Phylum distributions of top blast hit species in the invertebrate protein database.

<p>The percentages of phylum for top blast hit species are shown for the results with a cutoff e-value of 1e-10.</p

<em>De Novo</em> Sequencing and Transcriptome Analysis of the Central Nervous System of Mollusc <em>Lymnaea stagnalis</em> by Deep RNA Sequencing

<div><p>The pond snail <em>Lymnaea stagnalis</em> is among several mollusc species that have been well investigated due to the simplicity of their nervous systems and large identifiable neurons. Nonetheless, despite the continued attention given to the physiological characteristics of its nervous system, the genetic information of the <em>Lymnaea</em> central nervous system (CNS) has not yet been fully explored. The absence of genetic information is a large disadvantage for transcriptome sequencing because it makes transcriptome assembly difficult. We here performed transcriptome sequencing for <em>Lymnaea</em> CNS using an Illumina Genome Analyzer IIx platform and obtained 81.9 M of 100 base pair (bp) single end reads. For <em>de novo</em> assembly, five programs were used: ABySS, Velvet, OASES, Trinity and Rnnotator. Based on a comparison of the assemblies, we chose the Rnnotator dataset for the following blast searches and gene ontology analyses. The present dataset, 116,355 contigs of <em>Lymnaea</em> transcriptome shotgun assembly (TSA), contained longer sequences and was much larger compared to the previously reported <em>Lymnaea</em> expression sequence tag (EST) established by classical Sanger sequencing. The TSA sequences were subjected to blast analyses against several protein databases and <em>Aplysia</em> EST data. The results demonstrated that about 20,000 sequences had significant similarity to the reported sequences using a cutoff value of 1e-6, and showed the lack of molluscan sequences in the public databases. The richness of the present TSA data allowed us to identify a large number of new transcripts in <em>Lymnaea</em> and molluscan species.</p> </div

Comparison of <i>de novo</i> assembly with a distinct k-value.

<p>Three assembly programs, ABySS, Velvet and OASES, were tested with a distinct k-mer from 31 to 95. In each assembly program, the number of contigs, the N50 length, and the average and maximum contig length were calculated using the assembled contigs longer than 100 bp (black), 200 bp (orange), 300 bp (blue), 400 bp (green) and 500 bp (pink).</p

Protein sequence alignment and phylogenetic tree of dopa decarboxylase.

<p>Protein sequence alignment of dopa decarboxylase (A) and phylogenetic analyses (B) were conducted by the neighbor-joining method using the MEGA4 program with the <i>C. elegans</i> tyrosine decarboxylase as outgroup. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches. The scale bars indicate the estimated evolutionary distance in the units of the number of amino acid substitutions per site.</p

Protein sequence alignment and phylogenetic tree of tyramine beta hydroxylase.

<p>Protein sequence alignment of tyramine beta hydroxylase (A) and phylogenetic analyses (B) were conducted by the neighbor-joining method using the MEGA4 program with the <i>C. elegans</i> of tyramine beta hydroxylase as outgroup. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches. The scale bars indicate the estimated evolutionary distance in the units of the number of amino acid substitutions per site.</p

Blast hits of the <i>Lymnaea</i> TSA to different protein databases.

<p>Blast hits of the <i>Lymnaea</i> TSA to different protein databases.</p

Comparison of <i>de novo</i> assembly quality among the different programs.

<p>(A) Overall comparison of the results from five assembly programs. The bars indicate the number of contigs longer than 200 bp (left axis). The red lines indicate the N50 length and the black lines indicate average contig length in bp (right axis). (B) Blast requests of LymCREB2 CDS (1,140 bp) for differently assembled contigs. The black line above represents the query sequence and the colored lines below represent the results of the similarity of hit contigs in the databases. The alignment scores are indicated by five colors in the label at the top.</p

Comparison of the present <i>Lymnaea</i> TSA and the previously reported <i>Lymnaea</i> EST.

<p>Comparison of the present <i>Lymnaea</i> TSA and the previously reported <i>Lymnaea</i> EST.</p

Comparison of the <i>Lymnaea</i> TSAs and <i>Aplysia</i> EST.

<p>Comparison of the <i>Lymnaea</i> TSAs and <i>Aplysia</i> EST.</p

Gene ontology distribution for the <i>Lymnaea</i> TSA.

<p>Gene ontology distribution of the <i>Lymnaea</i> TSA derived from BLAST2GO. The results are summarized as molecular functions, biological processes and cellular components. The x-axis represents the percentage of contigs divided by the total number of cells counted with the given level 2 GO terms.</p