHd.2.3.1.aug.gff.gz - Gene model GFF file as predicted by Augustus for nHd.2.3 genome assembly. This is Augustus run as a second pass annotation (using transcriptome assembly as evidence) after a first pass Maker (see below)</p> <p>3. nHd.2.3.1.aug.proteins.fasta.gz - Protein fasta file generated by Augustus for nHd.2.3 genome assembly.</p> <p>4. nHd.2.3.1.aug.transcripts.fasta.gz - Transcript CDS fasta file generated by Augustus for nHd.2.3 genome assembly.</p> <p><br> Edinburgh genome assembly and annotation - intermediate files<br> =============================================================</p> <p>1. nHd.1.0.contigs.cov.fna.gz - Preliminary assembly of all data, without any contamination screening</p> <p>2. maker1.gff3.gz - Gene model GFF file as generated by MAKER run as a first pass to generate enough genes to train genefinders more thoroughly</p> <p>3. all.maker.proteins.edit.fasta.gz - Protein fasta file generated by MAKER run as a first pass.</p> <p>4. all.maker.transcripts.edit.fasta.gz - Transcript CDS file generated by MAKER run as a first pass.</p> <p>Blob plots<br> ==========</p> <p>1. nHd.2.3.nHd_lib350-cov.BlobDB.json.gz - A blobDB (a JSON file generated using the blobtools package) which contains mapping, assembly and taxonomic information for the Edinburgh assembly and our read data. http://drl.github.io/blobtools/</p> <p>2. nHd.1.0.BlobDB.json.gz - A blobDB (a JSON file generated using the blobtools package) which contains mapping, assembly and taxonomic information for the Edinburgh preliminary assembly nHd.1.0 and Edinburgh read data. http://drl.github.io/blobtools/</p> <p>3. unc.TG-cov.BlobDB.json.gz - A blobDB (a JSON file generated using the blobtools package) which contains mapping, assembly and taxonomic information for the UNC assembly and their read data.  http://drl.github.io/blobtools/</p> <p>4. unc.nHd-cov.uniref.nt.BlobDB.json.gz - A blobDB (a JSON file generated using the blobtools package) which contains mapping, assembly and taxonomic information for the UNC assembly and the Edinburgh read data. http://drl.github.io/blobtools/</p> <p>5. tardi_RNASeq.vs.unc.bam.reads_cov.catcolour.txt.gz - Space delimited text file with classification of each UNC scaffold by avg coverage of each base by PolyA-selected RNAseq reads</p> <p>6. tardi_RNASeq.vs.nHd.2.3.bam.reads_cov.catcolour.txt.gz - Space delimited text file with classification of each Edinburgh scaffold by avg coverage of each base by PolyA-selected RNAseq reads</p> <p>H dujardini transcriptome data<br> ==============================</p> <p>1. Trinity.fasta.c99.gz - Preliminary transcriptome assembly by Itai Yanai's lab. Please do not use in any publications without checking with yanailab.technion.ac.il first</p> <p> </p> <p>Abstract of bioRxiv paper at http://dx.doi.org/10.1101/033464</p> <p>====================================== <br> The genome of the tardigrade Hypsibius dujardini <br> ======================================</p> <p>Background: Tardigrades are meiofaunal ecdysozoans that may be key to understanding the origins of Arthropoda. Many species of Tardigrada can survive extreme conditions through adoption of a cryptobiotic state. A recent high profile paper suggested that the genome of a model tardigrade, Hypsibius dujardini, has been shaped by unprecedented levels of horizontal gene transfer (HGT) encompassing 17% of protein coding genes, and speculated that this was likely formative in the evolution of stress resistance. We tested these findings using an independently sequenced and assembled genome of H. dujardini, derived from the same original culture isolate. </p> <p>Results: Whole-organism sampling of meiofaunal species will perforce include gut and surface microbiotal contamination, and our raw data contained bacterial and algal sequences. Careful filtering generated a cleaned H. dujardini genome assembly, validated and annotated with GSSs, ESTs and RNA-Seq data, with superior assembly metrics compared to the published, HGT-rich assembly. A small amount of additional microbial contamination likely remains in our 135 Mb assembly. Our assembly length fits well with multiple empirical measurements of H. dujardini genome size, and is 120 Mb shorter than the HGT-rich version. Among 23,021 protein coding gene predictions we found 216 genes (0.9%) with similarity to prokaryotes, 196 of which were expressed, suggestive of HGT. We also identified ~400 genes (<2%) that could be HGT from other non-metazoan eukaryotes. Cross-comparison of the assemblies, using raw read and RNA-Seq data, confirmed that the overwhelming majority of the putative HGT candidates in the previous genome were predicted from scaffolds at very low coverage and were not transcribed. Crucially much of the natural contamination in both projects was non-overlapping, confirming it as foreign to the shared target animal genome. </p> <p>Conclusions: We find no support for massive horizontal gene transfer into the genome of H. dujardini. Many of the bacterial sequences in the previously published genome were not present in our raw reads. In construction of our assembly we removed most, but still not all, contamination with approaches derived from metagenomics, which we show are very appropriate for meiofaunal species. We conclude that HGT into H. dujardini accounts for 1-2% of genes and that the proposal that 17% of tardigrade genes originate from HGT events is an artefact of undetected contamination.</p