Transcriptome characterization via 454 pyrosequencing of the annelid Pristina leidyi, an emerging model for studying the evolution of regeneration
© Nyberg et al.; licensee BioMed Central Ltd. 2012
Received: 23 February 2012
Accepted: 29 June 2012
Published: 29 June 2012
The naid annelids contain a number of species that vary in their ability to regenerate lost body parts, making them excellent candidates for evolution of regeneration studies. However, scant sequence data exists to facilitate such studies. We constructed a cDNA library from the naid Pristina leidyi, a species that is highly regenerative and also reproduces asexually by fission, using material from a range of regeneration and fission stages for our library. We then sequenced the transcriptome of P. leidyi using 454 technology.
454 sequencing produced 1,550,174 reads with an average read length of 376 nucleotides. Assembly of 454 sequence reads resulted in 64,522 isogroups and 46,679 singletons for a total of 111,201 unigenes in this transcriptome. We estimate that over 95% of the transcripts in our library are present in our transcriptome. 17.7% of isogroups had significant BLAST hits to the UniProt database and these include putative homologs of a number of genes relevant to regeneration research. Although many sequences are incomplete, the mean sequence length of transcripts (isotigs) is 707 nucleotides. Thus, many sequences are large enough to be immediately useful for downstream applications such as gene expression analyses. Using in situ hybridization, we show that two Wnt/β-catenin pathway genes (homologs of frizzled and β-catenin) present in our transcriptome are expressed in the regeneration blastema of P. leidyi, demonstrating the usefulness of this resource for regeneration research.
454 sequencing is a rapid and efficient approach for identifying large numbers of genes in an organism that lacks a sequenced genome. This transcriptome dataset will be a valuable resource for molecular analyses of regeneration in P. leidyi and will serve as a starting point for comparisons to non-regenerating naids. It also contributes significantly to the still limited genomic resources available for annelids and lophotrochozoans more generally.
The process of regeneration, or the replacement of lost body parts, has long captured the interest of biologists. While early experiments on crayfish  and Hydra demonstrated the remarkable abilities of some animals to develop lost parts anew, it is also clear that many animals, including humans, do not possess such abilities. The ability to regenerate is thought to have been lost over the course of evolution in many animal lineages [3–5]. Despite recent advances in knowledge of the molecular and developmental basis of regeneration in a variety of animal systems [6–8], little is currently known about the developmental and evolutionary mechanisms that drive loss of regeneration ability . Understanding this phenomenon requires a comparative approach and the identification and development of animal systems that show variation in regeneration ability among closely related species.
The naid annelids are among a small number of documented groups in which regeneration ability varies among close relatives [5, 9–14], making them a good model for studying the loss of regeneration. Naids (the minimal clade including both the Naidinae and Pristininae) are a group of small aquatic oligochaete worms, many of which can reproduce asexually by fission . Many naids, including Pristina leidyi, possess excellent regeneration abilities, being able to regrow both their heads and tails after amputation. Following amputation, tissues at the wound site actively proliferate and form a regeneration blastema (a mass of undifferentiated cells) which ultimately differentiates to give rise to regenerated structures . The ability to regenerate anteriorly and posteriorly is thought to be ancestral for the clade. However, recent experiments indicate that head regeneration ability has been lost at least three times within the naids, allowing multiple independent comparisons between regenerating and non-regenerating species [9, 10, 17]. The degree of loss of the regeneration machinery can vary between lineages, suggesting that different developmental mechanisms may underlie independent evolutionary losses of regeneration . Thus, in the naids, evolution has crafted an ideal experiment for investigating loss of regeneration.
Much recent work on the developmental basis of regeneration has focused on the role of signaling pathways, such as the Wnt pathway, in recruiting stem cells and promoting morphogenesis in regenerated tissues [18–29]. In order to investigate the role of signaling pathways and other molecules in variation of regeneration ability, genomic resources are needed for the naids. Recent advances in high-throughput sequencing and bioinformatic analyses have made transcriptome sequencing feasible for discovering novel genes in non-model systems.
454 pyrosequencing, with sequence reads now approaching the length of traditional Sanger sequences, is ideal for transcriptome sequencing in a model that lacks a sequenced genome [30, 31]. While the sequencing depth of 454 is modest compared to that of other deep sequencing technologies, 454 does offer depth orders of magnitude above what can be obtained via Sanger sequencing . In addition, recent versions of the Newbler assembler from 454 allow for assembling sequences from cDNA, grouping presumptive gene isoforms together. Here, we describe the sequencing and assembly of a full run of 454 GS FLX sequencing with Titanium reagents from the regenerating annelid P. leidyi.
Results and discussion
Genome size estimation
We estimated the genome size of P. leidyi and four other naid species currently used in comparative regeneration studies . Using Feulgen densitometry analysis, we estimated a C-value of 1.37 pg for P. leidyi and C-values ranging from 0.54 to 1.09 pg for the four other naid species (Additional file 1). Previously published estimates for two naid species are 1.53 and 3.23 pg, and the mean of values reported for oligochaetes is ~1.6 pg (range: 0.43 to 7.64) [33, 34]. Thus, the genome size of P. leidyi is typical for this group.
Construction of a partially normalized cDNA library
RNA was extracted from whole worms at multiple time points between the initiation of regeneration and its completion and from unamputated worms that were actively undergoing fission. cDNA was synthesized with an oligo-dT primer and a MINT full-length reverse-transcription kit. PCR assays indicated that the dried Spirulina powder used as food was not metabolically active and could not be detected by RT-PCR in the cDNA sample (Additional file 2).
The overall amount of cDNA is greatly reduced during normalization, making PCR amplification necessary to produce a sufficient quantity of cDNA for 454 sequencing. Because PCR has its own biases, particularly against large amplicons, we pooled an unamplified, non-normalized sample with a normalized sample in a 1:2 ratio to increase the representation of longer transcripts (Figure 1). This pooled cDNA library was used for 454 pyrosequencing.
454 pyrosequencing and transcriptome assembly
Sequence and assembly output
454 sequencing results
mean read length
Newbler Assembler v2.3 assembly results
mean isotig length
mean max isotig length
fraction captured transcripts
gene discovery rate
33.21 reads/new gene
454 sequence reads were assembled using the Newbler Assembler v2.3 . Sequence output using the cDNA option of Newbler v2.3 differs from that of traditional genomic assemblers (e.g. SeqMan NGen 2.0, CAP3, Newbler 2.2) by taking into account the possibility that multiple isoforms (e.g. alternative splice variants) of a gene may be present. Overlapping sequence reads are assembled into contigs, much like traditional assemblers. However, if multiple isoforms are present, a sequence read may contain a portion that aligns perfectly with the previously constructed contig and a portion that does not (with the point of divergence being, for example, an exon-exon junction). When this occurs, Newbler v2.3 breaks up the aligned sequences into multiple contigs. Sequences shared between multiple isoforms are retained as unique contigs, and any adjacent variant sequences are split off as their own unique contigs. Thus, a single gene isoform might be assembled into multiple contigs, and the same contig might be shared across multiple isoforms. Each putative isoform identified by Newbler v2.3 is termed an isotig, and the multiple isoforms for each gene are organized into isogroups, representing putative gene loci.
BLAST analysis of 454 isotigs
Annotation of isotigs
EST2uni BLAST results
64,522 (max isotig per isogroup)
11,388 (17.7%, E-value < e-10)
Taxonomic identity of top UniProt hit
Total BLAST hits
Isotig matches to UniProt database
UniProt ref. size
5’ + 3’
BLAST results suggest that our efforts to minimize environmental contamination were successful. BLAST searches against the P. leidyi isotig dataset using either a cnidarian or human 16 S sequence [Hydra magnipapillata: GenBank:NC_011220|:307-2044; Homo sapiens: GenBank: FJ794693.1|:1673-3230] retrieve isotigs matching to Pristina 16S as the only hits with any reasonable significance (E-value < 0.1), suggesting no metazoan contamination. Furthermore, only a small number of isotigs matched prokaryotic genes (1.4%) (Table 2) and BLAST searches of the P. leidyi isotigs using bacterial 16S RNA from either the proteobacterium Escherichia coli [GenBank:4924485] or the cyanobacterium Arthrospira platensis (Spirulina) [GenBank:FJ798612.1] return a very limited number of isotigs (only nine, and the same nine, isotigs for both searches; E-values < e-2). Interestingly, four of these isotigs match 16S from bacterial genera known to be common endosymbionts in animal intestines (gammaproteobacteria Edwardsiella/Xenorhabdus/Photorhabdus; bacteroidetes Paenicardinium/Cardinium). Thus, some bacterial sequences present in the transcriptome may represent the endemic gut flora of P. leidyi.
To assess how well 5’ and 3’ ends were captured in our dataset, isotigs with significant BLAST hits were compared to their counterparts in UniProt (Table 3). The proportion of isotigs with captured ends (within 10 amino acids of the corresponding end of the UniProt sequence) varies with the length of the coding sequence in UniProt. Isotigs matching shorter UniProt sequences are more likely to be complete on both the 5’ and 3’ ends than isotigs matching longer UniProt sequences. 31.7% of isotigs matching UniProt sequences of less than 250 amino acids are complete on both ends, while only a single isotig matching UniProt sequences greater than 750 amino acids is complete. In total, 6.5% of isotigs can be considered complete, 13.0% have captured the 5’ end, 16.1% have captured the 3’ end, and 64.3% have no matches against either end. While we estimate that we have captured the vast majority of transcripts in the original cDNA library (Table 1) and that there is no strong bias towards either 5’ or 3’ ends, it is clear that most of our unigenes consist of only partial transcript sequences. Further sequencing, either in a high-throughput manner or on a targeted basis with genes of interest, will be necessary to fill in these gaps.
Gene ontology analysis of 454 isotigs
Identification of candidate regeneration genes
BLAST results for candidate regeneration genes
07867, 10773, 21383, 44961, 47307
03233, 13291, 15038, 40749, 49382
03101, 07423, 11748, 64463
00856, 52001, 54688
Independent confirmation of assembled transcripts
The utility of this transcriptome will ultimately be determined by whether these assembled sequences can be independently confirmed and manipulated for further studies. Because 454 sequencing has a nebulization step and is not performed using intact transcripts, Newbler v2.3 is unable to reconstruct with complete accuracy the actual gene isoforms present in vivo. Therefore, isotigs should be treated as predicted gene isoforms that require independent confirmation, such as by PCR assay. Contigs, on the other hand, already are well supported via the original sequencing and are expected to be true contiguous sequence and thus amplifiable by PCR.
Expression of wnt/β-catenin pathway genes during regeneration
To further demonstrate the utility of our sequencing effort for regeneration studies, we examined the expression patterns of two genes present in the transcriptome, homologs of frizzled (fz) and β-catenin (β-cat). These genes were chosen because they are components of the Wnt/β-catenin pathway, an important cell signaling pathway implicated in numerous developmental processes, including regeneration [18–29]. We identified from our transcriptome several homologs of fz (a Wnt ligand receptor) and a single homolog of β-cat (a multifunctional protein that acts as a transcription factor when Wnt signaling is activated). We examined expression of one of these fz homologs, Pl-fzA, and the homolog of β-cat Pl-β-cat, by whole mount in situ hybridization of regenerating and fissioning P. leidyi.
Our results provide the first expression data for frizzled or β-catenin genes during annelid regeneration and strongly implicate Wnt signaling in P. leidyi regeneration. They also add to the accumulating data showing a close developmental relationship between regeneration and fission in these animals. More broadly, these findings demonstrate that sequences from this transcriptome can provide new insights into annelid development, setting the stage for future comparative studies of annelid regeneration.
Applications for further regeneration research
This transcriptome dataset provides a valuable new resource for regeneration research in annelids. The work described here already demonstrates the utility of this dataset: our GO analysis suggests that a large number of genes relevant for regeneration research are represented, our sequence confirmation assays show that putative transcripts from our assembly can be independently validated, and our expression studies demonstrate that genes expressed during regeneration are indeed present in this transcriptome and can provide new insight into annelid regeneration.
This transcriptome resource promises to accelerate regeneration research in P. leidyi and provides a stepping-stone to studies of regeneration failure in closely related, non-regenerating naid species. This dataset greatly facilitates gene discovery, allowing genes of interest to be quickly identified and characterized by RT-PCR or in situ hybridization. Importantly, this resource can also provide a reference transcriptome for larger, genome-scale studies, such as high-throughput analyses of gene expression by microarrays or RNA-Seq [78–80]. Extending these approaches to closely related regenerating and non-regenerating naid species holds great promise for elucidating the genetic basis of both regeneration success and failure.
This transcriptome sequencing project has produced the first genomic-type sequence data for any of the naid annelids, a promising group for understanding regeneration loss. This dataset also represents the first regeneration-based, large-scale sequence database for annelids as a whole and thus provides a valuable resource for regeneration research more broadly. Our approach of using mixed-stage starting material and combining normalized/non-normalized cDNA pools was successful in producing a transcriptome with high gene representation. Based on BLAST searches for known regeneration genes and relevant GO analyses matches, we conclude that our methods captured a significant number of genes that may be involved in regeneration. This transcriptome resource enabled gene expression studies that have provided novel insight into annelid regeneration, yielding the first evidence suggesting that a cell-signaling pathway important in other regenerating systems, Wnt/β-catenin signaling, is initiated during annelid regeneration. Thus, this dataset promises to be instrumental in determining which genes are involved in regeneration processes in P. leidyi and will subsequently inform evolution of regeneration studies in the naids as a whole.
The development of genomic resources for the Lophotrochozoa (the large clade of bilaterians including platyhelminths, annelids, and molluscs, among others) has lagged considerably behind that of other major groups of animals. With the advent of less expensive sequencing technologies and an increased appreciation of the value of non-traditional model systems, genomic resources for this group are finally becoming available. Transcriptomes have recently been generated for a range of lophotrochozoan taxa [81–87], including the highly regenerative planarians [88, 89]. The growing number of genomic resources for lophotrochozoans promises to help fuel research on a broad range of questions in this large and diverse clade.
Genome size measurement
The genome sizes of five naid species were estimated using the Feulgen image analysis densitometry method . Individuals were obtained from laboratory cultures of the following species: P. leidyi (Carolina Biological Supply Company), Allonais paraguayensis (Wards Natural Science), Dero digitata (originally collected from Edwards Lake, University of Maryland at College Park, USA), Dero furcata (Connecticut Valley Biological Supply), and Paranais litoralis (originally collected from Herrington Bay, Deale, MD, USA). Fifty or more nuclei were measured from each sample. The Integrated Optical Density of the sample was converted to a genome size value (in picograms) using Gallus gallus domesticus (1.25 pg) as a standard.
Worm culture, sampling, and RNA extraction
To generate material for this sequencing effort, we established twelve replicate lab cultures of a single clonal line of Pristina leidyi (PRIle(cbs)cloneA). Each culture was initially started with 100 worms and was maintained at room temperature in 20 cm glass bowls filled with ~1 liter of commercially purchased Poland Spring Water (PSW). To ensure purity of the samples, worm cultures were rinsed frequently to remove algae and debris and cultures were routinely inspected visually for the presence of small metazoans (e.g. rotifers). Worms were fed dried Spirulina powder twice weekly, and water was changed at least 1-2 times per week.
Possible contamination by the dried Spirulina food source was assayed via PCR, using cDNA samples derived from live Arthrospira platensis (Spirulina) as a reference. RNA was extracted using TRIReagent (Applied Biosystems), and cDNA was constructed using random oligos and Superscript III reverse transcriptase (Invitrogen). Primers were constructed against the large subunit of rubisco (rbcL) [GenBank:AY147205.1] and c-phycocyanin (cpc) [GenBank:AF164139.1] genes of A. platensis (Additional file 5).
Worms were collected at a range of stages of regeneration and fission (Figure 1). For the fission material, 1,000 worms that were actively growing and fissioning were collected and starved for 24 hours. To generate regenerating material, 3,485 worms were amputated anteriorly and posteriorly and allowed to regenerate for various lengths of time before collection. Most worms were actively fissioning and consisted of chains of linked zooids at time of amputation. A cut was made 2 body segments anterior to the most anterior fission zone to elicit posterior regeneration. A second cut was made after the 6th body segment of the most posterior zooid to elicit anterior regeneration. If a worm did not consist of at least two nearly formed zooids, a single cut after the 6th body segment was made to elicit anterior regeneration. Because the initiation of regeneration processes holds particular significance for future studies, 1,985 of the regenerating worms in the sample were allowed to regenerate between 0 and 24 hours, which is roughly coincident with the start of blastema formation. Batches of 250 worms were also collected at 1.25 days post-amputation (dpa), 1.75 dpa, 2 dpa, 2.5 dpa, 3 dpa, and 3.5 dpa, when differentiation of adult morphology is nearly complete.
Fissioning and regenerating worms were washed 5x in PSW prior to RNA extraction. RNA was extracted using TRIReagent (Applied Biosystems), and RNA from all samples was then pooled together.
cDNA library construction
We constructed a pooled cDNA library consisting of a normalized fraction to capture lowly expressed transcripts and a non-normalized fraction to capture large transcripts that might be lost during the PCR amplification steps of the normalization process.
First-strand cDNA (F.S. cDNA) was made using a MINT full-length cDNA synthesis kit (Evrogen) and manufacturer’s instructions. A modified oligo-dT primer with breaks in the homopolymer-T run was used to minimize the negative effects of an extensive homopolymer run on 454 sequence quality (Additional file 5). A portion of the F.S. cDNA was incubated for 2 hours at 15°C with NEB Buffer 2, DNA Polymerase I (New England Biolabs), and RNase H in order to make full-length double-strand cDNA. A fraction of F.S. cDNA was then normalized with Evrogen’s duplex-specific nuclease (DSN). F.S. cDNA-RNA duplexes in hybridization buffer were denatured at 98° for 3 minutes and then allowed to hybridize at 70°C for 5 hours. Preheated DSN at 1/4x concentration was then added and incubated for 20 minutes at 70°C. DSN stop solution was then added, and the sample was incubated for 5 minutes at 70°C. Normalized cDNA was then PCR amplified using an Encyclo PCR kit (Evrogen). PCR conditions were: 1 cycle × 95°C-1 min.; 17 cycles × 95°C-15 sec., 66°C-20 sec., 72°C-3 min.; 1 cycle × 66°C-15 sec., 72°C-3 min. Normalization efficiency was assayed via gel smear and qPCR of genes with known relative abundance (Additional file 5). qPCR analysis was performed using LinRegPCR [91, 92].
The non-normalized and normalized cDNA libraries were then pooled in a 1:2 ratio (Figure 1).
Five μg of pooled cDNA library was sent to the Roy J. Carver Biotechnology Center at the University of Illinois for sequencing. The cDNA library was sheared to 500-800 bp in length, 454 sequencing adaptors were ligated onto ends (Additional file 5), and the library was then converted to a single-stranded template library. Three titration runs (each of 1/16 lane) were performed to optimize sequencing conditions. A full plate was then sequenced on a Roche/454 GS FLX Sequencer using Titanium reagents.
Assembly of 454 sequence reads
Reads from the full plate and three titration runs were assembled using the Newbler Assembler v2.3 (Roche) using default parameters under the cDNA option. Prior to assembly, specified primers and adaptors were trimmed, namely the oligo-dT primer, the MINT PlugOligo adapter and PCR primer (Evrogen), and the 454 sequencing adaptors (Additional file 5).
Determination of fraction of captured transcripts
The coverage statistic developed by Susko and Roger (2004) estimates the proportion of genes from a cDNA library that is actually represented in the sequence data . Using this method, the unbiased estimate of coverage was calculated for our transcriptome with the equation = 1 – n 1 /n, where n 1 is the number of singletons in the assembly and n is the total number of reads [39, 40]. The new gene discovery rate was estimated using the term 1/(1 – ).
Annotation and analysis of BLAST hits
The set of 95,644 isotigs was input into the EST2uni annotation pipeline using default parameters, but with PCR marker integration, microarray printing, reciprocal BLAST for orthologues, Gene Ontology, and RFLP integration options turned off . Within EST2uni, the CAP3 assembly parameters were adjusted to –f 2 –g 100 –p 100 –d 110 to produce an assembly of all singletons, thereby preserving the isotigs produced by Newbler. Isotigs were annotated if they produced BLASTX matches against UniProtKB Release 2010_04 (23-Mar-2010) with E-values less than e-10. Parsing of BLAST data for Table 2 was done with custom Perl scripts (available upon request).
Completeness of annotated isotigs in Table 3 was performed using only the largest isotig from each isogroup as a representative for its putative gene locus. A Perl script utilizing BioPerl modules (available upon request) was used for completeness analysis. An isotig was considered complete on either end if it matched within ten amino acids of the corresponding end of the UniProt sequence .
The set of max isotigs was also used to identify Gene Ontology (GO) designations using the program Blast2GO [44, 45]. Results from BLAST searches against the UniProt database were imported from EST2uni, and GO annotation in Blast2GO was performed with default parameters (E-value threshold of e-6).
Identification of candidate regeneration genes
TBLASTX was used to search the P. leidyi isotig dataset for homologs of genes implicated in animal regeneration in the literature. A reciprocal TBLASTX search was then performed against UniProt or the nr database via NCBI to verify the putative identity of candidate regeneration genes in P. leidyi.
Validation of transcript assembly
Transcript validation assays were performed for two isogroups, isogroup08478 (a putative piwi-like homolog) and isogroup03233 (a putative frizzled homolog). Isotigs of each isogroup were aligned together using ClustalX v2.1 with manual editing by Seaview v4.0 [93, 94]. PCR was then performed to verify contigs and isotigs of each isogroup (Additional file 5). PCR amplicons of the expected size were either sequenced directly or cloned into the pGEM-T Easy vector (Promega) prior to sequencing. Sequencing was performed using an Applied Biosystems 3730 × l DNA Analyzer.
Transcript assembly was also verified for four previously known P. leidyi genes. BLAST searches against the 454 dataset were performed using the published gene sequences of Pl-en [GenBank: AF336055.1], Pl-otx1 [GenBank: AF336056.1], Pl-otx2 [GenBank: AF336057.1], and Pl-nos [GenBank: GQ369728.1]. GenBank sequences were aligned to transcriptome sequences using Sequencher v.4.7 (Gene Codes Corporation).
Analysis of gene expression by whole mount in situ hybridization
A ~1250 bp fragment of Pl-fzA (isogroup23343) and a ~1300 bp fragment of Pl-β-cat (isogroup01340) were amplified by PCR (Additional file 5). Synthesis of sense and antisense riboprobes and in situ hybridization were performed as previously described .
We thank Yinon Bentor for help with PERL and other programming issues, the University of Illinois’ Roy J. Carver Biotechnology Center for performing 454 sequencing and helping with subsequent analyses, Karen L. Carleton for assistance with DSN normalization and qPCR, Duygu Özpolat for assistance with in situ hybridizations, the Delwiche Lab for live cultures of Arthrospira platensis, and Thomas D. Kocher for providing bioinformatic resources. This work was funded by NSF grant IOS-0920502 to AEB.
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