Illumina sequencing

The normalized RNA pool yielded 16,923,850 reads with a median of 61 nucleotides in length and a maximum read length of 101bp. The raw sequence data are available at NCBI Short Read Archive (SRP005151). The 13 mtDNA genome gene sequences of R. balthica were used as reference to calculate transcript coverage, which was found to range between 5x for the ND4L gene up to 989x for COIII, with a mean coverage of 137x. One means to asses normalization success, is to determine whether the average number of ESTs per ribosomal gene contigs does not exceed 5x the average number of ESTs for all other contigs [3]. Using the NGEN assembly, there was an average of 487 ESTs per ribosomal gene contig vs. 541 for the non-ribosomal genes, indicating that normalization was successful.

Indirect contig quality assessment

Velvet

The VELVET assembly resulted in a total of 220,153 contigs ranging in size between 61bp to 2257bp, with a mean of 120bp in length (Table 1, Figure 1). Only 9% of these contigs were longer than 200bp. A BLAST X against the UniProt database with a cutoff value of < e^-5 yielded 115,649 putative gene homologies, which resulted in 5956 unique gene hits (UniGens) after removing redundant hits (Figure 2). A cutoff value of < e^-10 decreased the number of hits by over one third to 4736, leading to 1671 UniGens, of which 962 are above 200bp. A BLAST N against the RefSeq Invertebrate database with a cutoff value < e^-5 yielded 6127 putative gene similarities, which after removing redundant hits resulted in 3907 UniGens (Figure 3). Most of these hits matched to the sea urchin Strongylocentrotus purpuratus (10%), followed by the sea anemone Nematostella vectensis (7%) and Hydra magnipapillata (7%). In total, gene homologies were identified across 32 different species, of which 23 were insect species. Applying a cutoff value of < e^-10 the BLAST N against the RefSeq Invertebrates database resulted in 2010 hits of which 1465 are UniGen hits and 475 are larger than 200bp. A BLAST X against Biomphalaria glabrata ESTs yielded 10,750 hits with < e^-5 and 5269 UniGens (Figure 4). Decreasing the cutoff value to < e^-10 rendered 6055 hits with 3621 UniGens and 1866 longer than 200bp.

	all contigs					> 200bp contigs
	Velvet	NGen	ORK21	ORK31	Meta	Velvet	NGen	ORK21	ORK31	Meta
Min	61	31	55	52	61	200	200	200	200	200
Max	2257	2435	2070	2551	2667	2257	2435	2070	2551	2667
Mean	120	303	267	309	432	398	428	442	443	536
Median	93	217	179	213	341	326	338	359	359	442
Total	220,153	57,986	52,477	41,590	54,450	20,346	31,898	23,816	22,089	39,941

(ORK21/31: OASES k-mer = 21/31)

NGen

The NGEN assembly resulted in a total of 57,986 contigs with mean length 303bp (Table 1, Figure 1). Fifty-five percent of the total contigs were found to be larger than 200bp. Figure 5 depicts the relationship of average coverage versus contig size. Contig length increased with coverage depth and reached an asymptote approximately at an average coverage of about 30. A BLAST X against the UniProt database with a cutoff value of < e^-5 yielded 10,072 putative gene homologies, which resulted in 5059 UniGens after removing redundant hits (Figure 2). Using a cutoff value of < e^-10 resulted in 7630 hits and 4166 UniGens, of which 3810 were larger than 200bp. A BLAST N against the RefSeq Invertebrate database with a cutoff value < e^-5 yielded 1835 putative gene similarities, which after removing redundant hits resulted in 974 UniGens (Figure 3). Most of the hits matched to Hydra magnipapillata (16%), followed by the sea urchin Strongylocentrotus purpuratus (12%), and the tick Ixodes scapularis (9%). Overall homologies to 44 different species were detected, of which 21 belonged to insect species, but also 12 mitochondrial homologies were found belonging to 11 snail species. Applying a cutoff value of < e^-10 decreased the number of RefSeq hits to 1091 of which 606 are UniGen hits and 441 were larger than 200bp. A BLAST X against Biomphalaria glabrata ESTs yielded 10,569 hits with < e^-5 and 5490 UniGens (Figure 4). Decreasing the cutoff value to < e^-10 rendered 7913 hits with 4513 UniGens and 3671 lager than 200bp.

Assessing UniProt hits

In the absence of genomic sequence for R. balthica, contigs from the different assemblies were compared among each other. Only contigs having UniProt BLAST X gene hits were used. Pairwise comparisons of single gene hits (contigs > 200bp) revealed higher a similarity between the NGEN and OASES contig sets (both k-mer sizes), than between VELVET and any of the latter (Figure 6). This was true for both cutoff values. For example, NGEN shared 2973 hits with OASES k-mer = 21 with a cutoff value of < e^-5, but only 1269 with VELVET. With a cutoff value of < e^-10NGEN shared 2359 hits with OASES k-mer = 21, and 628 with VELVET.

Direct contig quality assessment via mitochondrial genes

Contig assembly was also assessed by looking at the average aligned contig length vs. the total contig length (Figure 7, left panels). VELVET contigs are all very short and show little relationship between increasing contig length and increased alignment length, while this relationship is very clear for the other assemblies. Directly related to this is the ratio of the aligned region to the full length of a given contig, plotted against decreasing cutoff value alignment score (Figure 7, right panel). All but the meta-assembly show an increase in this ratio as alignment score increases (cutoff value decreasing), with the meta-assembly having several outlier contigs with good alignment scores but low ratio. These outliers appear to be chimeric contigs, consisting of two previous contigs incorrectly joined together.

Functional annotation of ESTs

Using BLAST2GO (version 2.4.4), we were able to assign gene ontology classes to 2270 of the 2405 BlastX hits identical between the NGEN and OASES k-mer = 31 assemblies. There were a total of 30,464 gene ontology (GO) terms on all GO-levels associated with the 2270 identified genes. Of these, assignments to level two GO-terms of the highest order category Biological Processes made up the majority (5652) followed by Cellular Components (4231) and Molecular Function (3124) (Additional file 2).

Repeat motifs

As shown previously, transcriptome sequences are useful to identify repeat motifs (i.e. microsatellites), which due to their high levels of variation are widely used for studies such as genome mapping, parentage analysis, analysis of gene flow and genetic structure [e.g. 1, 14, 19-21]. Using the program Sputnik [22], we identified 552 di-, tri-, tetra-, and penta-nucleotide repeats with a minimum repeat length of six, respectively (Additional file 3).

Indirect contig quality assessment

Although the VELVET assembly had the largest number of contigs and the greatest number of hits against various databases, these are due to the poor assembly of contigs. Importantly, our ability to gain this insight is dependent upon the reference database used for BLAST searches and thus requires careful attention. In the BLAST comparisons against the UniProt database, the number of UniGen hits for the VELVET contigs is substantially smaller compared to the other assemblies with a cutoff value of < e^-10 and also contigs > 200bp (Figure 2). The discrepancy between total and UniGen hits derives most probably from the incomplete assembly of contigs by the VELVET assembler, resulting in many independent contigs each hitting similar genes, while these are joined together by the other programs and thus constitute single hits to given genes for the NGEN and OASES assemblies. Additionally, the long contig assemblies by the other programs generate more high quality BLAST hits than those found for VELVET (Figure 2). However, BLAST results against RefSeq indicate a much greater number of UniGen hits by the VELVET assembly than the other two methods (Figure 3). This result arises due to the highly redundant nature of the RefSeq database, as it contains unique sets of genes for numerous species. The RefSeq database should therefore be used with caution since the number of unique types of genes should not differ significantly from those identified using the UniProt database.

In the BLAST comparison against a database consisting of a single closely related species, B. glabrata, the NGEN assembly resulted in the highest number of UniGens, and VELVET showing especially poor performance when considering assembled contigs > 200bp in length.

Combining all of these assemblies into the meta-assembly resulted in contigs that outperformed the other assemblies in the BLAST X searches against the UniProt and B. glabrata databases in all but one category.

An additional means of assessing contig assembly performance is to compare the actual hits identified by the different assemblies. Similar hits indicate similar contig sequence and accuracy. Comparisons were made between assemblies for the BLAST X search versus the UniProt database (cutoff value < e^-10, contig length > 200bp), which showed that the proportion of contigs leading to identical gene hits was highest between the NGEN and OASES contig sets. This again strengthened our interpretation that the quality of the NGEN and OASES contigs exceeded those of VELVET.

Direct contig quality assessment

The different contig assemblies were directly assessed by comparing their performance among the 13 mitochondrial genes of R. balthica[24]. In general, VELVET contigs had the highest number of hits against these genes due to these contigs being much shorter. The other assemblies had longer and fewer contigs, which had higher average alignment length, with the meta-assembly showing the best performance (i.e. fewest number of contigs with highest average aligned contig length (Table 2). Longer contigs had a lower identity match to the mtDNA genes, which likely arises due to genetic differences in the samples used for this and the published mtDNA genome for R. balthica and potentially sequencing errors (which have a higher probability to occur in long contigs compared to short ones). We identified some contigs whose middle region did not resemble the reference sequence and we identified these as assembly errors. In addition, most contigs of the NGEN and OASES assembly had a 20-30bp extension attached at the beginning of the contig that does not match the mtDNA genome. For the NGEN assembly, this extension was identified as the Illumina sequencing adaptor not removed during filtering due to low identity matching. For the OASES contigs we currently lack an explanation for the attachment origin. As the extensions seem to be an almost systematic error, cutting the first 30bp of each contig sequence is one means to solve this problem (although some good quality sequence may be lost).

Despite these differences, coverage of the mtDNA genes was quite similar among the contig assemblies, averaging around 50 - 55% (Table 2). Pooling all the contigs from the assemblies covered 79% of the mt genes. Thus, even though contigs of the three assemblers overlap to a large extent, each contig set covers some parts which are missed by the others, with at least 24% of the available bp information is not used by any of the three assemblers. We identified 27 clusters with 2 to 25 overlapping, and to a large extent identical VELVET contigs. In contrast, among the NGEN contigs not more than two contigs with more than 30bp overlap were found. As the two main reasons for insufficient assembly visual inspection of the mt genome alignments revealed insufficient read overlap and missed assemblies, even though identical and sufficient overlap was present. This might be traced back to the use of RNA from several pooled individuals, which leads to a larger number of SNP variants, and thus might hamper assembly [11]. In our study we identified 6.3 SNPs per thousand base pairs (n = 52), similar to the 6.7 identified SNPs in the Vera et al. [11] study. The estimated number of sequencing errors is almost identical (n = 51), and results in a sequencing error rate of 0.6%. Obviously SNP variation and sequencing errors affect the VELVET assembly, but do not appear to influence the other two assemblers. The meta-assembly combined the short VELVET SNP containing contigs into one, thus largely eliminating redundancy (Additional file 4). However, although the meta-assembly decreased the number of contigs from 560 to 82, this only resulted in a modest improvement in net coverage compared to VELVET (58% vs. 55% respectively).

Two other important observations merit discussion. First, contigs giving a hit against the mt genes can be split in two groups. One group of the contigs shows a clear relation between alignment length of the contig to total contig length. The other group consists of contigs that passed the cutoff value < e^-5, but only have a very short alignment length to the reference sequence and are therefore due effectively due to random, non-homologous matches (Figure 7). Second, while a clear relationship between cutoff value and alignment length is visible for the NGEN and OASES contigs, both the VELVET and meta-assembly contigs have clear outliers that may be assembly errors. These are contigs near the cutoff value with low alignment length, and with very high stringency cutoff values (e.g. < e^-65).

Comparison to other studies

The number of UniGen matches against the UnipProt database found in other transcriptome studies of non-model organisms based on the 454/Roche platform is roughly similar to the 5380 meta-assembly matches detected in this study, at a cutoff value of < e^-5 (e.g. [11]Melitaea cinxia: 6122 at < e^-5 ). However, given our increased sequencing effort compared to previous studies (total quality data produced: 976 Mbp vs. 66 Mbp, i.e. 14 times higher compared to the M. cinxia study [11]), we expected to identify more genes. Previous observations of low blast results in mollusk species can be traced back to three main factors [25, 26]. First, the low amount of hits can be explained by the lack of EST datasets from mollusk species in Genbank [25, 26], and the general paucity of mollusk genetic data compared to insects and fish. Second, a large proportion of genes in mollusk species do not share orthologous relationships, but rather represent novel gene families [26]. Third, the high level of amino acid divergence to other, better studied invertebrate lineages and evolutionary distance to other organisms reduces the probability and quality of BLAST hits [26, 27]. These points highlight the need for more genomic data from molluscs to increase our knowledge and facilitate genomic studies in this phylum.

Sampling of snails

Radix balthica were caught from a highly inbred population (F _IS = 0.53) from a small lake in Bavaria, Germany and kept for several generations in the laboratory. Snails were put in 500 ml jars under varying conditions (Additional file 5). Different aeration, life stages, temperatures, feeding conditions, salinity and temperature shock treatments were employed to cover a wide range of temperature related transcription patterns useful for later temperature involved studies. Two individuals per treatment were selected and pooled for cDNA synthesis (n = 24).

cDNA library construction

RNA was isolated following the RNeasy maxi kit protocol (Qiagen) (Additional file 6 Figure S1). cDNA production and normalization was performed by GenXPro GmbH (Frankfurt, Germany). In order to optimize the recovery of full-length transcripts, Calf Intestinal Phosphatase (CIP, Ambion) was used to remove the 5'-phosphate from all RNA species except those with cap structure. Tobacco Acid Phosphatase (TAP, Ambion) was used to remove the cap structure, leaving RNA molecules with a 5' phosphate. An RNA oligonulceotide ("SceI-site") was then ligated to the afore capped RNAs. First strand synthesis was performed using SuperScript^® cDNA Synthesis System as described by the manufacturers, with SuperScript III as reverse transcriptase (Invitrogen) and a biotinylated oligodT with a T7 recognition site at the 5' end. The first strand product was used as template for a 20 cycles PCR reaction using primers specific for the T7 and the SceI-site. The PCR product was then normalized using the kamtchatka Crab Double Strand Specific Nuclease (DSN; Evrogen), as described by Zhulidov et al.[28] (Additional file 6 Figure S2). The normalized product was again amplified by PCR with 15 cycles and used as template for T7 -RNA polymerase amplification (Fermentas) for 5 hours. In order to select for longer transcripts, RNA > 600 bps was gel-selected and purified from the agarose gel (Additional file 6 Figure S3). The recovered RNA was fragmented by Zinc treatment. Illumina GA-II sequencing adapters were ligated to the fragments, as described by Illumina's Paired-End Sample Preparation Guide (catalogue number PE-930-1001). A 300-400 bp smear containing the cDNA fragments flanked by Illumina PE adapters was cut from an agarose gel and cDNA purified. Sequencing was performed on an Illumina GA-II using the Chrysalis 36 cycles v 3.0 sequencing kit, with one lane of 2 × 101 bp reads from both ends of the fragments ("paired ends") with an insert length of > 180 bp. The PhiX control lane revealed error rates between 3.16% for Read 1 and 4.07% for Read 2. Data basecall was performed using the Solexa GAPipeline 1.4.0. We chose to use the paired-end sequencing approach as it bears some additional advantages compared to normal one way sequencing. For one, information on two sequence reads being located in close vicinity is obtained and their position to one another known, second, if long enough these reads might be joined to one single read, longer than the usual read length.

EST analysis and bioinformatics

The raw sequence data was quality checked and trimmed if five consecutive bases were of quality score "B", these plus the subsequent bases at the 3' end of the low-quality-base reads were discarded. T7 and Illumina adaptors were removed using an inhouse script.

EST-contigs were assembled using three different assemblers: a) VELVET short read assembler, Version 0.7.55 [29] as commonly used short read assembler, with following parameters: k-mer hash length 31, coverage = 1, read category = shortPaired; b) OASES short read assembler, Version 0.1.11 has recently been released as extension for Velvet, optimized for EST assembly (available at http://www.ebi.ac.uk/~zerbino/oases/). It uses preliminary VELVET assemblies with reduced setting options as input. Two additional VELVET assemblies were created with k-mer sizes 21 and 31, with read category set to shortPaired of the raw read data. The same k-mer sizes were additionally applied to the previously trimmed sequence set, which in both cases resulted in less contigs of smaller size (data not shown), and were thus excluded from further analysis.; c) SeqMan NGEN program (hereafter called NGEN) from Lasergene DNAStar software package (Madison, WI; http://www.dnastar.com/t-products-seqman-ngen.aspx) k-mer size = 31, Illumina reads and PairedEnd option turned on for the first contig assembly, with the Illumina raw reads. If not otherwise stated default parameters were used. Assemblies were run on computers with 32 Gb RAM, and finished within 24h.

As contigs created from the different assemblers differed in their read length, number of BLAST hits and position along a transcript, we additionally investigated whether a meta-assembly, thus the combined assembly of all four contig sets might increase contig information. The approach of combined assembly of several contig sets has already proven useful for 454-contigs obtained by different assemblers [30]. As our first-order contig analyses revealed adaptor remains on some contigs, all first-order contig sets were BLAST ed against the T7 and Illumina adaptors to ensure high quality meta-assembly. If similarities were detected the according bases plus subsequent bases to the nearest sequence end were trimmed (this affected 3-7% for the Oases and Velvet as well as 35% of the NGen contigs). We chose to use NGEN for this task as it gave good results in our study but especially also for long read assembly [30]. Default parameters were used for the "other reads" option with k-mer size = 25.

Contigs from all assemblies can be found in Additional files 7, 8, 9, 10 and 11.

After evaluating the results of the above mentioned methods, we came to the conclusion that the best quality contigs obtained are the ones assembled by NGEN. We therefore produced an overview graph of contig average coverage versus contig length, and the gene ontology annotation (see below) for this contig set only. Contig average coverage (ac) was calculated by summing up the lengths of all sequences (ssl) building up one contig, divided by the contig length (cl), thus ac = ssl/cl.

Gene ontology annotation

Assignment of gene ontology (GO) terms to the 2405 genes identified by both the NGEN and OASES k-mer 31 assembled contigs > 200bp was performed by importing the GO-numbers obtained by the above mentioned BlastX search against the UniProt database into BLAST2GO (version 2.4.4; http://www.blast2go.org/). BLAST2GO is an automated tool for the assignment of gene ontology terms and was designed for use with novel sequence data [32]. Categorization of the BLAST matches and construction of pie charts was conducted using the standard graph configurations including level two GO-terms only.

SNP and sequencing error estimation

Reads of VELVET contigs that resulted in high quality hits against the mitochondrial genome of R. balthica were assembled against the latter using Geneious Pro 5.4.2 (http://www.geneious.com). SNP calling parameters were set to minimum coverage = 5, and minimum variant frequency = 0.2. Sites with at least 10x coverage and the presence of one single deviating nucleotide were scored as sequencing error.

Repeat motifs

Microsatellite repeat motifs were identified using the program SPUTNIK (http://espressosoftware.com/sputnik/; [22]). SPUTNIK is a program based on C language which searches DNA sequence files for microsatellite repeats using a recursive algorithm. We then extracted di-, tri-, tetra-, and penta-nucleotide repeats with a minimum repeat length of six, respectively.