Table 1.
Summary of RNA-sequencing statistics and assembly results.
Fig 1.
General composition of P. nigriventer venom gland transcriptome sequenced by NGS.
Unique sequences were searched against UniProt database and classified as ‘putative venom components’ or ‘cellular function proteins’. Left graph shows relative proportions expressed as percentages of unique sequences. Most of the unique sequences (66%) did not match any sequence from UniProt database (e-value < 1e-5). Right graph shows relative proportions expressed as percentages of abundance (FPKM) of transcripts belonging to each category.
Fig 2.
Diversity and abundance of putative venom components from P. nigriventer venom gland transcriptome sequenced by NGS.
Unique sequences were searched against UniProt database and classified into known toxin subfamilies. Left graph shows relative proportions expressed as percentages of unique entries. Right graph shows relative proportions expressed as percentages of abundance (FPKM) of transcripts belonging to each subfamily.
Fig 3.
Relative abundance, expressed as FPKM, of subfamilies of the putative venom components found in the NGS analysis of P. nigriventer venom glands.
A) Cysteine-rich peptide toxins and B) Other families of venom components. Unique sequences were classified into known toxin subfamilies according to UniProt database. Bars represent the sum of FPKM for each transcript belonging to the described groups.
Fig 4.
Conventional sequencing annotation.
Annotation distribution of unique sequences (A) and ESTs (B) against UniProt. C) Distribution of EST annotation per Contig size (number of ESTs used to generate contig sequences). Numbers inside the bars are the raw numbers of ESTs per annotation class.
Fig 5.
P. nigriventer venom composition analyzed by MudPIT proteomic technique.
Left graph shows the proportion of components detected by venom analysis. The peptide sequences found were searched against the NGS transcriptomic database and classified according to their UniProt annotation as ‘putative venom components’ or ‘cellular functions’. Eighteen percent of the retrieved proteins did not match any sequence from the database. Right graph shows putative venom components divided into subfamilies of putative toxins. The proportion of each category was calculated by the sum of the emPAI.
Fig 6.
Venn diagram representing the total number of unique cysteine-rich peptide toxins found in P. nigriventer venom by each technique used.
Table 2.
Classification of the cysteine-rich peptide toxins identified, according to their cysteine frameworks.
Fig 7.
Sequence alignments of cysteine-rich peptide toxin precursors from group I.
Alignment was performed with MUSCLE, Signal peptide is highlighted in yellow, propeptide is highlighted in green and processing quadruplet motif (PQM) is highlighted in cyan. Conserved cysteines are marked in blue. Percentage of identity (ID%) with the reference protein was calculated using the tool EMBOSS Stretcher for pairwise sequence alignment using either the complete or processed mature sequence. U23-cntx-Pn1a (UniProt: P84015), PRTx26An0C3 (UniProt: P86418), U13-cntx-Pn1a (UniProt: P83894), U14-cntx-Pn1a (UniProt: P83998) and U5-cntx-Pn1a (UniProt: P29426), from P. nigriventer, were used as references.
Fig 8.
Sequence alignments of cysteine-rich peptide toxin precursors from group II.
Alignment was performed with MUSCLE, Signal peptide is highlighted in yellow, propeptide is highlighted in green and processing quadruplet motif (PQM) is highlighted in cyan. Conserved cysteines are marked in blue. Percentage of identity (ID%) with the reference protein was calculated using the tool EMBOSS Stretcher for pairwise sequence alignment using either the complete or processed mature sequence. CSTX-10 (UniProt: B3EWT0), from C. salei spider; U7-cntx-Pn1a (UniProt: P81791), U10-cntx-Pn1a (UniProt: P0C2S9), U6-cntx-Pn1a (UniProt: P81793), ω-cntx-Pn1a (UniProt: O76201), κ-cntx-Pn1a (UniProt: O76200), U9-cntx-Pn1a (UniProt: P0C2S6), from P. nigriventer spider; and U1-cntx-Pk1a (UniProt: P83895), from P. keyserlingi spider were used as references.
Fig 9.
Sequence alignments of cysteine-rich peptide toxin precursors from groups III-V.
Alignment was performed with MUSCLE, Signal peptide is highlighted in yellow, propeptide is highlighted in green and processing quadruplet motif (PQM) is highlighted in cyan. Conserved cysteines are marked in blue. Percentage of identity (ID%) with the reference protein was calculated using the tool EMBOSS Stretcher for pairwise sequence alignment using either the complete or processed mature sequence. A) Group III alignment, using U20-lctx-Ls1d (UniProt: B6DCY1), from L. singoriensis spider, as reference. B) Group IV alignment, using U19-cntx-Pn1a (UniProt: P83997), from P. nigriventer spider, as reference. C) Group V alignment, using U2-cntx-Pn1a (UniProt: P29423), δ-cntx-Pn2c (UniProt: O76199), δ-cntx-Pn2a (UniProt: P29425), δ-cntx-Pn1a (UniProt: P59368), γ-cntx-Pn1a (UniProt: P59367), U1-cntx-Pn1a (UniProt: P61229), from P. nigriventer spider, and U4-cntx-Pk1a (UniProt: P83896), from P. keyserlingi spider, as references.
Fig 10.
Sequence alignments of cysteine-rich peptide toxin precursors from groups VI-IX.
Alignment was performed with MUSCLE, Signal peptide is highlighted in yellow, propeptide is highlighted in green and processing quadruplet motif (PQM) is highlighted in cyan. Conserved cysteines are marked in blue. Percentage of identity (ID%) with the reference protein was calculated using the tool EMBOSS Stretcher for pairwise sequence alignment using either the complete or processed mature sequence. A) Group VI alignment, using U9-agtx-Ao1a (UniProt: Q5Y4U3), from A. orientalis spider, as reference. B) Group VII alignment, using U12-cntx-Pn1a (UniProt: P0C2S8), ω-cntx-Pn1a (UniProt: O76201), U11-cntx-Pn1a (UniProt: P0C2S7), from P. nigriventer spider, as references. C) Group VIII alignment, using ω-cntx-Pn3a (UniProt: P81790), μ-cntx-Pn1a (UniProt: P17727), U20-cntx-Pn1a (UniProt: P84093) from P. nigriventer spider, as references. D) Group IX alignment, using ω-agtx-1A (UniProt: P15969), from A. aperta spider, as reference.
Table 3.
List of the main families of molecules for the venom components found in NGS transcriptomic analysis.