Mapping qualities of Illumina datasets mapped onto transcriptome assembly.

<p>MapQ 0: several equally best hits, MapQ 37: maximum MapQ for single end reads in BWA.</p

Transcriptome assembly contig distributions.

<p>(A) Final assembly contig length distribution. (B) Illumina data assembly contig length distribution before and after error correction using Reptile (red: decrease, green: increase).</p

Towards Decrypting Cryptobiosis—Analyzing Anhydrobiosis in the Tardigrade <i>Milnesium tardigradum</i> Using Transcriptome Sequencing

<div><p>Background</p><p>Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade <i>Milnesium tardigradum</i>.</p><p>Results</p><p>A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage.</p><p>Conclusions</p><p>Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in <i>M</i>. <i>tardigradum</i> that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair.</p></div

DESeq differential transcript expression analysis.

<p>(A–C) Log ratio versus abundance plots (MA-plot) for each of the anhydrobiotic stages versus active stage (A: active vs. dehydration; B: active vs. inactive tun; C: active vs rehydration; red dots: significantly regulated at FDR of 0.1, red triangle regions pointed by arrow: significantly regulated without counts in one of the stages and outliers). (D) Venn diagram of differentially expressed transcripts compared to active stage shared between stages (generated using <a href="http://bioinformatics.psb.ugent.be/webtools/Venn/" target="_blank">http://bioinformatics.psb.ugent.be/webtools/Venn/</a>).</p

Workflow for the de novo hybrid assembly of the <i>M. tardigradum</i> transcriptome.

<p>(A) Hybrid assembly of 454 and Illumina using ABySS (AbySS_hyb). (B) Illumina assembly using (AbySS_sol). (C) Hybrid assembly of Sanger, 454 and ABySS contigs using MIRA. (D) Final clustering and assembly using TGICL.</p

Annotation of CEGs identified in <i>M. tardigradum</i> transcriptome.

<p>(A) Databases from which annotations were retrieved. (B) Distribution of GO levels. P: protein, F: function, and C: component.</p

Representative results of proteome coverage analysis using est2assmbly.

<p>Reference cDNA and proteome data were from <i>Anopheles gambiae</i> (<i>An.g.</i>), <i>Caenorhabditis elegans</i> (<i>C.e.</i>), <i>Daphnia pulex</i> (<i>D.p.</i>), <i>Drosophila melanogaster</i> (<i>D.m.</i>), <i>Ixodes scapularis</i> (<i>I.s.</i>), <i>Pristionchus pacificus</i> (<i>P.p</i>.), <i>Strigamia Maritima</i> (<i>S.m</i>.) and <i>Tetranychus urticae</i> (<i>T.u</i>.). (A) Unique hits on reference proteomes aligned to the <i>M. tardigradum</i> transcriptome (open square) and <i>T.u</i>. cDNA (solid square), respectively; and the reference proteome coverages when aligned to the <i>M. tardigradum</i> transcriptome (open circle) and <i>T.u</i>. CDNA (solid circle). (B) Ratio of redundancy over coverage for reference proteomes aligned to <i>An.g</i>. cDNA (solid square), <i>D.m</i>. cDNA (solid circle) and the <i>M. tardigradum</i> transcriptome (solid diamond). (C) Proteins identified in relation to sequence identity in <i>An.g</i>. (solid square), <i>D.m</i>. (solid circle) and <i>I.s</i>. (solid diamond) proteomes compared to <i>M. tardigradum</i>. (D) Distribution of query HSPs covered by <i>An.g</i>. (solid square), <i>D.m</i>. (solid circle) and <i>I.s</i>. (solid diamond) proteomes respectively.</p

Annotated differentially expressed transcripts in all three anhydrobiotic stages.

<p>(“∞” is used for undetectable expression in one of the samples/lack of fold change).</p

Filtering of reference transcripts for quantification.

<p>(A) Theoretical representation of original transcript as fragmented contigs in assembly. (B) Filtering of reference contigs by mapping window (top) or basal expression across all tested conditions (bottom).</p

Comparative proteome analysis of proteins identified in different states.

<p>The Venn diagram illustrates the number of protein identifications in EES, AS and TS. A total of 1301 proteins were found in all three states, 472 proteins are found only in EES (a) and 680 proteins are found only in adult tardigrades in TS and AS (f). Proteins which are non-overlapping (a, b, c) or partially overlapping (d, e, f) between the different states are analyzed using Blast2GO program to determine the involved biological processes. The ten major biological processes for non-overlapping proteins are listed in 2a-2c and for partially overlapping proteins in 2d–2f.</p