The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins

Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake. Our assembly has a scaffold N50 of 223.35 Mb, with 19 scaffolds containing 95% of the genome. Of the 23,248 predicted protein-coding genes, 12,346 venom-gland-expressed genes constitute the ‘venom-ome’ and this included 139 genes from 33 toxin families. Among the 139 toxin genes were 19 ‘venom-ome-specific toxins’ (VSTs) that showed venom-gland-specific expression, and these probably encode the minimal core venom effector proteins. Synthetic venom reconstituted through recombinant VST expression will aid in the rapid development of safe and effective synthetic antivenom. Additionally, our genome could serve as a reference for snake genomes, support evolutionary studies and enable venom-driven drug discovery.


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Nature GeNetics components is poorly understood. This is further exacerbated by the lack of access to antivenom and its high cost in many developing countries 14 . Although several alternative approaches have been proposed, large animal-based antivenom production using extracted snake venom as the antigen continues to be the standard practice [15][16][17][18] .
High-quality genomes of venomous snakes, combined with transcriptomics, will enable generation of a comprehensive catalog of venom-gland-specific toxin genes that can be used for the development of synthetic antivenom of defined composition using recombinant technologies. Thus far, only a few snake genomes have been published. A majority of these were generated primarily using short-read sequencing resulting in highly fragmented assemblies, thus limiting their utility for creating a complete catalog of venomrelevant toxin genes [19][20][21][22][23][24][25] . The 'big four' medically important snakes from India, including N. naja, are no exception and have not been well characterized at either the genome or transcriptome level. Only nine N. naja toxin gene sequences and 38 toxins, some of which probably represent the same gene, have been reported using mass spectrometry 26 .
In this study, using a number of genomic technologies, we have generated a de novo near-chromosome level reference genome assembly of N. naja, the Indian cobra. This high-quality genome allowed us to study various aspects of snake venom biology, including venom gene genomic organization, genetic variability, evolution and expression of key venom genes. Our integrated genome-transcriptome analysis identified a minimal set of 19 VST genes that constitute the core venom toxins. Targeting these core toxins should lead to the development of a safe and effective humanized antivenom.

Results
Near-chromosomal de novo genome assembly. Using flow cytometry, we estimated the size of the Indian cobra haploid genome at 1.48-1.77 Gb (Extended Data Fig. 1). Cytogenetic analysis revealed a diploid karyotype of 2n = 38, comprising seven pairs of macrochromosomes (MACs), one pair of sex chromosomes (ZZ male or ZW female) and 11 pairs of microchromosomes (MICs), consistent with a previous report 27 (Extended Data Fig. 2).
DNA from an adult male Indian cobra (NN01) was used to generate long-read (PacBio and Oxford Nanopore Technologies (ONT)), short-read (Illumina), Chicago 28 , Hi-C 29 and optical mapping (Bionano Genomics (BNG)) data (Supplementary Table 1a,b). Additionally, we generated linked-read 10x Genomics, BNG and short-read Illumina sequence data for a female animal (NN05; Supplementary Note and Supplementary Table 1a,b). Our sequential assembly approach ( Fig. 1 and Supplementary Note) resulted in a 1.79-Gb Nana_v5 genome with a scaffold N50 of 223. 35 Mb and a BUSCO 30 genome completeness score of 94.3% (Table 1 and  Supplementary Table 2a-c).
To assign scaffolds from Nana_v5 to chromosomes, we used complementary DNA (cDNA) chromosome marker sequences from a colubrid, the Japanese rat snake Elaphe quadrivirgata 31 (Fig. 1e), synteny information from Anolis carolinensis (the green anole lizard genome 32 ) and single-chromosome sequencing data (SChromseq; Fig. 1f, Supplementary Table 3a-c and Supplementary Note). We also generated a hybrid 10x BNG genome assembly to identify a 52.1-Mb female-specific W chromosome-linked scaffold (Supplementary Table 3d and Supplementary Note).
Comparison of the N. naja de novo genome assembly to the human 33 and goat 34 genomes showed that the scaffold N50 of the N. naja genome was 2.5× (87.27 versus 223.35 Mb) and 3.3× (67.79 versus 223.35 Mb) greater than the goat and human genomes previously assembled using the involved, expensive, time-consuming physical and cytogenetic maps developed over a decade or more ( Table 2). Compared with the king cobra genome 19 , also an elapid, the Indian cobra genome contained far fewer scaffolds (296,399 versus 1,897, respectively), and had 929-fold better contiguity (scaffold N50 of 0.24 versus 223. 35 Mb, respectively). Also, compared to the 7,034 scaffolds and 179.89-Mb scaffold N50 reported recently for the prairie rattlesnake genome (Crotalus viridis) 35 , the Indian cobra genome had a higher scaffold N50 and fewer scaffolds ( Table 2).
Genome features. The average DNA base (GC) content of the N. naja genome was 40.46%. While MACs, representing 88% of the genome, had a GC content of 39.83%, that of MICs was 43.50% despite their containing only 12% of the genome (Welch's twosample t-test, two-sided P < 0.0001; Fig. 2a). Analysis of the repeat content in the Indian cobra genome in relation to other squamate reptile genomes revealed that 43.22% of the genome was repetitive (~760 Mb; Fig. 2a, Supplementary Table 4a,b, Extended Data Fig. 3 and Supplementary Note).
Whole-genome synteny comparison between the Indian cobra and prairie rattlesnake genomes revealed large syntenic blocks between the macro-, micro-and Z-chromosomes (Fig. 2b,c). We observed several fusion/fission events consistent with the difference in chromosome number between these two genomes. In particular, chromosome 4 of the Indian cobra shared syntenic regions with rattlesnake chromosomes 3 and 5, indicating a possible fusion event. In contrast, Indian cobra chromosomes 5 and 6 were syntenic to rattlesnake chromosome 5, indicating a possible fission event (Fig.  2b,c). Comparison of the Indian cobra genome to that of the more distantly related green anole lizard also showed regions of synteny and chromosomal rearrangements (Fig. 2c). Lizard chromosome 2 contained syntenic regions corresponding to chromosomes 4, 5 and 6 in the Indian cobra genome (Fig. 2c). Our synteny analysis also showed that the lizard chromosome 6 is homologous to the Z-chromosome of the Indian cobra ( Fig. 2c and Supplementary  Table 3b) 36 . Despite an estimated divergence time of ~280 Ma between snake and chicken (Gallus gallus), we observed synteny between several macro-and microchromosomes. Several regions of chicken chromosomes 1 and 2 showed syntenic blocks across Indian cobra macro-, micro-and Z-chromosomes (Fig. 2c). This indicated large-scale changes in macro-and microchromosome organization between squamate and avian genomes during evolution 31,37 .
Gene prediction and annotation. We used the MAKER pipeline 38 to annotate the genome using protein homology information, in combination with gene expression data from 14 different tissues (n = 26 samples; Figs. 2d and 3, Supplementary Table 1a and Supplementary Note). Overall, we predicted 23,248 protein-coding genes and 31,447 transcripts that included alternatively spliced products encoding 31,036 predicted proteins (Fig. 2d). Of the 23,248 genes, we found 22,116 (95%) on the 19 largest scaffolds corresponding to the numbered chromosomes. A total of 26,216 of the 31,036 predicted proteins (84.4%) contained a canonical start and stop codon. We identified 3,265 of 26,216 proteins (~12.5%) with an N-terminal secretion signal sequence, a feature important for venom gland toxin secretion (Supplementary Table 5). We performed extensive functional annotation of the 31,036 predicted proteins and found that 17,019 (54.8%) had a corresponding ortholog in either the Human Gene Nomenclature Committee database, NCBI's nonredundant database or the TrEMBL (https://www.ebi.ac.uk/uniprot) database (Supplementary Tables 5 and 7b and Supplementary Note). Comparison of our annotated proteome to that of the king cobra 19 , prairie rattlesnake 35 and green anole lizard genomes 32 identified 26,323, 25,505 and 11,820 orthologs in those genomes, respectively.
We comprehensively annotated venom-gland-relevant genes by combining the predicted gene models with long-read sequencing data (Supplementary Table 6a and Supplementary Note), toxin gene hidden Markov models (HMM) and manual curation to identify 139 toxin genes from 33 gene families 39 that included 19 three-finger toxins (3FTxs), 8 snake venom metalloproteinases (SVMPs) and 6 cysteine (Cys)-rich secretory venom proteins Articles Nature GeNetics (CRISPs) (Supplementary Table 6b). Near-chromosomal assembly also allowed us to assess the genomic organization of gene families that encoded enzymatic and non-enzymatic toxin proteins involved in venom gland function. In the N. naja genome, 16 major toxin gene families were organized on MACs (Fig. 4a and Supplementary  Table 6c). This is in contrast to the genomes of viperids C. viridis (prairie rattlesnake) and Protobothrops flavoviridis (Amami habu), where a majority of the venom gland genes were found on MICs 23  Long-read (PacBio and ONT) and short-read (lllumina) data (a) were used to build contigs that were then combined with Chicago 28 chromatin interaction data (b) to generate scaffolds. c, Scaffolds from BNG optical mapping de novo assembly were combined with those from the previous step to generate super-scaffolds. d, Hi-C sequencing data were used to refine the assembly. e, Electronic fluorescence in situ hybridization (eFISH) was performed using cDNA FISH marker sequences from the Japanese rat snake, E. quadrivirgata. f, SChrom-seq data were used to assign scaffolds to chromosomes.

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Of the 19 full-length 3FTx genes identified in the genome, 14 were clustered within a 6.3-Mb region on chromosome 3 (Fig.  4b). One 3FTx gene (Nana001KS) was located on chromosome 4 and the remaining four were found on an unassigned scaffold, ScVE01q_1072 (Supplementary Table 6c). Additionally, we identified 10 3FTx pseudogenes that lacked parts of the coding region and were not expressed. The second largest toxin gene family encoded by the N. naja genome consisted of eight SVMPs that were clustered on a MIC 1 (Fig. 4c). A cluster of six CRISPs were found on chromosome 1 (Fig. 4d). Other toxin genes, including natriuretic peptide, C-type lectin, snake venom serine proteinase (SVSP), Kunitz and venom complement-activating gene families, were found to be distributed across the 19 chromosomes while two group I Phospholipase A2 (PLA2) genes and one cobra venom factor (CVF) gene were located on an unassigned scaffold (ScVE01q_344; Supplementary Table 6c). Comparisons of venom gland genes between the C. viridis genome and that of the Indian cobra identified 15 toxin gene families that were unique to the Indian cobra. This included phospholipase B-like toxins and cathlecidins 40,41 (Supplementary Table  6d). Assessment of the 139 Indian cobra venom gland toxin genes for orthologs in the king cobra showed that, while 96 genes had a match, 43 did not (Supplementary Table 6e). Although some of the 43 toxins are likely to be unique to the Indian cobra, a majority were not annotated in the king cobra genome probably due to its highly fragmented assembly (Table 2).
Synteny comparisons of the major toxin gene families (3FTx, SVMP and CRISP) in genomes of the Indian cobra, prairie rattlesnake and green anole lizard revealed multiple duplication events in each family involving a paralog of non-venomous origin, leading to co-option/recruitment and expression of the duplicated gene in the venom gland (Fig. 4e,f and Extended Data Fig. 4 Fig. 4g and Supplementary Note). These 19 VST genes are likely to encode the core venom effector toxin proteins, consisting of six neurotoxins, one cytotoxin, one cardiotoxin, one muscarinic toxin, six SVMPs, nerve growth factor (NGF-β), two venom Kunitz serine proteases and a CRISP (Supplementary Table 6b, column L). Additionally, we confirmed the presence of 16 of the 19 VSTs at the protein level using mass spectrometry (Supplementary Table 8).
Functionally diverse 3FTxs. Three-finger toxins are short polypeptides (60-90 amino acids) that belong to a superfamily of nonenzymatic proteins found primarily in elapid snakes 43 . These small proteins are known to primarily target neuronal receptors including nicotinic acetylcholine receptors (nAChRs), muscarinic acetylcholine receptors, calcium channels and other proteins 43 . Structurally, they fold into an outstretched, three-finger-like structure where each finger contains a β-hairpin loop that extends from a disulfide bond-stabilized hydrophobic core 44,45 . 3FTxs typically contain four conserved disulfide bridges, with some containing a fifth disulfide bridge 46 . Functionally, 3FTxs are broadly classified as neurotoxins, cytotoxins, cardiotoxins and anticoagulants.
While expression of all 19 annotated N. naja 3FTxs was detected in the venom gland, 9 were specific to the venom gland. Of these 19 3FTxs, 14 were classified as conventional 3FTxs with 8 conserved Cys residues, while 4 3FTxs contained 10 Cys residues. Homologybased assessment enabled classification of the 3FTxs into seven neurotoxins, six cytotoxins, four cardiotoxins, one muscarinic toxin and one anticoagulant (Supplementary Table 9). The neurotoxins included two type I short-chain neurotoxins (Nana002KS and Nana005KS), known to interact with muscle nAChR, one type II long-chain neurotoxin (Nana012KS), known to target muscle and neuronal nAChRs, and three putative type III weak neurotoxins containing 10 conserved cysteines (Nana001KS, Nana003KS and Nana004KS), a characteristic feature of both non-conventional and prey-specific 3FTxs 47,48 . Nana018KS was structurally similar to   a, Circos plot of the reference genome assembly (NN01, a male adult (n = 1)) representing N. naja chromosomes (outermost track) from the Nana_v5 assembly, repeat content, gene density and GC content (%). Regions of the genome with GC content higher than average (40.46%) are shown in blue. Regions within the gene density of more than 10 genes are shown as red spikes, while those with 5 to 10 genes are indicated by yellow spikes. Green spikes represent regions with fewer than five genes. The average repeat content is indicated by the red line. All data were plotted in 100-kb windows. The female-specific W-linked scaffold obtained using NN05 DNA is shown on the right. b, Chromosome painting depicting synteny between Indian cobra and rattlesnake genomes. c, Dot-plots showing synteny of the Indian cobra genome with the prairie rattlesnake, chicken or green anole lizard genomes. d, Bar plot of the number of predicted genes and corresponding transcripts observed in Nana_v5. Dashed and solid lines denote average number of genes and transcripts detected in each chromosome along 100-Mb windows, respectively. MICs were combined into one group. Unp, unplaced scaffolds (n = 1,878) containing predicted genes.

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haditoxin from the king cobra and known to block α7-nAChRs 49 . Further assessment by structural modeling classified the Indian cobra 3FTxs into four groups (Fig. 5, Extended Data Fig. 6 and Supplementary Table 10). The aromatic residue (Tyr25 or Phe27), crucial for proper folding 50 and stability of the antiparallel β-sheet structure, was found to be conserved in all 19 3FTxs (Fig. 5a). Three of the four 10-Cys-residue-containing 3FTxs were non-conventional 3FTxs containing a pair of additional cysteines in loop I,

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Nature GeNetics resulting in a longer N terminus loop that could potentially also play a role in stabilizing this loop and contribute to toxin function 47 (Fig. 5a,b). The other 10-Cys 3FTx was a long-chain neurotoxin that contained a pair of additional cysteines in loop II (Nana012KS). The charged amino acid residue Arg39, which typically stabilizes native toxin conformation by forming a salt bridge with the C terminus 51 ,  Chr 3 N a n a 0 0 2 K S N a n a 0 0 3 K S N a n a 0 0 4 K S N a n a 0 0 5 K S N a n a 0 0 6 K S N a n a 0 2 3 K S N a n a 0 1 9 K S N a n a 0 0 7 K S N a n a 0 0 8 K S N a n a 0 0 9 K S N a n a 2 8 3 2 4 N a n a 2 8 3 2 5 N a n a 2 8 3 2 6 N a n a 2 8 3 2 7 N a n a 2 8 3 2 8 N a n a 2 8 3 2 9 N a n a 2 8 3 3 2 N a n a 2 8 3 3 4 N a n a 0 2 8 6 1 N a n a 0 2 8 6 2 N a n a 0 2 8 6 3 N a n a 0 2 8 6 5 N a n a 0 2 8 6 6 N a n a 0 2 8 6 8 N a n a 0 2 6 K S N a n a 0 2 7 K S N a n a 0 2 0 K S N a n a 0 1 0 K S N a n a 0 2 5 K S N a n a 0 1 1 K S N a n a 0 1 2 K S N a n a 0 2 1 K S N a n a 0 1 3 K S N a n a 0 1 4 K S N a n a 0 1 5 K S N a n a 0 2 4 K S N a n a 0 2 8 K S

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Nature GeNetics Nana002KS Dashed circles highlight the additional disulfide bonds in Nana001KS, Nana012KS and the unpaired Cys in Nana005KS. f, Superimposition of ribbon models of Nana001KS, Nana003KS and Nana010KS highlighting the differences in loop length and conformation between the distinct classes of 3FTx found in the Indian cobra genome. g, Analysis of evolutionary rates on 3FTx venom genes and their non-venom paralogs. K A and K S values were calculated according to the Nei-Gojobori method. K A and K S with values <1 were not included in further analysis. SNTX, short neurotoxin; LNTX, long neurotoxin; MTLP, muscarinic toxin-like; CTX, cardiotoxin or cytotoxin; Nonc, non-conventional toxin; WTX, weak neurotoxin. SV, snake venom; NV, non-venom.

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was conserved in all identified 3FTxs except in Nana012KS, where it was replaced by a leucine residue (Fig. 5a). The homology model of this protein indicated that Leu39 may exhibit van der Waals interactions and form part of a hydrophobic core comprising Ile35, Phe4, Thr22 and Arg68 (Fig. 5c). Two of the cytotoxins, Nana008KS and Nana010KS, had shorter loops I and III compared to the other 3FTxs (Fig. 5d).
Of note, Nana005KS contained nine Cys residues and such unusual 3FTxs have been found in only two other elapids, Micrurus lemniscatus and Micrurus altirostris 52 . This free Cys residue at position 16 that precedes the conserved second Cys in loop I (Fig. 5b) probably facilitates the formation of covalent homo-or heterodimeric 3FTxs (Supplementary Table 10). Nana005KS was closely related to the short-chain 3FTx Nana002KS and contained a majority of the residues required for activity against muscle nAChRs. For instance, the presence of the positively charged residues Lys25, Lys26 and Arg32 indicates that these toxins might be crucial for envenomation in mammals. In particular, the guanidyl group of Arg32 mimics acetylcholine forming a cation-π interaction with α-α and α-δ interfaces in the nAChRs 53 .
Acquired mutations affecting the neurotoxin binding site of nAChR have rendered certain species, such as the Egyptian mongoose (Herpestes ichneumon), immune to snake venom [54][55][56] . Comparison of N. naja nAChR sequence and H. ichneumon nAChR and other representative mammalian species showed that the N. naja nAChR carries a key p.Phe189Asn alteration in the α-neurotoxin site, known to result in decreased sensitivity to shortand long-chain neurotoxins 55,56 (Extended Data Fig. 7).
In the final group of 3FTxs, Nana013KS was structurally similar to AncTx-1 (ref. 57 ), a synthetic ancestral toxin known to interact with β-adrenergic G-protein-coupled receptors. The 3FTx encoded by Nana006KS was found to be a close homolog of ringhalexin, an inhibitor of the extrinsic tenase coagulation complex, from Hemachatus haemachatus, the African ringhals cobra 58 (Fig. 5e and Supplementary Table 9). Additionally, we found Nana017KS to be 97% similar to a recently reported Naja atra (μ-EPTX-Na1a) 59 Nav1.8 voltage-gated sodium channel inhibitor.
To understand the consequences of genetic variation in the 3FTx family, we assessed the rate of evolution of the major toxin families by computing the numbers of synonymous (K S ) and non-synonymous (K A ) nucleotide substitutions per site for each pairing of toxin gene and its non-toxin paralog. The K A /K S substitution ratio for the 3FTx toxin genes was 2.034 (±0.818), while that of the non-toxin paralogs (Ly-6/UPAR domain-containing genes) was 0.894 (±0.103; Fig. 5g). The observed high K A /K S ratio (>1) indicated diversifying selection leading to rapid divergence and functional diversification of the venom-gland-specific 3FTx genes.
Indian cobra SVMP, CRISPs, PLA2, CVF and growth factor genes. We detected six venom-gland-specific SVMPs that belonged to the P-III class of metalloproteinases (with metalloproteinase/disintegrin/ Cys-rich domains) known to be involved in the induction of hemorrhage, inflammation, apoptosis, prothrombin activation and inhibition of platelet aggregation 60 . The Indian cobra SVMPs were found to evolve less rapidly than 3FTx genes, because the K A /K S ratio for the venom-gland-specific SVMPs was 1.070 (±0.137) when compared to 0.998 (±0.049) observed for metalloprotease domain-containing paralogs (Extended Data Fig. 8). The Indian cobra SVMPs formed a separate cluster compared with the viperid SVMPs (Extended Data Fig. 9). In agreement with this, a seventh Cys residue in domain M12, involved in the disulfide bond exchange for autolysis during secretion or formation of the biologically active disintegrin/Cys-rich domain typical of Viperidae SVMP-PIIIs 60 , was absent (Extended Data Fig. 9).
We also detected six CRISPs in the venom gland transcriptome that were highly conserved across different snake species (Supplementary Fig. 1). Venom CRISPs have a wide variety of biological effects, including blockade of K + and/or Ca 2+ currents in neurons and blockage of vascular smooth muscle contraction 61 . Two of the five CRISPs were homologous to venom CRISPs from the Chinese cobra, Naja atra (natrin), and the monocled cobra, Naja kaouthia (UniProtKB: Q7T1K6). Expression of the N. naja natrin homolog (Nana02866) was venom gland specific, and it probably functions as a Kv1.3 potassium channel blocker 62 .
Additionally, two group I secretory acidic PLA2 genes (Nana39244 and Nana39246) that were highly expressed in the venom and salivary glands showed a high degree of similarity to other elapid venom PLA2s, and contained the characteristic calcium-binding (XCGXGG) and catalytic (DXCCXXHD) motifs 63 ( Supplementary Fig. 2 and Supplementary Note).
In addition to the major elapid toxin families described above, we detected transcripts from other toxin gene families including hyaluronidases, phospholipase B-like genes, cathelicidins, ohanin (known to induce hypolocomotion and hyperalgaesia) 64 and 5′ nucleotidases (Supplementary Note). We also detected l-amino acid oxidase (LAAO) (Nana07858), which is involved in platelet aggregation, edema and hemorrhage. Furthermore, we identified two full-length c-type natriuretic peptide genes, Nana20849 and Nana20852, in the venom-ome. Of the three Kunitz serine protease inhibitors detected in the venom-ome, two were VST gene products (Nana13192 and Nana13193) and these probably function to inhibit serine proteases acting in the hemostatic system 65 . In addition, two full-length cystatin genes, Nana15538 and Nana35841, were also found expressed in the venom-ome.
Cobra venom factor is a non-lethal protein that resembles the complement C3 protein in structure and function 66 . Previously, the complete structure of one CVF gene from N. Naja has been reported 67 . In the present study, we identified three CVF genes (Nana10828, Nana38416 and Nana10826) in the N. naja genome. Nana38416 and Nana10828 contained 40 exons and spanned ~118 and ~75 kb, respectively, on chromosome 2. Isoform sequencing (Iso-seq) data confirmed the expression of the full-length transcripts corresponding to Nana10828 and Nana38416. Though the 5′ genomic structure of Nana10826 was not fully resolved in the currently assembly, the expression of the full-length transcript of Nana10826 was confirmed by iso-seq. Protein sequence alignment showed that Nana38416 was 96% similar to CVF (UniProtKB: Q91132) from N. kaouthia, while Nana10828 was 99% similar to a previously characterized N. naja C3 complement component protein 68 .

Discussion
Much of our current understanding of snake venom is based on proteomic studies that have provided only a partial picture of its components [69][70][71] . A comprehensive catalog of venom proteins, their expression and coding sequence is fundamental to developing a safe and effective antivenom 15,72 . Also, such a detailed catalog of venom components will be valuable for drug candidate prospecting.
Using next-generation sequencing in combination with emerging genomic technologies, we have generated a de novo high-quality N. naja reference genome 73 . The near-chromosomal assembly revealed regions of synteny between reptilian and avian genomes, consistent with their evolutionary trajectories. The high contiguity of our genome enabled visualization of the striking differences in venom gene organization between elapid and viperid snake Overall, we found evidence for expression of 12,346 genes that constitute the venom-ome, and this included 139 toxin genes of which 19 were designated as VSTs based on their venom-glandspecific expression. Additionally, well-known modulators of venom function such as CVF, coagulation factors, protein disulfide isomerases, natriuretic peptides, hyaluronidases, PLA2s, phospholipase B-like genes, LAAO, vascular endothelial growth factor (VEGF) and 5′ nucleotidases were also found to be highly expressed in the venom gland. It is likely that these genes, together with the 19 VSTs, form the core toxic effector components of the venom and induce a wide range of symptoms including cardiovascular dysfunction, muscular paralysis, nausea, blurred vision and systemic effects such as hemorrhage 6 (Fig. 6). We propose that neutralization of these core venom effectors using antibodies would be an effective therapeutic strategy. Furthermore, given the variation in venom composition, cataloging the venom gland gene repertoire and its variation (Extended Data Fig. 10 and Supplementary Note), both within and across different snake species, will be important for developing a broadly efficacious antivenom 74,75 .
Snake antivenom is made by immunizing large mammals, such as horses, with extracted snake venom as antigen 12 . Such horsederived antibodies show variation in efficacy due to expected differences in the horse antibody response following antigen challenge 6,13 . Moreover, the heterologous nature of the antibodies leads to adverse treatment-related side effects 6,13 . Starting with a complete catalog of VSTs, synthetic venom of a defined composition can be generated using recombinant protein expression technologies 76 . Such a cocktail of recombinant core venom VST proteins can be used to raise

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specific antibodies in horses, tested for neutralizing activity, rapidly cloned/synthesized and humanized to produce the next generation of antivenoms 14,16,77 . Alternatively, recombinant venom proteins can be used as baits against antibody phage libraries to obtain toxinneutralizing, activity-tested humanized synthetic antibodies 16,72,78 .
Genome-guided recombinantly produced venom proteins can be also used to characterize and improve existing horse-derived antivenom. In this paradigm, antibodies can be raised against recombinant venom toxins that are non-immunogenic in horses and used to supplement current antivenom cocktails to improve their efficacy. Alternatively, single-cell sequencing of B cell repertoires from horses immunized with extracted or synthetic venom can be performed to identify putative neutralizing antibodies 79 . This information can be used to rapidly synthesize, humanize and identify toxin-neutralizing antibodies for the generation of synthetic antivenom.
As more high-quality snake genomes are completed and venom gland VSTs are cataloged, synthetic antibodies directed against key species-specific toxins identified from such genome initiatives can be combined to create potent broad-spectrum antivenom 80 . Furthermore, antibodies targeting different epitopes on recombinant toxins can be developed using phage display. These recombinant toxin-directed antibodies can be combined to generate an antivenom that is likely to be more efficacious [81][82][83][84] .
Primary cultures of venom gland cells 85  We believe the Indian cobra reference genome and the analysis presented here will facilitate innovations in antivenom development. The genome and the associated predicted proteome will serve as a powerful platform for evolutionary studies of venomous organisms. More importantly, the comprehensive catalog of the venom proteins presented here should enable drug development, in particular to treat hypertension, pain and other disorders [86][87][88] .

Online content
Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41588-019-0559-8.

Methods
Karyotyping. Briefly, blood from a female animal (NN03) was collected in a sodium-heparin vacutainer (Becton-Dickinson) and used for short-term (72-h) lymphocyte cultures as previously described 89 . We used phytohemagglutinin (PHA from Phaseolus vulgaris, 20 μg ml -1 , Sigma Aldrich) as the mitogen. Additionally, we established primary fibroblast cultures under sterile conditions using small pieces (0.5 mm 2 ) of ovarian tissue from NN03. The fibroblasts were incubated in MEM alpha containing nucleosides and GlutaMax (Thermo Fisher), supplemented with 20% fetal bovine serum (Atlanta Biologicals) and antibiotic-antimycotic (Thermo Fisher) at 30 °C with 5% CO 2 . Metaphase chromosomes were obtained from both lymphocyte and fibroblast cultures by arresting cells with demecolcine (KaryoMax, Thermo Fisher; final concentration, 0.1 μg ml -1 ), followed by hypotonic treatment with Optimal Hypotonic Solution (Rainbow Scientific) and fixation in methanol/acetic acid (3/1). Metaphase spreads were prepared on precleaned wet glass slides at room temperature. Chromosomes were stained with 5% Giemsa (karyoMax, Thermo Fisher) in GURR buffer (Gibco). At least 30 metaphase spreads were captured and analyzed for karyotyping using an Axioplan2 microscope (Zeiss) and Ikaros (MetaSystems) software.
Flow cytometry-based genome size estimation. We estimated genome size by flow cytometry using a previously described protocol 90 . Whole blood cells of the Indian cobra (NN03) were cultured for 4 d in a medium consisting of MEM alpha with nucleosides and GlutaMax (Thermo Fisher), 30% fetal bovine serum (Atlanta Biologicals) and 1% PHA (1 mg ml -1 , Sigma Aldrich) at 30 °C. Blood cells were washed six times in sterile water and then fixed in cold ethanol. Horse (Equus caballus) lymphocytes from a female were separated from erythrocytes with a Lymphoprep (Stemcell Technologies) before being cleaned and fixed in the same manner as the Indian cobra cells. Next, both cell types were diluted to a concentration of 1 × 10 6 cells ml -1 and stained with propidium iodide (PI, Thermo Fisher). After staining, the Indian cobra and horse cells were mixed 1/1 and analyzed on a Becton-Dickinson Accuri C6 personal flow cytometer. Two peaks were observed based on the amount of PI absorbed by the cells of either species, and genome size was estimated using the formula: genome size (cobra) = PI (cobra)/PI (horse) × genome size (horse), where PI (cobra) denotes the median amount of PI absorbed by cobra cells, PI (horse) denotes the median amount of PI absorbed by horse cells, and genome size (horse) is the expected size of the horse genome of 2.5 Gb (EquCab3, GCA_002863925.1) 91 .

Samples and nucleic acid preps.
A total of six animals-two from Kerala, India (NN01 and NN02) and four unrelated animals from the Kentucky Reptile Zoo, KY (NN03, NN04, NN05 and NN06)-were part of the study (Supplementary Table  1a). Two animals from India were fresh road kills that were submitted for postmortem analysis to the Wayanad wildlife sanctuary forest veterinary officer, and were permitted to be used for this study under order no. WL10-12401/2017 from the Kerala forest and wildlife chief warden. A summary of tissues collected and processed from each animal is shown in Supplementary Table 1a. Genomic DNA was prepared using the Qiagen Magattract HMW DNA kit (Qiagen). RNA was extracted using the Qiagen RNeasy kit (Qiagen).
Genome sequencing. Libraries for ONT, PacBio SMRT and Illumina sequencing were constructed as per the manufacturers' instructions using high-molecular weight (HMW) DNA extracted from animal NN01 liver and kidney. A total of 71.23-Gb (~40×) PacBio RSII/Sequel, 61.8 -Gb (~34×) ONT and 117.5-Gb (~64×) Illumina (2 × 150-base pairs (bp)) data were generated. Additionally, paired-end Illumina sequencing data (2 × 150 bp) were generated for each study animal as indicated in Supplementary Table 1b. BNG data generation. Purified DNA extracted from muscle tissue from animal NN01 was embedded in a thin agarose layer, labeled and counterstained following the nick, label, repair and stain (NLRS) or direct label and stain (DLS) Reagent Kit protocol (BNG). Samples were then loaded onto Saphyr chips and run on the Saphyr imaging instrument (BNG). A total of 814 and 580 Gb of optical map data were generated using the NLRS or DLS protocol, respectively. De novo genome assembly using the BNG Access software for the NLRS or DLS optical map data produced assemblies consisting of 3,921 and 1,477 scaffolds with scaffold N50 of 0.88 and 16.33 Mb, respectively. Additionally, we performed BNG DNA labeling using DNA extracted from the ovary of NN05 for use in scaffolding with the 10x Genomics-derived de novo assembly. A total of 400 Gb of BNG data were generated for this animal using the DLS protocol, resulting in a de novo optical map assembly consisting of 1,338 scaffolds and a scaffold N50 of 15.92 Mb.
Chicago library preparation and sequencing. Three Chicago libraries were prepared as described previously 28 using DNA from muscle tissue corresponding to animal NN01 (Dovetail Genomics). Briefly, for each library, ~500 ng of HMW gDNA (mean fragment length, 75 kb) was reconstituted into chromatin in vitro and fixed with formaldehyde. Fixed chromatin was digested with DpnII, the 5′ overhangs filled in with biotinylated nucleotides and free blunt ends were then ligated. After ligation, cross-links were reversed and the DNA purified from protein. Purified DNA was treated to remove biotin that was not internal to ligated fragments. The DNA was then sheared to ~350 bp mean fragment size and sequencing libraries were generated using NEBNext Ultra enzymes and Illuminacompatible adapters. Biotin-containing fragments were isolated using streptavidin beads before PCR enrichment of each library. The libraries were sequenced on an Illumina platform. We produced 2 × 150-bp reads and about 525 million, 489 million and 516 million reads for libraries 1, 2 and 3, respectively. Together, these Chicago library reads provided ~250× (459-Gb) physical coverage of the genome (1-50 kb pairs).
Hi-C library preparation and sequencing. Two Dovetail Genomics Hi-C libraries were prepared as described previously 29 using muscle tissue from animal NN01. Briefly, for each library, chromatin was fixed in place with formaldehyde in the nucleus and then extracted. Fixed chromatin was digested with DpnII, the 5′ overhangs filled in with biotinylated nucleotides and then free blunt ends were ligated. After ligation, cross-links were reversed and the DNA purified to remove proteins. Purified DNA was treated to remove biotin that was not internal to ligated fragments. The DNA was then sheared to ~350-bp mean fragment size and sequencing libraries were generated using NEBNext Ultra enzymes and Illuminacompatible adapters. Biotin-containing fragments were isolated using streptavidin beads before PCR enrichment of each library. The libraries were sequenced on an Illumina platform to generate 242 million and 291 million 2 × 150-bp reads for libraries 1 and 2, respectively. Together, these Dovetail Hi-C library reads provided ~89× (159 Gb) physical coverage of the genome (1-50 kb pairs).
Scaffold to chromosome mapping. We used the synteny between Elaphe chromosomes and the N. naja Nana_v5 genome to assign scaffolds to chromosomes. Briefly, 105 cDNA sequences corresponding to E. quadrivirgata chromosomes 31 were downloaded from the NCBI nucleotide database. The cDNA sequences were aligned to the N. naja Nana_v5 genome assembly using Exonerate 92 . The chromosome number corresponding to the Elaphe cDNA marker was assigned to the best matching Nana_v5 scaffold based on the highest sequence alignment score.

Snake chromosome laser capture microdissection and SChrom-seq.
Chromosome slides for laser microdissection were prepared on Leica 0.9-µm POL-membrane frame steel frame slides (Leica, no. 11505188) following the manufacturer's recommended protocol. About 10 µl of fixed and previously tested metaphase cell suspensions from ovarian (NN03) fibroblasts was dropped on a dry POL-membrane slide, allowed to spread by gravity and dried overnight. Airdried slides were placed in 50 ml of Giemsa solution (1 ml of 5% Giemsa staining solution per 50 ml of GURR buffer (Gibco)) for 10 min, washed three times in sterile water and air-dried. Laser confocal microscopy-enabled dissection of individual chromosomes was done with the Leica LMD 6 Laser Microdissection microscope (150× dry-immersion objective; HC PL FLUOTAR ×150/0.9 numerical aperture) and software. A total of 104 dissections were performed. Each sample tube containing microdissected chromosomes was lysed to release genomic DNA, and whole-genome amplification performed to amplify the DNA and create libraries with the SMARTer PicoPLEX DNA-seq kit (Takara Bio). The resulting 250-bp single-end libraries were further amplified exponentially with primers containing unique Illumina dual-index barcodes suitable for Illumina sequencing. Data from each library were mapped to a combined reference genome including GRCh38 and snake (Nana_v5) using Burrows-Wheeler aligner (BWA) 93 with default options. Mapped reads were sorted and duplicates marked with PicardTools (http://broadinstitute.github.io/picard/). Reads mapping to multiple loci, GRCh38, a combined bacterial sequence database or those with a mapping quality below 10 were discarded. All remaining mapped reads were then used to calculate total counts per chromosome. Coverage data were generated with GATK 94 and binned using 100-kb windows.
Synteny mapping and dot-plot analysis. The repeat-masked C. viridis (prairie rattlesnake) and A. carolinensis genomes were aligned to Nana_v5 chromosomes with CoGe's SynMap program using LAST 95 . The 10x-BNG hybrid scaffolds and Nana_v5 scaffolds (>1 Mb) were aligned with Symap (v.4.2) using default parameters 96 . Chromosome painting of N. naja chromosomes with prairie rattlesnake chromosomes was performed using the SatsumaSynteny2 script with default parameters 97 .

Nature GeNetics
PolyA RNA-sequencing (RNA-seq) libraries were prepared with 1 µg RNA from each tissue using the Illumina TruSeq stranded messenger RNA kit and sequenced on HiSeq (Illumina).
Iso-seq analysis. Total RNA from venom gland was used to generate cDNA and PacBio iso-seq libraries as per the manufacturer's instructions. Size selection of libraries was performed using the BluePippin system (Sage Science). Sequencing was performed on a PacBio RSII/Sequel (Supplementary Table 5a). Full-length consensus isoform sequences were generated using PacBio's SMRT portal and SMRTLink for the RSII and Sequel data, respectively. Arrow (https://github.com/ PacificBiosciences/GenomicConsensus) was used to polish the consensus isoforms. In total, 101,761 transcript isoforms were processed by the pipeline. Full-length transcripts (that contained a complete open reading frame) were then aligned to the genome using GMAP with parameters specific to long-read alignment 98 . Complete open reading frames were directly annotated with tBLASTx (v.2.2.29+) 99 against NCBI NR and TrEMBL using the same pipeline as done with wholegenome annotation. We used these data to manually verify and correct venomgland-specific toxin gene annotations (Supplementary Note).

Repeat element identification.
We identified the repetitive elements in the genome by combining both homology-based and de novo predictions. Next, we used a previously described reptile-specific repeat library 20 with RepeatMasker (v.4.0.7) (http://www.repeatmasker.org) to annotate repetitive elements in the Indian cobra genome. We then used RepeatModeler (v.1.0.11) (http://www. repeatmasker.org/RepeatModeler.html) to construct the species-specific repeat sequence libraries for the Indian cobra, and then used these as a query to identify repetitive elements using RepeatMasker. Finally, we retrieved a non-redundant annotation for each species after combining all the annotation results using the reptile-specific libraries and de novo repeat library.
Venom gene synteny analysis. BLASTn was used to identify homologs of all N. naja toxin genes (Supplementary Table 6b) in the Anolis and C. viridis genomes. BLASTn hits were then filtered using the following parameters: query coverage ≥70% and identity ≥80%. Synteny was then plotted using a publicly available Python script for gene synteny visualization (https://github.com/ biopython/biopython/blob/master/Doc/examples/Proux_et_al_2002_Figure_6.py).
Differential gene expression analysis. After adapter trimming and quality filtering, reads were aligned to Nana_v5 using STAR (v.2.6.0a) 100 with default parameters. Gene counts per gene across all samples were calculated using featureCounts 101 . Raw gene counts were then normalized between samples using the CPM normalization method from the EdgeR package 102 (Supplementary  Table 7b-h). Differential expression analysis was then performed using EdgeR (fold change >2 and 1% FDR), and differentially upregulated genes (DUGs) were filtered for further analysis for each tissue that had a replicate. Gene Ontology-term enrichment analysis of DUGs was performed using EnrichR 103,104 (Supplementary Note).

Liquid chromatography-tandem mass spectrometry analysis of venom.
Pooled Indian cobra venom samples obtained from Kentucky Reptile Zoo (10 μg per lane) were reduced with DTT (10 mM, 60 min at 37 °C), alkylated with iodoacetamide (20 mM, 15 min at room temperature) and separated by SDS-PAGE for ~1 cm on a 4-12% Bis-Tris gel. The gel was Coomassie stained with SimplyBlue (Invitrogen) and each lane cut into two segments based on the ~70-kDa molecular weight marker. Gel pieces were de-stained in MilliQ water twice and dehydrated with NH 4 HCO 3 /50% acetonitrile (ACN) (20 min) and 100% ACN (2 × 5 min). Digestion was performed overnight at 37 °C with trypsin (20 ng µl -1 in 50 mM NH 4 HCO 3 ). Peptides were extracted twice in 1% formic acid (FA)/50% ACN, once with 100% ACN and then dried to completion. The sample was then reconstituted in solvent A (2% ACN/0.1% FA) and injected onto a 0.1 × 100-mm 2 Waters Symmetry 1.7-mm BEH-130 C18 column via an auto-sampler for separation by reverse-phase chromatography on a NanoAcquity UPLC system (Waters). A dual-stage linear gradient with a flow rate of 1 μl min -1 was applied for peptide separation, where solvent A was 0.1% FA/2% ACN/ water and solvent B was 0.1% FA/2% water/ACN. Solvent B was increased from 2 to 25% over 35 min and then from 25 to 60% over 2 min, with a total analysis time of 60 min. Peptides eluting from the column were analyzed on an LTQ Orbitrap Elite mass spectrometer (Thermo Fisher) equipped with an Advance CaptiveSpray ionization source (Michrom). MS1 precursor ions were scanned in Fourier transform mass spectroscopy at 60,000 resolution and tandem mass spectrometry (MS/MS) data acquired on the 15 most abundant ions in LTQ linear ion trap mass spectrometer in data-dependent mode. MS/MS spectra were searched using the Mascot algorithm (Matrix Sciences) against a concatenated target-decoy database comprised of the FASTA sequences from the N. naja predicted proteome, Swiss-Prot entries from the Elapinae family (https://www. uniprot.org/taxonomy/42168) and common contaminant proteins, as well as the reversed versions of each sequence. Peptide spectral matches were filtered using a linear discriminant function to a peptide FDR of 5% to identify those that mapped to the toxin proteins identified from the genome. 3FTx structural modeling. Three-dimensional structural models were generated using MOE software (Chemical Computing Group, v.0101) and homology modeling was performed using the AMBER10 EHT forcefield 105 . Representative sequences were first searched in the protein database and the closest hits based on percentage sequence identity were chosen as templates for construction of structural homology models and for root mean square deviation calculations (Supplementary Table 10).

Molecular evolutionary analysis.
For pairwise comparisons of both venomexpressed and non-venom-expressed genes, the number of nucleotide substitutions per synonymous (K S ) and non-synonymous site (K A ) for each pair of protein-coding genes was computed according to the Nei-Gojobori method 106 using Sqdif Plot (http://www.gen-info.osaka-u.ac.jp/~uhmin/study/sqdifPlot/ index.html).

Identification of ZRS limb enhancer deletion.
To identify the ZRS enhancer region, sequences corresponding to this region were downloaded from ref. 107 . BLASTn (v.2.2.29+) 99 was used to identify the orthologous N. naja sequence. Multiple sequence alignment was performed using Clustal 108 .

Variant analysis.
We first performed phylogenetic analysis using 1,654 polymorphisms identified in the mitochondrial genomes using shortread whole-genome sequence data for each of the six study animals. Briefly, adapter and quality trimmed reads were aligned to the N. naja mitochondrial genome (GenBank: DQ343648.1) using BWA 93 . Variant calling was performed using SAMtools 109 . The resulting alignments of all six animals were compared to a reference 110 . Multiple sequence alignment of the consensus mitochondrial sequence from the study animals, generated using MUSCLE 108 , was used to construct the phylogenetic tree using FigTree v.4.2.
For the genome-wide protein-coding variant analysis, whole-genome sequencing data for the six study animals were mapped to the Nana_v5 reference genome assembly using BWA 93 with default options. Mapped reads were sorted and duplicates marked with PicardTools. Local realignment, germline variant calling and joint genotyping were performed using GATK (v.4.1.0.0) 94,111 . Germline variants were decomposed and normalized with vt 112 and functionally annotated with snpEff 113 against the annotated genome. Germline variants that had genotype calls in all samples were used to calculate percentage identity values between samples.
Genome heterozygosity estimation. K-mers from short-read sequencing data for each of the six animals used in this study were counted using Jellyfish (v.2.2.6) 114 for several values of K: 15, 17 and 21. Each computed K-mer histogram was then analyzed with GenomeScope 115 .
Reporting Summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
Sequence data (DNA and RNA) can be accessed at NCBI under BioProject accession no. PRJNA527614. The MS data are available under accession no. MSV000084564.

October 2018
Sex: female Age: 6 years old Wild animals Two animals used in this study from India were fresh road kills and were approved for use in the study as described in the methods section.

Field-collected samples
This study did not include field-collected samples

Ethics oversight
This study did not require ethics approval.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots. Gating strategy is provided in Supplementary Fig. 1a Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.