Antennal transcriptome profiles of anopheline mosquitoes reveal human host olfactory specialization in Anopheles gambiae

BMC Genomics

Volumn 14, Issue 1, 2013, Pages

  Rinker, David C ^a   Zhou, Xiaofan ^b   Pitts, Ronald J ^b   Rokas, Antonis ^a,b   Zwiebel, Laurence J ^a,b   Neafsey, Daniel E ^c   Berlin, Aaron M ^c   Christophides, George K ^c   Collins, Frank H ^c   Emrich, Scott J ^c   Fontaine, Michael C ^c   Gelbart, William ^c   Hahn, Matthew W ^c   Howell, Paul I ^c   Kafatos, Fotis C ^c   Lawson, Daniel ^c   Muskavitch, Marc A T ^c   Young, Sarah ^c   Waterhouse, Robert ^c   Besansky, Nora J ^c  

^a VANDERBILT UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b VANDERBILT UNIVERSITY   (United States)

^c Institutes of Chemical Biology and Global Health ^*

Author keywords

Anopheles; Antenna; Host seeking; Malaria; Molecular evolution; Mosquito; Odorant receptor; Olfaction; RNAseq; Transcriptome

Indexed keywords

MESSENGER RNA; TRANSCRIPTOME; VOLATILE AGENT; OLFACTORY RECEPTOR;

ANOPHELES GAMBIAE; ANTENNA (ORGAN); ARTICLE; CHEMOSENSORY GENE; CONTROLLED STUDY; GENE; GENETIC POLYMORPHISM; HOST PARASITE INTERACTION; MULTIGENE FAMILY; NONHUMAN; OLFACTORY RECEPTOR; OLFACTORY SYSTEM; PHENOTYPE; QUALITATIVE ANALYSIS; QUANTITATIVE ANALYSIS; RNA SEQUENCE; SIBLING; SPECIES DIVERSITY; ANIMAL; FEMALE; GENETICS; GENOME; HUMAN; METABOLISM; MOLECULAR EVOLUTION; MOLECULAR GENETICS; SEQUENCE ANALYSIS;

ANOPHELES GAMBIAE; GAMBIAE SPECIES COMPLEX;

ANIMALS; ANOPHELES GAMBIAE; ARTHROPOD ANTENNAE; EVOLUTION, MOLECULAR; FEMALE; GENOME; HUMANS; MOLECULAR SEQUENCE ANNOTATION; RECEPTORS, ODORANT; SEQUENCE ANALYSIS, RNA; TRANSCRIPTOME;

EID: 84886742079     PISSN: None     EISSN: 14712164     Source Type: Journal    
DOI: 10.1186/1471-2164-14-749     Document Type: Article

References (83)

1. Coetzee M, Hunt RH, Wilkerson R. Anopheles coluzzii and Anopheles amharicus, new members of the Anopheles gambiae complex. Zootaxa 2013, 3619:246-274.
2. Scott TW, Takken W. Feeding strategies of anthropophilic mosquitoes result in increased risk of pathogen transmission. Trends Parasitol 2012, 28:114-121. 10.1016/j.pt.2012.01.001, 22300806.
3. Takken W, Eling W, Hooghof J, Dekker T, Hunt R, Coetzee M. Susceptibility of Anopheles quadriannulatus Theobald (Diptera: Culicidae) to Plasmodium falciparum. T Roy Soc Trop Med H 1999, 93:578-580.
4. Habtewold T, Povelones M, Blagborough AM, Christophides GK. Transmission Blocking Immunity in the Malaria Non-Vector Mosquito Anopheles quadriannulatus Species A. PLoS Pathog 2008, 4:e1000070. 10.1371/journal.ppat.1000070, 2374904, 18497855.
5. Kamali M, Xia A, Tu Z, Sharakhov IV. A new chromosomal phylogeny supports the repeated origin of vectorial capacity in malaria mosquitoes of the Anopheles gambiae complex. PLoS Pathog 2012, 8:e1002960. 10.1371/journal.ppat.1002960, 3464210, 23055932.
6. White G. Comparative studies on sibling species of the Anopheles gambiae Giles complex (Dipt., Culicidae). III. The distribution, ecology, behaviour and vectorial importance of species D in Bwamba County, Uganda, with an analysis of biological, ecological, morphological and cytogenetical relationships of Ugandan species D*. Bull Entomol Res 1973, 63:65-97.
7. Dekker T, Takken W. Differential responses of mosquito sibling species Anopheles arabiensis and An-quadriannulatus to carbon dioxide, a man or a calf. Med Vet Entomol 1998, 12:136-140. 10.1046/j.1365-2915.1998.00073.x, 9622366.
8. Pates H, Takken W, Curtis C, Huisman P, Akinpelu O, Gill G. Unexpected anthropophagic behaviour in Anopheles quadriannulatus. Med Vet Entomol 2001, 15:293-298. 10.1046/j.0269-283x.2001.00310.x, 11583447.
9. Ayala F, Coluzzi M. Chromosome speciation: Humans, Drosophila, and mosquitoes. Proc Natl Acad Sci U S A 2005, 102:6535-6542. 10.1073/pnas.0501847102, 1131864, 15851677.
10. Coluzzi M. A polytene chromosome analysis of the Anopheles gambiae species complex. Science 2002, 298:1415-1418. 10.1126/science.1077769, 12364623.
11. Takken W, Knols BG. Odor-mediated behavior of Afrotropical malaria mosquitoes. Annu Rev Entomol 1999, 44:131-157. 10.1146/annurev.ento.44.1.131, 9990718.
12. Takken W, Verhulst NO. Host preferences of blood-feeding mosquitoes. Annu Rev Entomol 2013, 58:433-453. 10.1146/annurev-ento-120811-153618, 23020619.
13. Pitts RJ, Fox AN, Zwiebel LJ. A highly conserved candidate chemoreceptor expressed in both olfactory and gustatory tissues in the malaria vector Anopheles gambiae. Proc Natl Acad Sci U S A 2004, 101:5058-5063. 10.1073/pnas.0308146101, 387373, 15037749.
14. Melo ACA, Rützler M, Pitts RJ, Zwiebel LJ. Identification of a chemosensory receptor from the yellow fever mosquito, Aedes aegypti, that is highly conserved and expressed in olfactory and gustatory organs. Chem Senses 2004, 29:403-410. 10.1093/chemse/bjh041, 15201207.
15. Kwon H-W, Lu T, Rützler M, Zwiebel LJ. Olfactory responses in a gustatory organ of the malaria vector mosquito Anopheles gambiae. Proc Natl Acad Sci USA 2006, 103:13526-13531. 10.1073/pnas.0601107103, 1569196, 16938890.
16. Schymura D, Forstner M, Schultze A, Kröber T, Swevers L, Iatrou K, Krieger J. Antennal expression pattern of two olfactory receptors and an odorant binding protein implicated in host odor detection by the malaria vector Anopheles gambiae. Int J Biol Sci 2010, 6:614-626. 2962264, 20975820.
17. Jones WD, Nguyen T-AT, Kloss B, Lee KJ, Vosshall LB. Functional conservation of an insect odorant receptor gene across 250 million years of evolution. Curr Biol 2005, 15:R119-R121.
18. Xia Y, Zwiebel LJ. Identification and characterization of an odorant receptor from the West Nile virus mosquito, Culex quinquefasciatus. Insect Biochem Mol Biol 2006, 36:169-176. 10.1016/j.ibmb.2005.12.003, 3100213, 16503478.
19. Qiu YT, van Loon JJA, Takken W, Meijerink J, Smid HM. Olfactory Coding in Antennal Neurons of the Malaria Mosquito, Anopheles gambiae. Chem Senses 2006, 31:845-863. 10.1093/chemse/bjl027, 16963500.
20. Takken W, van Loon J, Adam W. Inhibition of host-seeking response and olfactory responsiveness in Anopheles gambiae following blood feeding. J Insect Physiol 2001, 47:303-310. 10.1016/S0022-1910(00)00107-4, 11119776.
21. Fox AN, Pitts RJ, Robertson HM, Carlson JR, Zwiebel LJ. Candidate odorant receptors from the malaria vector mosquito Anopheles gambiae and evidence of down-regulation in response to blood feeding. Proc Natl Acad Sci USA 2001, 98:14693-14697. 10.1073/pnas.261432998, 64743, 11724964.
22. Hill CA, Fox AN, Pitts RJ, Kent LB, Tan PL, Chrystal MA, Cravchik A, Collins FH, Robertson HM, Zwiebel LJ. G Protein-Coupled receptors in Anopheles gambiae. Science 2002, 298:176-178. 10.1126/science.1076196, 12364795.
23. Lu Q, Lu T, Qiu YT, Wang G, Kwon JY, Rutzler M, Kwon HW, Pitts RJ, van Loon JJA, Takken W, Carlson JR, Zwiebel LJ. Odor coding in the Maxillary Palp of the malaria vector mosquito Anopheles gambiae. Curr Biol 2007, 17:1533-1544. 10.1016/j.cub.2007.07.062, 3113458, 17764944.
24. Xia Y, Wang G, Buscariollo D, Pitts RJ, Wenger H, Zwiebel LJ. The molecular and cellular basis of olfactory-driven behavior in Anopheles gambiae larvae. Proc Natl Acad Sci USA 2008, 105:6433-6438. 10.1073/pnas.0801007105, 2359781, 18427108.
25. Carey AF, Wang G, Su C-Y, Zwiebel LJ, Carlson JR. Odorant reception in the malaria mosquito Anopheles gambiae. Nature 2010, 1-38.
26. Wang G, Carey AF, Carlson JR, Zwiebel LJ. Molecular basis of odor coding in the malaria vector mosquito Anopheles gambiae. Proc Natl Acad Sci USA 2010, 107:4418-4423. 10.1073/pnas.0913392107, 2840125, 20160092.
27. Pitts RJ, Rinker DC, Jones PL, Rokas A, Zwiebel LJ. Transcriptome profiling of chemosensory appendages in the malaria vector Anopheles gambiae reveals tissue- and sex-specific signatures of odor coding. BMC Genomics 2011, 12:271. 10.1186/1471-2164-12-271, 3126782, 21619637.
28. Rinker DC, Pitts RJ, Zhou X, Suh E, Rokas A, Zwiebel LJ. Blood meal-induced changes to antennal transcriptome profiles reveal shifts in odor sensitivities in Anopheles gambiae. Proc Natl Acad Sci USA 2013, 110:1-9.
29. Hittinger CT, Johnston M, Tossberg JT, Rokas A. Leveraging skewed transcript abundance by RNA-Seq to increase the genomic depth of the tree of life. Proc Natl Acad Sci USA 2010, 107:1476-1481. 10.1073/pnas.0910449107, 2824393, 20080632.
30. Kopp A, Barmina O, Hamilton AM, Higgins L, McIntyre LM, Jones CD. Evolution of gene expression in the Drosophila olfactory system. Mol Biol Evol 2008, 25:1081-1092. 10.1093/molbev/msn055, 3299402, 18296696.
31. Xu PX, Zwiebel LJ, Smith DP. Identification of a distinct family of genes encoding atypical odorant-binding proteins in the malaria vector mosquito, Anopheles gambiae. Insect Mol Biol 2003, 12:549-560. 10.1046/j.1365-2583.2003.00440.x, 14986916.
32. Liu C, Pitts RJ, Bohbot JD, Jones PL, Wang G, Zwiebel LJ. Distinct Olfactory Signaling Mechanisms in the Malaria Vector Mosquito Anopheles gambiae. PLoS Biol 2010, 8:e1000467. 10.1371/journal.pbio.1000467, 2930861, 20824161.
33. Croset V, Rytz R, Cummins S, Budd A, Brawand D, Kaessmann H, Gibson TJ, Benton R. Ancient Protostome Origin of Chemosensory Ionotropic Glutamate Receptors and the Evolution of Insect Taste and Olfaction. PLoS Genet 2010, 6:e1001064. 10.1371/journal.pgen.1001064, 2924276, 20808886.
34. Rinker DC, Jones PL, Pitts RJ, Rützler M, Camp G, Sun L, Xu P, Dorset DC, Weaver D, Zwiebel LJ. Novel high-throughput screens of Anopheles gambiae odorant receptors reveal candidate behaviour-modifying chemicals for mosquitoes. Physiol Entomol 2012, 37:33-41.
35. Hallem EA, Fox AN, Zwiebel LJ, Carlson JR. Olfaction: mosquito receptor for human-sweat odorant. Nature 2004, 427:212-213. 10.1038/427212a, 14724626.
36. Meijerink J, Braks M, Brack AA, Adam W, Dekker T, Posthumus MA, Beek TAV, Van Loon JJA. Identification of Olfactory Stimulants for Anopheles gambiae from Human Sweat Samples. J Chem Ecol 2000, 26:1367-1382.
37. Meijerink J, Braks MA, Van Loon JJ. Olfactory receptors on the antennae of the malaria mosquito Anopheles gambiae are sensitive to ammonia and other sweat-borne components. J Insect Physiol 2001, 47:455-464. 10.1016/S0022-1910(00)00136-0, 11166310.
38. Braks MA, Takken W. Incubated Human Sweat but not Fresh Sweat Attracts the Malaria Mosquito Anopheles gambiae sensu stricto. J Chem Ecol 1999, 25:663-672.
39. Thorne N, Chromey C, Bray S, Amrein H. Taste perception and coding in Drosophila. Curr Biol 2004, 14:1065-1079. 10.1016/j.cub.2004.05.019, 15202999.
40. Dekker T, Geier M, Cardé RT. Carbon dioxide instantly sensitizes female yellow fever mosquitoes to human skin odours. J Exp Biol 2005, 208:2963-2972. 10.1242/jeb.01736, 16043601.
41. Ai M, Min S, Grosjean Y, Leblanc C, Bell R, Benton R, Suh GSB. Acid sensing by the Drosophila olfactory system. Nature 2010, 468:691-695. 10.1038/nature09537, 3105465, 21085119.
42. Biessmann H, Nguyen QK, Le D, Walter MF. Microarray-based survey of a subset of putative olfactory genes in the mosquito Anopheles gambiae. Insect Mol Biol 2005, 14:575-589. 10.1111/j.1365-2583.2005.00590.x, 16313558.
43. Pelletier J, Guidolin A, Syed Z, Cornel AJ, Leal WS. Knockdown of a Mosquito Odorant-binding Protein Involved in the Sensitive Detection of Oviposition Attractants. J Chem Ecol 2010, 36:245-248. 10.1007/s10886-010-9762-x, 2837830, 20191395.
44. Biessmann H, Andronopoulou E, Biessmann MR, Douris V, Dimitratos SD, Eliopoulos E, Guerin PM, Iatrou K, Justice RW, Kröber T, Marinotti O, Tsitoura P, Woods DF, Walter MF. The Anopheles gambiae odorant binding protein 1 (AgamOBP1) mediates indole recognition in the antennae of female mosquitoes. PLoS ONE 2010, 5:e9471. 10.1371/journal.pone.0009471, 2830424, 20208991.
45. Zhou X, Slone JD, Rokas A, Berger SL, Liebig J, Ray A, Reinberg D, Zwiebel LJ. Phylogenetic and Transcriptomic Analysis of Chemosensory Receptors in a Pair of Divergent Ant Species Reveals Sex-Specific Signatures of Odor Coding. PLoS Genet 2012, 8:e1002930. 10.1371/journal.pgen.1002930, 3431598, 22952454.
46. Robertson HM, Warr CG, Carlson JR. Molecular evolution of the insect chemoreceptor gene superfamily in Drosophila melanogaster. Proc Natl Acad Sci USA 2003, 100(Suppl 2):14537-14542. 304115, 14608037.
47. Sánchez-Gracia A, Vieira FG, Almeida FC, Rozas J. Comparative Genomics of the Major Chemosensory Gene Families in Arthropods 2001, John Wiley & Sons, Ltd: Chichester, UK.
48. Gilad Y, Wiebe V, Przeworski M, Lancet D, Pääbo S. Loss of Olfactory receptor genes coincides with the acquisition of full trichromatic vision in primates. PLoS Biol 2004, 2:e5. 10.1371/journal.pbio.0020005, 314465, 14737185.
49. Stensmyr M, Dekker T, Hansson B. Evolution of the olfactory code in the Drosophila melanogaster subgroup. P Roy Soc Lond B Bio 2003, 270:2333-2340.
50. Dekker T, Ibba I, Siju KP, Stensmyr MC, Hansson BS. Olfactory shifts parallel superspecialism for toxic fruit in Drosophila melanogaster siblingD. sechellia. Curr Biol 2006, 16:101-109. 10.1016/j.cub.2005.11.075, 16401429.
51. Mcbride CS, Arguello JR, O'meara BC. Five Drosophila genomes reveal nonneutral evolution and the signature of host specialization in the chemoreceptor superfamily. Genetics 2007, 177:1395-1416. 10.1534/genetics.107.078683, 2147975, 18039874.
52. McBride CS. Rapid evolution of smell and taste receptor genes during host specialization in Drosophila sechellia. Proc Natl Acad Sci USA 2007, 104:4996-5001. 10.1073/pnas.0608424104, 1829253, 17360391.
53. Torre AD, Costantini C, Besansky NJ, Caccone A, Petrarca V, Powell JR, Coluzzi M. Speciation within Anopheles gambiae--the glass is half full. Science 2002, 298:115-117. 10.1126/science.1078170, 12364784.
54. Obbard DJ, Maclennan J, Kim K-W, Rambaut A, O'Grady PM, Jiggins FM. Estimating divergence dates and substitution rates in the Drosophila phylogeny. Mol Biol Evol 2012, 29:3459-3473. 10.1093/molbev/mss150, 3472498, 22683811.
55. Bernays EA, Chapman RE. Patterns of Host-Plant Use. Host-Plant Selection by Phytophagous Insects 1994, 4-13. New York: Chapmand and Hall.
56. Smith NGC. Are radical and conservative substitution rates useful statistics in molecular evolution?. J Mol Evol 2003, 57:467-478. 10.1007/s00239-003-2500-z, 14708579.
57. Hanada K, Shiu SH, LI WH. The nonsynonymous/synonymous substitution rate ratio versus the radical/conservative replacement rate ratio in the evolution of mammalian genes. Mol Biol Evol 2007, 24:2235-2241. 10.1093/molbev/msm152, 17652332.
58. Nichols AS, Luetje CW. Transmembrane segment 3 of drosophila melanogaster odorant receptor subunit 85B contributes to ligand-receptor interactions. J Bio Chem 2010, 285:11854-11862.
59. Nakagawa T, Pellegrino M, Sato K, Vosshall LB, Touhara K. Amino acid residues contributing to function of the heteromeric insect olfactory receptor complex. PLoS ONE 2012, 7:e32372. 10.1371/journal.pone.0032372, 3293798, 22403649.
60. DeGennaro M, McBride CS, Seeholzer L, Nakagawa T, Dennis EJ, Goldman C, Jasinskiene N, James AA, Vosshall LB. orco mutant mosquitoes lose strong preference for humans and are not repelled by volatile DEET. Nature 2013, 498:487-491. 10.1038/nature12206, 23719379.
61. Larsson MC, Domingos AI, Jones WD, Chiappe ME, Amrein H, Vosshall LB. Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction. Neuron 2004, 43:703-714. 10.1016/j.neuron.2004.08.019, 15339651.
62. Pitts RJ, Zwiebel LJ. Antennal sensilla of two female anopheline sibling species with differing host ranges. Malar J 2006, 5:26. 10.1186/1475-2875-5-26, 1532926, 16573828.
63. Cork A, Park KC. Identification of electrophysiologically-active compounds for the malaria mosquito, Anopheles gambiae, in human sweat extracts. Med Vet Entomol 1996, 10:269-276. 10.1111/j.1365-2915.1996.tb00742.x, 8887339.
64. Bohbot JD, Jones PL, Wang G, Pitts RJ, Pask GM, Zwiebel LJ. Conservation of indole responsive odorant receptors in mosquitoes reveals an ancient olfactory trait. Chem Senses 2010, 36:149-160. 3020388, 20956733.
65. Bernier UR, Kline DL, Barnard DR, Schreck CE, Yost RA. Analysis of human skin emanations by gas chromatography/Mass spectrometry. 2. identification of volatile compounds that are candidate attractants for the yellow fever mosquito (Aedesaegypti). Anal Chem 2000, 72:747-756. 10.1021/ac990963k, 10701259.
66. Hughes DT, Pelletier J, Luetje CW, Leal WS. Odorant receptor from the Southern House mosquito narrowly tuned to the Oviposition attractant Skatole. J Chem Ecol 2010, 36:797-800. 10.1007/s10886-010-9828-9, 2908433, 20623327.
67. Pelletier J, Hughes DT, Luetje CW, Leal WS. An odorant receptor from the southern house mosquito Culex pipiens quinquefasciatus sensitive to oviposition attractants. PLoS ONE 2010, 5:e10090. 10.1371/journal.pone.0010090, 2851645, 20386699.
68. Millar JG, Chaney JD, Mulla MS. Identification of oviposition attractants for Culex quinquefasciatus from fermented Bermuda grass infusions. J Am Mosquito Contr 1992, 8:11-17.
69. Kostelc JG, Preti G, Zelson PR, Stoller NH, Tonzetich J. Salivary volatiles as indicators of periodontitis. J Periodont Res 1980, 15:185-192. 10.1111/j.1600-0765.1980.tb00273.x, 6445980.
70. Pates H, Takken W, Stuke K, Curtis C. Differential behaviour of Anopheles gambiae sensu stricto (Diptera Culicidae) to human and cow odours in the laboratory. Bull Entomol Res 2001, 91:289-296. 10.1079/BER200198, 11587625.
71. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389-3402. 10.1093/nar/25.17.3389, 146917, 9254694.
72. Birney E. GeneWise and Genomewise. Genome Res 2004, 14:988-995. 10.1101/gr.1865504, 479130, 15123596.
73. Katoh K, Standley DM. MAFFT multiple sequence alignment software Version 7: Improvements in performance and usability. Mol Biol Evol 2013, 30:772-780. 10.1093/molbev/mst010, 3603318, 23329690.
74. Capella-Gutierrez S, Silla-Martinez JM, Gabaldon T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 2009, 25:1972-1973. 10.1093/bioinformatics/btp348, 2712344, 19505945.
75. Stamatakis A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 2006, 22:2688-2690. 10.1093/bioinformatics/btl446, 16928733.
76. Yang Z. PAML 4: Phylogenetic analysis by maximum likelihood. Mol Biol Evol 2007, 24:1586-1591. 10.1093/molbev/msm088, 17483113.
77. Zhang J. Rates of conservative and radical nonsynonymous nucleotide substitutions in mammalian nuclear genes. J Mol Evol 2000, 50:56-68.
78. Bernsel A, Viklund H, Hennerdal A, Elofsson A. TOPCONS: consensus prediction of membrane protein topology. Nucleic Acids Res 2009, 37(Web Serve):W465-W468. 2703981, 19429891.
79. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotech 2011, 29:644-652.
80. Li L, Stoeckert CJ, Roos DS. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res 2003, 13:2178-2189. 10.1101/gr.1224503, 403725, 12952885.
81. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature Publishing Group 2012, 7:562-578.
82. Feng J, Meyer CA, Wang Q, Liu JS, Liu SX, Zhang Y. GFOLD: a generalized fold change for ranking differentially expressed genes from RNA-seq data. Bioinformatics 2012, 28:2782-2788. 10.1093/bioinformatics/bts515, 22923299.
83. Mortazavi A, Williams BA, Mccue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Meth 2008, 5:621-628.