- Open Access
Identification and characterization of alternative splicing in parasitic nematode transcriptomes
© Abubucker et al.; licensee BioMed Central Ltd. 2014
- Received: 17 January 2014
- Accepted: 14 March 2014
- Published: 1 April 2014
Alternative splicing (AS) of mRNA is a vital mechanism for enhancing genomic complexity in eukaryotes. Spliced isoforms of the same gene can have diverse molecular and biological functions and are often differentially expressed across various tissues, times, and conditions. Thus, AS has important implications in the study of parasitic nematodes with complex life cycles. Transcriptomic datasets are available from many species, but data must be revisited with splice-aware assembly protocols to facilitate the study of AS in helminthes.
We sequenced cDNA from the model worm Caenorhabditis elegans using 454/Roche technology for use as an experimental dataset. Reads were assembled with Newbler software, invoking the cDNA option. Several combinations of parameters were tested and assembled transcripts were verified by comparison with previously reported C. elegans genes and transcript isoforms and with Illumina RNAseq data.
Thoughtful adjustment of program parameters increased the percentage of assembled transcripts that matched known C. elegans sequences, decreased mis-assembly rates (i.e., cis- and trans-chimeras), and improved the coverage of the geneset. The optimized protocol was used to update de novo transcriptome assemblies from nine parasitic nematode species, including important pathogens of humans and domestic animals. Our assemblies indicated AS rates in the range of 20-30%, typically with 2-3 transcripts per AS locus, depending on the species. Transcript isoforms from the nine species were translated and searched for similarity to known proteins and functional domains. Some 21 InterPro domains, including several involved in nucleotide and chromatin binding, were statistically correlated with AS genetic loci. In most cases, the Roche/454 data explored in this study are the only sequences available from the species in question; however, the recently published genome of the human hookworm Necator americanus provided an additional opportunity to validate our results.
Our optimized assembly parameters facilitated the first survey of AS among parasitic nematodes. The nine transcriptome assemblies, their protein translations, and basic annotations are available from Nematode.net as a resource for the research community. These should be useful for studies of specific genes and gene families of interest as well as for curating draft genome assemblies as they become available.
- Parasitic nematodes
- Alternative splicing
- Next-generation sequencing
Alternative splicing (AS) is a post-transcriptional, mRNA modification process that allows a single gene to give rise to multiple protein isoforms [1, 2]. These spliced isoforms can have distinct molecular functions and biological roles and may be differentially expressed among tissues, life cycle stages or environmental conditions , resulting in involvement in more genetic interactions and biochemical pathways compared to non-AS genes . Therefore, AS provides a significant boost to genomic complexity without necessitating a proportional increase in genome size. AS takes place to some extent in most eukaryotic organisms [5–7], and has been studied extensively in humans and model species, including Caenorhabditis elegans[8–12], but has not received much attention in parasitic nematodes. In fact, information on AS in parasitic nematodes is extremely sparse, and existing reports have focused on a few or single representative gene(s) [13–16].
High throughput cDNA sequencing is the preferred method for detecting and quantifying AS. Today’s most prevalent sequencing protocols (e.g., 454, Illumina, Ion Torrent, etc.) involve fragmentation of nucleic acid molecules, construction of sequencing libraries, and generation of many thousands to millions or even billions of short reads. In the absence of a well-curated genome for comparison, these reads must be re-assembled de novo into contiguous sequences (contigs) that faithfully represent the full-length transcripts from which they were derived. Graph-based assembly algorithms have been developed to maintain associations between transcripts with shared contigs, making it possible to identify different isoforms of the same gene . However, this procedure is computationally challenging, and various studies have shown that de novo cDNA assemblers typically overestimate the number of isoforms associated with a given locus and that many of the predicted isoforms are illegitimate [18–21]. Care must be taken to optimize assembly parameters to minimize errors and maximize accuracy and coverage.
cDNA sequencing is a cost-effective means of gene discovery in non-model organisms, so it often serves as the first line of investigation into an organism’s genetic complement. Thus, the transcriptomes of many parasitic nematodes (often including multiple sexes and life cycle stages) have been sequenced and relevant datasets are readily available . Several de novo transcriptome assemblies have been reported [23–30], but most were generated with software that did not account for AS (e.g., Newbler prior to version 2.3, CAP3, etc.). Revisiting existing datasets with a cDNA-specific, splice-aware, assembly protocol would provide a far more accurate impression of AS in parasitic nematodes, a factor that can have important practical implications with respect to pathogenesis, drug susceptibility/resistance, vaccine development, etc. [31, 32]. For example, the broad-spectrum anthelmintic drug ivermectin is known to bind tightly to one isoform of the α3 glutamate gated chloride channel subunit but not another, and these isoforms appear to be differentially expressed in susceptible versus resistant strains of the cattle parasites Cooperia oncophora and Ostertagia ostertagi[13, 33].
In this study, we used cDNA data from the well-characterized model organism Caenorhabditis elegans to define a set of optimized parameters for high-confidence splice isoform prediction using the Newbler assembler. The optimized protocol was then applied to existing and novel cDNA sequences from a diverse array of parasitic nematodes, including Ancylostoma caninum, C. oncophora, Dictyocaulus viviparus, Necator americanus, Oesophagostomum dentatum, Onchocerca flexusoa, O. ostertagi, Teladorsagia circumcincta, and Trichostrongylis colubriformis[23–30] in the first broad survey of AS among parasitic worms. Our assemblies offer a better impression of genetic and transcriptional complexity in these non-model species and will aid in studies on specific genes/gene families and for annotating and curating draft genomes as they become available.
454/Roche library construction, sequencing and data cleaning
One splice-leader (SL1) and four oligo(dT) cDNA libraries were constructed from DNase treated C. elegans (Bristol N2) RNA according to previously described methods . Libraries were sequenced with a GS 454 FLX pyrosequencer using a standard protocol , and raw reads were deposited in the NCBI sequence read archive (SRA) under project number SRP003926. Parasitic nematode sequences were mostly obtained from previous studies, but novel sequences were produced and submitted to the SRA in the same manner (see Additional file 1: Table S1 & [23–30]).
Raw reads were edited and filtered prior to assembly. Relevant adapter sequences were removed with Cutadapt , and reads with an overall quality score less than 20 and an overall dust score less than seven were removed using seq_crumbs software (http://bioinf.comav.upv.es/seq_crumbs/). The remaining reads were aligned to rRNA [36, 37] and bacterial  sequence databases with Bowtie2 (version 2.1.0, default parameters ) and to the human (hs37) genome and relevant host genomes with Tophat2 (version 2.0.8, default parameters ) for contaminant removal. Host genomes, obtained from GenBank, included: Canis lupus famliaris (CanFam3.1) for A. caninum; Bos taurus (Btau4.6.1) for C. oncophora, D. viviparus, and O. ostertagi; Sus scrofa (Sscrofa10.2) for O. dentatum; Ovis aries (Oar3.1) for T. colubriformis and T. circumcincta. O. flexuosa, a parasite of European red deer (Cervus elaphus), and N. americanus, maintained in golden hamsters (Mesocricetus auratus), were screened against Bos taurus (Btau4.6.1) and the GenBank rodent database (gbrod, downloaded April 24, 2013), respectively, as close substitutes for the unavailable host genomes.
Cleaned C. elegans Roche/454 reads were mapped to C. elegans coding sequences (WormBase  release WS236) with Bowtie2 (version 2.1.0, default parameters ) in order to assess the scope of the dataset prior to assembly. The coverage of each feature was assessed using RefCov version 0.3 (http://gmt.genome.wustl.edu/gmt-refcov/) and coverage was reported in Additional file 2: Table S2.
Assembly and evaluation
Caenorhabditis elegans test assemblies 1
cDNA -icl 10
cDNA -icl 30
cDNA -icl 50
cDNA -het -icl 30 -mi 95 -ml 100
% Aligned reads
Ave. isotigs per AS isogroup
BLASTN v. CDSs
Isotigs with match4
Isogroups with match5
C. elegans genes represented
BLASTN v. CDS + UTR
Isotigs with match4
Isogroups with match5
C. elegans genes represented
Parasitic nematode transcriptomes were assembled with parameters that showed the optimal performance on C. elegans data (minimum overlap of 100 bp and 95% identity, minimum contig length of 30, and heterozygosity specified). Large or complex datasets were reduced to a manageable size by digital read normalization prior to assembly using khmer with a word size of 31 bp (http://ged.msu.edu/papers/2012-diginorm/). N. americanus isotigs were compared with transcript isoforms reported along with the genome of N. americanus by BLASTN (cutoff of ≥90% sequence identity over ≥75% length of the isotig in a single high-scoring segment pair).
Annotation of parasitic nematode transcriptomes
Parasite isotigs were searched against the GenBank non-redundant protein database (downloaded July 9, 2013), and non-overlapping top hits with e-value ≤1e-5 were recorded (Additional files 4, 5, 6, 7, 8, 9, 10, 11 and 12: Tables S4-S12). Prot4EST  was used to generate translations from the parasite isotigs, and InterPro protein domains and gene ontology terms were predicted from translated proteins using InterProScan [45, 46]. Transcript sequences, peptide translations, and annotations are reported in Additional files 4, 5, 6, 7, 8, 9, 10, 11 and 12: Tables S4-S12 and are available from Nematode.net .
Enrichment of AS isogroups associated with functional domains
The number of alternatively spliced and non-alternatively spliced isogroups associated with each InterPro domain was counted (Additional file 13: Table S13), and a non-parametric binomial distribution test was applied to each InterPro domain to test for enrichment of AS isogroups using the following input parameters: (i) the background frequency of AS isogroups across all species (40.5%); (ii) the number of AS isogroups associated with the InterPro domain across all species (i.e., number of “successes”); (iii) the total number of isogroups associated with the InterPro domain across all species (i.e., number of “trials”). In order to reduce false positives resulting from poorly represented domains, domains represented by fewer than ten isogroups or fewer than four species were ignored, reducing total number of domains considered from 5,190 to 3,141 (a 39.5% reduction; Additional file 14: Figure S1). P values calculated for each domain were population corrected using False Discovery Rate (FDR) correction , and a significance threshold of 0.01 on the corrected P values was used to determine which InterPro domains were significantly more often associated with AS isogroups than non-AS isogroups.
Optimization of assembly parameters
The Newbler assembler, distributed by 454 Life Sciences, is considered the gold standard for Roche/454 read assembly. Using the cDNA option, Newbler identifies regions of shared sequence, termed contigs, and compiles them into full-length transcripts, termed isotigs. Isotigs with shared contigs, theoretically derived from AS of the same gene, are clustered into isogroups representing distinct genetic loci. We tested various combinations of program parameters in order to reduce assembly errors and increase the percentage of isotigs and isogroups that accurately represent known C. elegans sequences (Table 1). The best results were obtained with a contig length of 30 bp, minimum read overlap of 100 bp, minimum sequence identity of 95% (Table 1, last column). The heterozygous mode had little effect on our C. elegans assemblies, but we chose to invoke this option to accommodate the genetic heterogeneity of our parasitic worm datasets. Using these parameters, 96.7% of the clean reads were assembled into 15,940 isogroups containing 16,772 isotigs. Some 691 (4.3%) of these isogroups are associated with more than one isotig, with an average of 2.2 isotigs per AS isogroup (Table 1). Approximately 17% of the C. elegans genes reported in WormBase build WS236 are associated with more than one CDS isoform with an average of 2.6 isoforms per AS gene , and a 2011 study reported that at least 25% of all C. elegans genes undergo AS . The relatively low rate of AS detected in our test assembly is probably a reflection of the clonal worm population that, despite being mixed-stage, was dominated at the tissue level by the relatively large adult hermaphrodites. Sampling each sex and life cycle stage independently could have provided greater resolution of AS events; however, the aim of this exercise was to optimize assembly protocols, not to explore AS in the model worm.
By adjusting assembly parameters, we were able to increase the number and percentage of isotigs and isogroups that accurately reflected known CDSs, increase the coverage of the gene set, and reduce the rates of misassembled transcripts (i.e., cis- and trans-chimeras). In the best version of our transcriptome assembly, 38.07% of the isotigs were matched to 7,027 distinct CDS isoforms from 5,727C. elegans genes (Table 1, last column). Match rates increased when isotigs were compared to coding transcripts (CDSs plus untranslated regions) rather than CDSs, indicating that a portion of our sequence data corresponds to untranslated regions at the extreme ends of the transcript.
Caenorhabditis elegans assembly statistics 1
Unique CDS isoforms
Altogether, 12,265 of the 16,772 isotigs included in our best transcript assembly (73.1%) were verified either by a match to previously reported transcript isoforms included in WormBase or by our orthologous sequencing chemistries. Given the limitations presented by today’s sequencing technologies and assembly software, no combination of parameters will provide a perfect assembly. Some rate of error is to be expected given the challenges presented by complex, dynamic eukaryotic transcriptomes (e.g., varying expression rates, RNA half-life, secondary structure, AS, etc.). However, the error rates we detect are lower than those reported in other studies (particularly those involving shorter reads and deBruijn graph assemblers [50, 51]). Clearly, we were able to show improvement over default program parameters using our test dataset, and we expect that the impact of parameter optimization could prove even more vital as the size and complexity of the dataset increases.
Assembly of down-sampled Caenorhabditis elegans read sets
Average read length
% aligned reads
Average isotigs per AS isogroup
BLASTN v. CDSs
Isotigs with match1
Isogroups with match2
C. elegans genes matched
Parasitic nematode transcript assemblies
Parasitic nematode transcript assemblies
Genome BioProject ID
Egg, L1, L2, iL3, aL3, male, female
Egg, L1, L2, iL3, aL3, L4, male, female
Egg, L1, iL3, L5, male, female
iL3, mixed sex adults
L2, iL3, L4, male, female
Mixed sex adults
Egg, L1, L2, iL3, L4, mixed sex adults
Mixed sex adults
Mixed sex adults
Normalized or full assembly
Number of isotigs
Average isotig length
Number of isogroups
Number of AS Isogroups
5, 589 (24.24%)
Average isotigs per AS isogroup
Number of unique translations
Number of unique InterPro domains
Number of Unique GO terms
Parasitic nematode transcriptome assembly statistics are reported in Table 4. The datasets range in size and complexity from approximately one million reads derived from adult O. flexuosa to upwards of 7.5 million reads derived from eggs, larvae and adults of two geographically distinct strains of O. ostertagia. As previously discussed, there is a limit to the amount of data that can be processed by OLC assembly algorithms like those implemented by Newbler, so several datasets had to be reduced by digital read normalization prior to assembly (Table 4). Our tests seem to indicate that the complexity of the transcriptome has a greater impact on assembly efficiency than the absolute number of reads. For instance, we were able to assemble some 2.5 million reads from mixed sex adult T. colubriformis, whereas a full assembly of the 1.5 million reads derived from L3 and mixed sex adult N. americanus was not possible.
The number of isogroups obtained from each assembly ranged from 15,828 from O. flexuosa to 42,785 from the more thoroughly covered transcriptome of C. oncophora. Detected rates of AS, as measured by the number of isogroups associated with multiple isotigs, mostly fell within the 20-30% range, with a maximum AS rate of 34.65% in D. viviparus (Table 4). The AS rates seen in the parasitic nematodes were expected to be similar, as previous studies have shown that splice events are highly conserved among Caenorhabditis species despite hundreds of millions of years of evolutionary separation [53, 54]. It is also reasonable to expect that the parasitic nematodes, especially those with extremely complex life cycles like N. americanus and D. viviparus, would have higher rates of AS than free-living worms like C. elegans due to the increased genomic complexity that may be required to interact with multiple hosts/vectors, host/vector tissues, and environmental conditions. We did not make an effort to classify or compare the nature of these AS events (e.g., alternative starts and/or stops, intron retention, exon skipping, etc.), but we expect that this will be possible in future studies aimed at exploring AS profiles of particular species in greater detail.
It stands to reason that sampling and sequencing more life cycle stages would lead to increased resolution of AS events. Indeed, including more stages tended to increase the number of isogroups (i.e., genetic loci) identified, but overall AS rates and the average number of isotigs associated with each isogroup remained relatively consistent with the notable exception of T. colubriformis. The AS rate reported for T. colubriformis (11.68%) was much lower than AS rates reported for other species represented by a single cDNA library derived from mixed-sex adults (24.44% AS in O. flexuosa and 21.14% AS in T. circumcincta). This disparity may be due to decreased transcriptomic complexity in T. colubriformis, but there may be other explanations. In the case of T. colubriformis, material was obtained from an inbred laboratory strain , while O. flexuosa and T. circumcinta material were collected in the field [28, 29]. O. flexuosa nodules tend to be dominated by large, adult females . Likewise, T. circumcinta is a polymorphic species with sex ratios biased towards females . This is significant due to the fact that a patent female represents a broad survey of adult female tissues, embryos in various stages of development, and even stored sperm from males, all of which contribute to diversity in the transcript population. Sequencing additional life cycle stages of T. colubriformis or specifically studying adults of other species would provide additional data needed to better understand the obtained results.
The assembled sequences from these nine species, as well as their functional annotations and predicted translations are available from Nematode.net  for use by the wider community. Although genome sequencing projects are currently underway, the transcriptomes presented here represent a significant proportion of the sequence data available from these species at this time. These datasets are, therefore, a vital source of information on the genetic content and complexity of these parasites and will remain so even after draft genomes are published as genome sequencing does not, in and of itself, provide any information on AS. Historically, initial reports of draft genomes rarely comment on AS [57–64]. For instance, the draft genome of the well-studied filarial nematode B. malayi was published in 2007, but the report made no mention of AS . The most recent dataset available from WormBase (B. malayi WS236) includes multiple isoforms for 16% of the reported genes, but no comprehensive studies on the subject of AS in B. malayi have been reported despite an abundance of representative RNAseq data . The recently published N. americanus genome paper was unique in that it included an estimate of AS based on Illumina RNAseq data generated from L3 and adult worms. Multiple isoforms were identified from approximately 25% of the 19,151 predicted protein coding genes. Some 1,209 of the 3,354 AS isogroups from N. americanus match 1,114 genes reported as AS in the genome study (≥90% nucleotide sequence identity over ≥75% of the length of the isotigs in a single high-scoring segment pair) , while another 65 AS isogroups matched genes that previously lacked evidence for AS. Clearly our assemblies, performed with a special emphasis on AS, will be a useful complement to genome sequencing studies and transcriptome studies performed using orthologous sequencing and assembly approaches.
Protein domains associated with alternative splicing
Enrichment of InterPro protein domains among alternatively spliced isogroups
InterPro protein domain
Number of species with domain
Total number of Isogroups containing domain
Percentage of isogroups with domain that are AS*
P value for enrichment**
RNA recognition motif domain
Nucleotide-binding, alpha-beta plait
Acyl-CoA dehydrogenase, N-terminal
Acyl-CoA dehydrogenase/oxidase, N-terminal
AAA + ATPase domain
Aminoacyl-tRNA synthetase, class I, conserved site
Acyl-CoA oxidase/dehydrogenase, central domain
Acyl-CoA dehydrogenase/oxidase, N-terminal and middle domain
Pleckstrin homology-like domain
Helicase, superfamily 1/2, ATP-binding domain
Pyridoxal phosphate-dependent transferase, major region, subdomain 1
Chaperonin TCP-1, conserved site
RNA recognition motif domain, eukaryote
Chaperone tailless complex polypeptide 1 (TCP-1)
DNA/RNA helicase, DEAD/DEAH box type, N-terminal
Glycosyl transferase, family 8
De novo transcriptome assembly is a complicated procedure that is confounded by varied gene expression patterns, such AS of mRNA. Transcriptome assemblies benefit from the use of optimized parameters designed to increase accurate coverage of the gene set and minimize assembly error. The set of parameters we described was thoroughly tested with C. elegans data and verified using well-curated sequences available from WormBase as well as data from an unrelated sequencing chemistry. Our optimized parameters are offered as a guide to assist in the assembly of other nematode transcriptomes, and updated, annotated transcript assemblies from nine species of parasitic worms are offered as a resource to the research community. Rates of AS seem to be similar among the species studied, and 21 InterPro protein domains appear to be enriched among AS transcripts. This represents a first step in exploring AS among parasitic nematodes, an important and relevant topic that should be further investigated in future sequencing studies.
The authors acknowledge The Genome Institute production team for assistance with cDNA/RNA-seq library construction and sequencing and John Martin for providing technical support. This work was supported by NIH grant to MM.
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