- Email: [email protected]
An embryonic transcriptome of the pulmonate snail Radix balthica
Marine Genomics 24 (2015) 259–260
Contents lists available at ScienceDirect
Marine Genomics journal homepage: www.elsevier.com/locate/margen
Genomics/Technical resources
An embryonic transcriptome of the pulmonate snail Radix balthica Oliver Tills ⁎, Manuela Truebano, Simon Rundle Marine Biology and Ecology Research Centre, Marine Institute, School of Marine Science and Engineering, Plymouth University, Drake Circus, Plymouth PL4 8AA, UK
a r t i c l e
i n f o
Article history: Received 4 June 2015 Received in revised form 14 July 2015 Accepted 14 July 2015 Available online 18 August 2015 Keywords: Transcriptome Gastropod Radix balthica Predator cue
a b s t r a c t The pond snail, Radix balthica (Linnaeus 1758), is an emerging model species within ecological developmental biology. While its development has been characterised in detail, genomic resources for embryonic stages are lacking. We applied Illumina MiSeq RNA-seq to RNA isolated from pools of embryos at two points during development. Embryos were cultured in either the presence or absence of predator kariomones to increase the diversity of the transcripts assembled. Sequencing produced 47.2 M paired-end reads, assembled into 54,360 contigs of which 73% were successfully annotated. This transcriptome provides an invaluable resource to build a mechanistic understanding of developmental plasticity. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Radix balthica is a species of freshwater snail with a widespread distribution throughout North-western Europe where it inhabits a diverse range of freshwater and brackish water systems (Pfenninger et al., 2011; Schniebs et al., 2011; Tills et al., 2013a). R. balthica is an emerging model species in ecology (e.g. Jokela et al., 2006), climate change biology (e.g. Cordellier and Pfenninger, 2009) and ecological developmental biology (e.g. Rundle et al., 2011; Smirthwaite et al., 2007). The embryonic development of R. balthica occurs within transparent egg capsules and can be visualised non-invasively. Highthroughput automated bio-imaging of the entire embryonic development of R. balthica has revealed considerable intra-specific variation in the development of functional traits (Tills et al., 2013a), that appear to have some genetic basis (Tills et al., 2013b). However, the relative importance of drift vs selective processes during embryonic development remain unclear. An adult-stage transcriptome has been produced for R. balthica (Feldmeyer et al., 2011), but efforts to elucidate mechanisms underpinning variation in early development are hampered by the lack of molecular data for these early-life stages. Consequently, here we present an embryonic-stage transcriptome for R. balthica. 2. Data description 2.1. Organism culture Eggs were harvested from a laboratory R. balthica stock population (T = 20 °C, 12 h:12 h L:D, F0, collected from Cadover Bridge, Dartmoor, ⁎ Corresponding author. E-mail addresses: [email protected] (O. Tills), [email protected] (M. Truebano), [email protected] (S. Rundle).
http://dx.doi.org/10.1016/j.margen.2015.07.014 1874-7787/© 2015 Elsevier B.V. All rights reserved.
UK — 50° 27′ 54″ N, 4° 02′ 12″ W, in January 2014). Prior to undergoing the second cell division, eggs were extricated from egg masses, cleaned with moistened tissue paper and assigned to either a control (de-chlorinated tap water), or predator cue treatment (de-chlorinated water holding an individual tench, Tinca tinca, from a stock population, for 1 h). Embryos were cultured in 0.6 mL treatment water within a 48 well microtitre plate (25 eggs per well) housed within an incubation chamber (T = 20 ± 0.1 °C, OkoLab, Naples, Italy). Water was changed daily via manual pipetting. Embryos were monitored using low power microscopy (20×, Leica MZ12) to identify when they had reached either first heart function, or first crawling behaviour (Tills et al., 2010, 2011, 2013c). Once these developmental events had been reached, three replicate pools (n = 150)of each of the four treatments were fast frozen using liquid nitrogen and stored at −80 °C. 2.2. RNA isolation Total RNA was isolated (Qiagen RNeasy micro kit, Qiagen, Manchester, UK) and TruSeq RNA libraries (Illumina, San Diego, USA) were synthesised and sequenced using 250 base paired-end sequencing (MiSeq2, Illumina, San Diego, USA). Sequencing produced 23.6 M read pairs. Sequencing adapters and bases with a phred score of b 30 were removed using Cutadapt (version 1.3, minimum length = 50, quality cutoff = 30; Martin, 2011) after which reads were assembled using Trinity (version r20140412, seqType fq, JM 100G, CPU 20, no_run_quantifygraph, group_pairs_distance = 220, bflyHeapSpaceMax = 10G, bflyHeapSpaceInit 5G, no_run_butterfly; Grabherr et al., 2011). The assembly contained 116,167 contigs, representing 98,315 potential genes. Contigs were subsequently filtered to remove sequences that had either lower than 1% expression, compared with the expression from the Trinity component to which it
260
O. Tills et al. / Marine Genomics 24 (2015) 259–260
Table 1 Assembly and annotation statistics. Assembled bases
Number of contigs
Mean sequence length
Median sequence length
N50
GC content
75,068,156
54,360
1380
749
2474
37.32
SwissProt BlastX 30,651
SwissProt BlastP 18,070
GO
SignalP
Pfam
eggNOG
2630
17 009
11,025
Annotation statistics
18,610
belongs (IsoPct) and, fragments per kilobase of transcript effective length per million fragments mapped to all transcripts (FPKM) of less than 1. This filtering left 54,360 sequences, representing 45,946 potential genes. 2.3. Annotation Seventy three percent of contigs were successfully annotated using the Trinotate annotation pipeline (version r20140708, www. trinotate.github.io) with an e-value threshold of 1 × 10− 5. This pipeline assesses Blast homologies between the assembly and SwissProt using BlastX of the contig sequence, and BlastP of TransDecoder Predicted Proteins (Haas et al., 2013), run using Blast + (Altschul et al., 1990, Camacho et al., 2008. For full details of the Trinotate annotation pipeline see - http://trinotate.github.io (Table 1). 2.4. Data deposition Raw sequence data are available in the NCBI Sequences Read Archive (SRA) under the accession number PRJEB9533. The transcriptome assembly is available, upon request, from [email protected]. Acknowledgements This research was funded via a NERC NBAF Pilot Scheme Grant (NBAF698). RNA sequencing, transcriptome assembly and annotation was carried out at the NBAF GenePool genomics facility at the University of Edinburgh. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., Madden, T.L., 2008. BLAST+ architecture and applications. BMC Bioinf. 10, 421.
Transdecoder-predicted proteins 18 070
Cordellier, M., Pfenninger, M., 2009. Inferring the past to predict the future: climate modelling predictions and phylogeography for the freshwater gastropod Radix balthica (Pulmonata, Basommatophora). Mol. Ecol. 18, 534–544. Feldmeyer, B., Wheat, C.W., Krezdorn, N., Rotter, B., Pfenninger, M., 2011. Short read Illumina data for the de novo assembly of a non-model snail species transcriptome (Radix balthica, Basommatophora, Pulmonata), and a comparison of assembler performance. BMC Genomics 12, 317. Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., et al., 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644–652. Haas, B.J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P.D., et al., 2013. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc. 8, 1494–1512. Jokela, J., Wiehn, J., Kopp, K., 2006. Among- and within-population variation in outcrossing rate of a mixed-mating freshwater snail. Heredity 97, 275–282. Martin, M., 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet 17, 10–13. Pfenninger, M., Salinger, M., Haun, T., Feldmeyer, B., 2011. Factors and processes shaping the population structure and distribution of genetic variation across the species range of the freshwater snail Radix balthica (Pulmonata, Basommatophora). BMC Evol. Biol. 11, 135. Rundle, S.D., Smirthwaite, J.J., Colbert, M.W., Spicer, J.I., 2011. Predator cues alter the timing of developmental events in gastropod embryos. Biol. Lett. 209, 2362–2367. Schniebs, K., Glöer, P., Vinarski, M.V., Hundsdoerfer, A.K., 2011. Intraspecific morphological and genetic variability in Radix balthica (Linnaeus 1758) (Gastropoda: Basommatophora: Lymnaeidae) with morphological comparison to other European Radix species. J. Conchol. 40, 657. Smirthwaite, J.J., Rundle, S.D., Bininda-Emonds, O.R.P., Spicer, J.I., 2007. An integrative approach identifies developmental sequence heterochronies in freshwater basommatophoran snails. Evol. Dev. 9, 122–130. Tills, O., Spicer, J.I., Rundle, S.D., 2010. Salinity-induced heterokairy in an upper-estuarine population of the snail Radix balthica (Mollusca: Pulmonata). Aquat. Biol. 9, 95–105. Tills, O., Rundle, S.D., Salinger, M., Haun, T., Pfenninger, M., Spicer, J.I., 2011. A genetic basis for intraspecific differences in developmental timing? Evol. Dev. 13, 542–548. Tills, O., Rundle, S.D., Spicer, J.I., 2013a. Variance in developmental event timing is greatest at low biological levels: implications for heterochrony. Biol. J. Linn. Soc. 110, 581–590. Tills, O., Bitterli, T., Culverhouse, P., Spicer, J.I., Rundle, S.D., 2013b. A novel application of motion analysis for detecting stress responses in embryos at different stages of development. BMC Bioinf. 14, 37-36. Tills, O., Rundle, S.D., Spicer, J.I., 2013c. Parent-offspring similarity in the timing of developmental events: an origin of heterochrony? Proc. Roy. Soc. B. 280, 1479.
Contents lists available at ScienceDirect
Marine Genomics journal homepage: www.elsevier.com/locate/margen
Genomics/Technical resources
An embryonic transcriptome of the pulmonate snail Radix balthica Oliver Tills ⁎, Manuela Truebano, Simon Rundle Marine Biology and Ecology Research Centre, Marine Institute, School of Marine Science and Engineering, Plymouth University, Drake Circus, Plymouth PL4 8AA, UK
a r t i c l e
i n f o
Article history: Received 4 June 2015 Received in revised form 14 July 2015 Accepted 14 July 2015 Available online 18 August 2015 Keywords: Transcriptome Gastropod Radix balthica Predator cue
a b s t r a c t The pond snail, Radix balthica (Linnaeus 1758), is an emerging model species within ecological developmental biology. While its development has been characterised in detail, genomic resources for embryonic stages are lacking. We applied Illumina MiSeq RNA-seq to RNA isolated from pools of embryos at two points during development. Embryos were cultured in either the presence or absence of predator kariomones to increase the diversity of the transcripts assembled. Sequencing produced 47.2 M paired-end reads, assembled into 54,360 contigs of which 73% were successfully annotated. This transcriptome provides an invaluable resource to build a mechanistic understanding of developmental plasticity. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Radix balthica is a species of freshwater snail with a widespread distribution throughout North-western Europe where it inhabits a diverse range of freshwater and brackish water systems (Pfenninger et al., 2011; Schniebs et al., 2011; Tills et al., 2013a). R. balthica is an emerging model species in ecology (e.g. Jokela et al., 2006), climate change biology (e.g. Cordellier and Pfenninger, 2009) and ecological developmental biology (e.g. Rundle et al., 2011; Smirthwaite et al., 2007). The embryonic development of R. balthica occurs within transparent egg capsules and can be visualised non-invasively. Highthroughput automated bio-imaging of the entire embryonic development of R. balthica has revealed considerable intra-specific variation in the development of functional traits (Tills et al., 2013a), that appear to have some genetic basis (Tills et al., 2013b). However, the relative importance of drift vs selective processes during embryonic development remain unclear. An adult-stage transcriptome has been produced for R. balthica (Feldmeyer et al., 2011), but efforts to elucidate mechanisms underpinning variation in early development are hampered by the lack of molecular data for these early-life stages. Consequently, here we present an embryonic-stage transcriptome for R. balthica. 2. Data description 2.1. Organism culture Eggs were harvested from a laboratory R. balthica stock population (T = 20 °C, 12 h:12 h L:D, F0, collected from Cadover Bridge, Dartmoor, ⁎ Corresponding author. E-mail addresses: [email protected] (O. Tills), [email protected] (M. Truebano), [email protected] (S. Rundle).
http://dx.doi.org/10.1016/j.margen.2015.07.014 1874-7787/© 2015 Elsevier B.V. All rights reserved.
UK — 50° 27′ 54″ N, 4° 02′ 12″ W, in January 2014). Prior to undergoing the second cell division, eggs were extricated from egg masses, cleaned with moistened tissue paper and assigned to either a control (de-chlorinated tap water), or predator cue treatment (de-chlorinated water holding an individual tench, Tinca tinca, from a stock population, for 1 h). Embryos were cultured in 0.6 mL treatment water within a 48 well microtitre plate (25 eggs per well) housed within an incubation chamber (T = 20 ± 0.1 °C, OkoLab, Naples, Italy). Water was changed daily via manual pipetting. Embryos were monitored using low power microscopy (20×, Leica MZ12) to identify when they had reached either first heart function, or first crawling behaviour (Tills et al., 2010, 2011, 2013c). Once these developmental events had been reached, three replicate pools (n = 150)of each of the four treatments were fast frozen using liquid nitrogen and stored at −80 °C. 2.2. RNA isolation Total RNA was isolated (Qiagen RNeasy micro kit, Qiagen, Manchester, UK) and TruSeq RNA libraries (Illumina, San Diego, USA) were synthesised and sequenced using 250 base paired-end sequencing (MiSeq2, Illumina, San Diego, USA). Sequencing produced 23.6 M read pairs. Sequencing adapters and bases with a phred score of b 30 were removed using Cutadapt (version 1.3, minimum length = 50, quality cutoff = 30; Martin, 2011) after which reads were assembled using Trinity (version r20140412, seqType fq, JM 100G, CPU 20, no_run_quantifygraph, group_pairs_distance = 220, bflyHeapSpaceMax = 10G, bflyHeapSpaceInit 5G, no_run_butterfly; Grabherr et al., 2011). The assembly contained 116,167 contigs, representing 98,315 potential genes. Contigs were subsequently filtered to remove sequences that had either lower than 1% expression, compared with the expression from the Trinity component to which it
260
O. Tills et al. / Marine Genomics 24 (2015) 259–260
Table 1 Assembly and annotation statistics. Assembled bases
Number of contigs
Mean sequence length
Median sequence length
N50
GC content
75,068,156
54,360
1380
749
2474
37.32
SwissProt BlastX 30,651
SwissProt BlastP 18,070
GO
SignalP
Pfam
eggNOG
2630
17 009
11,025
Annotation statistics
18,610
belongs (IsoPct) and, fragments per kilobase of transcript effective length per million fragments mapped to all transcripts (FPKM) of less than 1. This filtering left 54,360 sequences, representing 45,946 potential genes. 2.3. Annotation Seventy three percent of contigs were successfully annotated using the Trinotate annotation pipeline (version r20140708, www. trinotate.github.io) with an e-value threshold of 1 × 10− 5. This pipeline assesses Blast homologies between the assembly and SwissProt using BlastX of the contig sequence, and BlastP of TransDecoder Predicted Proteins (Haas et al., 2013), run using Blast + (Altschul et al., 1990, Camacho et al., 2008. For full details of the Trinotate annotation pipeline see - http://trinotate.github.io (Table 1). 2.4. Data deposition Raw sequence data are available in the NCBI Sequences Read Archive (SRA) under the accession number PRJEB9533. The transcriptome assembly is available, upon request, from [email protected]. Acknowledgements This research was funded via a NERC NBAF Pilot Scheme Grant (NBAF698). RNA sequencing, transcriptome assembly and annotation was carried out at the NBAF GenePool genomics facility at the University of Edinburgh. References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., Madden, T.L., 2008. BLAST+ architecture and applications. BMC Bioinf. 10, 421.
Transdecoder-predicted proteins 18 070
Cordellier, M., Pfenninger, M., 2009. Inferring the past to predict the future: climate modelling predictions and phylogeography for the freshwater gastropod Radix balthica (Pulmonata, Basommatophora). Mol. Ecol. 18, 534–544. Feldmeyer, B., Wheat, C.W., Krezdorn, N., Rotter, B., Pfenninger, M., 2011. Short read Illumina data for the de novo assembly of a non-model snail species transcriptome (Radix balthica, Basommatophora, Pulmonata), and a comparison of assembler performance. BMC Genomics 12, 317. Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., et al., 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644–652. Haas, B.J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P.D., et al., 2013. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc. 8, 1494–1512. Jokela, J., Wiehn, J., Kopp, K., 2006. Among- and within-population variation in outcrossing rate of a mixed-mating freshwater snail. Heredity 97, 275–282. Martin, M., 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet 17, 10–13. Pfenninger, M., Salinger, M., Haun, T., Feldmeyer, B., 2011. Factors and processes shaping the population structure and distribution of genetic variation across the species range of the freshwater snail Radix balthica (Pulmonata, Basommatophora). BMC Evol. Biol. 11, 135. Rundle, S.D., Smirthwaite, J.J., Colbert, M.W., Spicer, J.I., 2011. Predator cues alter the timing of developmental events in gastropod embryos. Biol. Lett. 209, 2362–2367. Schniebs, K., Glöer, P., Vinarski, M.V., Hundsdoerfer, A.K., 2011. Intraspecific morphological and genetic variability in Radix balthica (Linnaeus 1758) (Gastropoda: Basommatophora: Lymnaeidae) with morphological comparison to other European Radix species. J. Conchol. 40, 657. Smirthwaite, J.J., Rundle, S.D., Bininda-Emonds, O.R.P., Spicer, J.I., 2007. An integrative approach identifies developmental sequence heterochronies in freshwater basommatophoran snails. Evol. Dev. 9, 122–130. Tills, O., Spicer, J.I., Rundle, S.D., 2010. Salinity-induced heterokairy in an upper-estuarine population of the snail Radix balthica (Mollusca: Pulmonata). Aquat. Biol. 9, 95–105. Tills, O., Rundle, S.D., Salinger, M., Haun, T., Pfenninger, M., Spicer, J.I., 2011. A genetic basis for intraspecific differences in developmental timing? Evol. Dev. 13, 542–548. Tills, O., Rundle, S.D., Spicer, J.I., 2013a. Variance in developmental event timing is greatest at low biological levels: implications for heterochrony. Biol. J. Linn. Soc. 110, 581–590. Tills, O., Bitterli, T., Culverhouse, P., Spicer, J.I., Rundle, S.D., 2013b. A novel application of motion analysis for detecting stress responses in embryos at different stages of development. BMC Bioinf. 14, 37-36. Tills, O., Rundle, S.D., Spicer, J.I., 2013c. Parent-offspring similarity in the timing of developmental events: an origin of heterochrony? Proc. Roy. Soc. B. 280, 1479.