The maternal and early embryonic transcriptome of the milkweed bug Oncopeltus fasciatus.
BACKGROUND
Most evolutionary developmental biology ("evo-devo") studies of emerging model organisms focus on small numbers of candidate genes cloned individually using degenerate PCR. However, newly available sequencing technologies such as 454 pyrosequencing have recently begun to allow for massive gene discovery in animals without sequenced genomes. Within insects, although large volumes of sequence data are available for holometabolous insects, developmental studies of basally branching hemimetabolous insects typically suffer from low rates of gene discovery.


RESULTS
We used 454 pyrosequencing to sequence over 500 million bases of cDNA from the ovaries and embryos of the milkweed bug Oncopeltus fasciatus, which lacks a sequenced genome. This indirectly developing insect occupies an important phylogenetic position, branching basal to Diptera (including fruit flies) and Hymenoptera (including honeybees), and is an experimentally tractable model for short-germ development. 2,087,410 reads from both normalized and non-normalized cDNA assembled into 21,097 sequences (isotigs) and 112,531 singletons. The assembled sequences fell into 16,617 unique gene models, and included predictions of splicing isoforms, which we examined experimentally. Discovery of new genes plateaued after assembly of ~1.5 million reads, suggesting that we have sequenced nearly all transcripts present in the cDNA sampled. Many transcripts have been assembled at close to full length, and there is a net gain of sequence data for over half of the pre-existing O. fasciatus accessions for developmental genes in GenBank. We identified 10,775 unique genes, including members of all major conserved metazoan signaling pathways and genes involved in several major categories of early developmental processes. We also specifically address the effects of cDNA normalization on gene discovery in de novo transcriptome analyses.


CONCLUSIONS
Our sequencing, assembly and annotation framework provide a simple and effective way to achieve high-throughput gene discovery for organisms lacking a sequenced genome. These data will have applications to the study of the evolution of arthropod genes and genetic pathways, and to the wider evolution, development and genomics communities working with emerging model organisms.[The sequence data from this study have been submitted to GenBank under study accession number SRP002610 (http://www.ncbi.nlm.nih.gov/sra?term=SRP002610). Custom scripts generated are available at http://www.extavourlab.com/protocols/index.html. Seven Additional files are available.].
Department of Organismic and Evolutionary Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.
Ben
B
Ewen-Campen
Nathan
N
Shaner
Kristen A
KA
Panfilio
Yuichiro
Y
Suzuki
Siegfried
S
Roth
Cassandra G
CG
Extavour
D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D020062:Heteroptera / Q000235:genetics*; D019476:Insect Proteins / Q000235:genetics*; D010053:Ovary / Q000378:metabolism
D018076:DNA, Complementary; D019476:Insect Proteins
BMC Genomics. 2008 Jun 30;9:312
18590545
Genome Res. 1998 Mar;8(3):195-202
9521923
Cell. 2003 Jun 27;113(7):853-65
12837244
BMC Genomics. 2010 Mar 16;11:180
20233449
Mol Phylogenet Evol. 2009 Dec;53(3):826-34
19695333
BMC Bioinformatics. 2009 Dec 24;10:447
20034392
Nature. 2008 Oct 30;455(7217):1193-7
18830242
Development. 2000 Sep;127(17):3683-94
10934013
Cold Spring Harb Protoc. 2009 Aug;2009(8):pdb.emo127
20147229
BMC Genomics. 2010 May 26;11:329
20504312
Cold Spring Harb Protoc. 2009 Aug;2009(8):pdb.prot5262
20147238
BMC Genomics. 2010 May 13;11:303
20462456
BMC Genomics. 2010 May 17;11:310
20478048
J Exp Biol. 1966 Jun;44(3):507-22
5962706
BMC Genomics. 2006 May 31;7:130
16737530
Insect Biochem Mol Biol. 2010 Jun;40(6):468-75
20403438
Nucleic Acids Res. 2009 Jan;37(Database issue):D583-7
19028744
Nucleic Acids Res. 2007 Jan;35(Database issue):D61-5
17130148
Bioinformatics. 2011 Apr 1;27(7):919-24
21335611
Dev Cell. 2008 Apr;14(4):605-15
18410735
BMC Genomics. 2007 Apr 25;8:109
17459168
Dev Biol. 2009 Oct 1;334(1):300-10
19580803
BMC Genomics. 2010 Oct 16;11:571
20950480
Dev Biol. 2009 Sep 15;333(2):297-311
19580800
Nat Rev Genet. 2009 Jun;10(6):416-22
19369972
Nat Genet. 2000 May;25(1):25-9
10802651
Gen Comp Endocrinol. 1982 Jan;46(1):42-52
7060934
Insect Biochem Mol Biol. 2003 Dec;33(12):1275-84
14599499
Science. 1987 Dec 18;238(4834):1675-81
3686007
Genome Biol. 2010;11(3):R25
20196867
Nat Rev Genet. 2007 Apr;8(4):311-9
17339879
Trends Ecol Evol. 2008 Oct;23(10):578-87
18715673
Genome Res. 2008 May;18(5):763-70
18212088
Nucleic Acids Res. 2009 Jan;37(Database issue):D555-9
18948289
Genome Res. 1998 Mar;8(3):186-94
9521922
Nucleic Acids Res. 2010 Jan;38(Database issue):D142-8
19843607
Trends Genet. 2008 Jul;24(7):353-60
18514356
Mol Reprod Dev. 2010 Jan;77(1):3-18
19790240
Trends Genet. 2007 Jan;23(1):16-20
17097187
BMC Genomics. 2010 Apr 19;11:251
20403183
BMC Genomics. 2008 Nov 15;9:542
19014603
BMC Genomics. 2009 May 12;10:219
19435504
Nat Rev Genet. 2002 Nov;3(11):838-49
12415314
Bioinformatics. 2005 Sep 15;21(18):3674-6
16081474
Biotechniques. 2001 Apr;30(4):892-7
11314272
Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14121-6
14614147
BMC Genomics. 2009 May 19;10:234
19454017
J Fish Biol. 2008 Jun 28;72(9):2354-2376
19936325
Cold Spring Harb Protoc. 2009 Aug;2009(8):pdb.prot5261
20147237
BMC Plant Biol. 2010 May 25;10:96
20500869
Insect Biochem Mol Biol. 2009 May-Jun;39(5-6):403-13
19364528
Dev Biol. 2004 Jul 15;271(2):306-21
15223336
Genome Res. 1999 Sep;9(9):868-77
10508846
BMC Genomics. 2009 Nov 24;10:555
19930683
Bioessays. 1995 May;17(5):451-5
7786291
PLoS Biol. 2010 Feb 23;8(2):e1000313
20186266
BMC Genomics. 2009 Jul 31;10:345
19646242
BMC Biotechnol. 2006 Jun 16;6:27
16776846
Genome Res. 1998 Mar;8(3):175-85
9521921
Dev Genes Evol. 2009 Dec;219(11-12):535-44
20127251
Appl Environ Microbiol. 2010 Jul;76(13):4521-9
20435767
BMC Bioinformatics. 2009 Dec 15;10:421
20003500
Mol Ecol. 2008 Apr;17(7):1636-47
18266620
Genome Res. 2010 Oct;20(10):1432-40
20693479
Mol Biosyst. 2008 Mar;4(3):205-12
18437263