BMC Genomics | |
Transcriptome profiling of pyrethroid resistant and susceptible mosquitoes in the malaria vector, Anopheles sinensis | |
Guiyun Yan2  Qi Gao1  Xuelian Chang2  Guofa Zhou2  Mei-Hui Wang2  Sui Xu1  Liang Bai1  Yaobao Liu1  Julin Li1  Huayun Zhou1  Jun Cao1  Daibin Zhong2  Guoding Zhu2  | |
[1] Jiangsu Institute of Parasitic Diseases, Key Laboratory of Parasitic Disease Control and Prevention (Ministry of Health), Jiangsu Provincial Key Laboratory of Parasite Molecular Biology, Wuxi, Jiangsu Province 214064, PR China;Program in Public Health, College of Health Sciences, University of California at Irvine, Irvine, CA 92697, USA | |
关键词: Anopheles sinensis; Gene expression; Pyrethroid resistance; Expressed sequence tag; Transcriptome; | |
Others : 1216643 DOI : 10.1186/1471-2164-15-448 |
|
received in 2013-11-06, accepted in 2014-05-28, 发布年份 2014 | |
【 摘 要 】
Background
Anopheles sinensis is a major malaria vector in China and other Southeast Asian countries, and it is becoming increasingly resistant to the insecticides used for agriculture, net impregnation, and indoor residual spray. Very limited genomic information on this species is available, which has hindered the development of new tools for resistance surveillance and vector control. We used the 454 GS FLX system and generated expressed sequence tag (EST) databases of various life stages of An. sinensis, and we determined the transcriptional differences between deltamethrin resistant and susceptible mosquitoes.
Results
The 454 GS FLX transcriptome sequencing yielded a total of 624,559 reads (average length of 290 bp) with the pooled An. sinensis mosquitoes across various development stages. The de novo assembly generated 33,411 contigs with average length of 493 bp. A total of 8,057 ESTs were generated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. A total of 2,131 ESTs were differentially expressed between deltamethrin resistant and susceptible mosquitoes collected from the same field site in Jiangsu, China. Among these differentially expressed ESTs, a total of 294 pathways were mapped to the KEGG database, with the predominant ESTs belonging to metabolic pathways. Furthermore, a total of 2,408 microsatellites and 15,496 single nucleotide polymorphisms (SNPs) were identified.
Conclusions
The annotated EST and transcriptome databases provide a valuable genomic resource for further genetic studies of this important malaria vector species. The differentially expressed ESTs associated with insecticide resistance identified in this study lay an important foundation for further functional analysis. The identified microsatellite and SNP markers will provide useful tools for future population genetic and comparative genomic analyses of malaria vectors.
【 授权许可】
2014 Zhu et al.; licensee BioMed Central Ltd.
【 预 览 】
Files | Size | Format | View |
---|---|---|---|
20150701202732571.pdf | 1374KB | download | |
Figure 7. | 29KB | Image | download |
Figure 6. | 58KB | Image | download |
Figure 5. | 82KB | Image | download |
Figure 4. | 103KB | Image | download |
Figure 3. | 41KB | Image | download |
Figure 2. | 88KB | Image | download |
Figure 1. | 70KB | Image | download |
【 图 表 】
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
【 参考文献 】
- [1]Rueda LM, Zhao TY, Ma YJ, Gao Q, Zhu GD, Khuntirat B, Sattabongkot J, Wilkerson RC: Updated distribution records of the Anopheles (Anopheles) hyrcanus species-group (Diptera: Culicidae) in China. Zootaxa 2007, 1407:43-55.
- [2]Zhou SS, Huang F, Wang JJ, Zhang SS, Su YP, Tang LH: Geographical, meteorological and vectorial factors related to malaria re-emergence in Huang-Huai River of central China. Malar J 2010, 9:337. BioMed Central Full Text
- [3]Zhu G, Xia H, Zhou H, Li J, Lu F, Liu Y, Cao J, Gao Q, Sattabongkot J: Susceptibility of Anopheles sinensis to Plasmodium vivax in malarial outbreak areas of central China. Parasit Vectors 2013, 6(1):176. BioMed Central Full Text
- [4]Kappe SHI, Vaughan AM, Boddey JA, Cowman AF: That was then but this is now: malaria research in the time of an eradication agenda. Science 2010, 328(5980):862-866.
- [5]The malERA Consultative Group on Vector Control: A research agenda for malaria eradication: vector control. PLoS Med 2011, 8(1):e1000401.
- [6]WHO: Global Insecticide use for Vector-Borne Disease Control. 5th edition. Geneva, Switzerland: WHO Pesticide Evaluation Scheme (WHOPES), World Health Organization; 2011.
- [7]WHO: Pesticides and Their Application for the Control of Vectors and Pests of Public Health Importance. Geneva, Switzerland: WHO Pesticide Evaluation Scheme (WHOPES), World Health Organization; 2006.
- [8]Chanda E, Hemingway J, Kleinschmidt I, Rehman AM, Ramdeen V, Phiri FN, Coetzer S, Mthembu D, Shinondo CJ, Chizema-Kawesha E, Kamuliwo M, Mukonka V, Baboo KS, Coleman M: Insecticide resistance and the future of malaria control in Zambia. PLoS One 2011, 6(9):e24336.
- [9]Githeko AK, Ototo EN, Guiyun Y: Progress towards understanding the ecology and epidemiology of malaria in the western Kenya highlands: opportunities and challenges for control under climate change risk. Acta Trop 2012, 121(1):19-25.
- [10]Ranson H, N’Guessan R, Lines J, Moiroux N, Nkuni Z, Corbel V: Pyrethroid resistance in African anopheline mosquitoes: what are the implications for malaria control? Trends Parasitol 2011, 27(2):91-98.
- [11]Maxmen A: Malaria surge feared. Nature 2012, 485(7398):293.
- [12]Cui F, Raymond M, Qiao C: Insecticide resistance in vector mosquitoes in China. Pest Manag Sci 2006, 62(11):1013-1022.
- [13]Qin Q, Li Y, Zhong D, Zhou N, Chang X, Li C, Cui L, Yan G, Chen X: Insecticide resistance of Anopheles sinensis and An. vagus in Hainan Island, a malaria-endemic area of China. Parasit Vectors 2014, 7(1):92. BioMed Central Full Text
- [14]Wang D, Xia Z, Zhou S, Zhou X, Wang R, Zhang Q: A potential threat to malaria elimination: extensive deltamethrin and DDT resistance to Anopheles sinensis from the malaria-endemic areas in China. Malar J 2013, 12(1):164. BioMed Central Full Text
- [15]Xu T, Zhong D, Tang L, Chang X, Fu F, Yan G, Zheng B: Anopheles sinensis mosquito insecticide resistance: comparison of three mosquito sample collection and preparation methods and mosquito age in resistance measurements. Parasit Vectors 2014, 7(1):54. BioMed Central Full Text
- [16]Zhong D, Chang X, Zhou G, He Z, Fu F, Yan Z, Zhu G, Xu T, Bonizzoni M, Wang M-H, Cui L, Zheng B, Chen B, Yan G: Relationship between knockdown resistance, metabolic detoxification and organismal resistance to pyrethroids in Anopheles sinensis. PLoS One 2013, 8(2):e55475.
- [17]Chang KS, Yoo DH, Shin EH, Lee WG, Roh JY, Park MY: Susceptibility and resistance of field populations of Anopheles sinensis (Diptera: Culicidae) collected from Paju to 13 insecticides. Osong Public Health Res Perspect 2013, 4(2):76-80.
- [18]Kang S, Jung J, Lee S, Hwang H, Kim W: The polymorphism and the geographical distribution of the knockdown resistance (kdr) of Anopheles sinensis in the Republic of Korea. Malar J 2012, 11(1):151. BioMed Central Full Text
- [19]Kim H, Baek JH, Lee W-J, Lee SH: Frequency detection of pyrethroid resistance allele in Anopheles sinensis populations by real-time PCR amplification of specific allele (rtPASA). Pestic Biochem Physiol 2007, 87(1):54-61.
- [20]Hemingway J, Hawkes NJ, McCarroll L, Ranson H: The molecular basis of insecticide resistance in mosquitoes. Insect Biochem Mol Biol 2004, 34(7):653-665.
- [21]Wood O, Hanrahan S, Coetzee M, Koekemoer L, Brooke B: Cuticle thickening associated with pyrethroid resistance in the major malaria vector Anopheles funestus. Parasit Vectors 2010, 3:67. BioMed Central Full Text
- [22]Ozsolak F, Milos PM: RNA sequencing: advances, challenges and opportunities. Nat Rev Genet 2011, 12(2):87-98.
- [23]Gibbons JG, Janson EM, Hittinger CT, Johnston M, Abbot P, Rokas A: Benchmarking next-generation transcriptome sequencing for functional and evolutionary genomics. Mol Biol Evol 2009, 26(12):2731-2744.
- [24]Gregory R, Darby AC, Irving H, Coulibaly MB, Hughes M, Koekemoer LL, Coetzee M, Ranson H, Hemingway J, Hall N, Wondji CS: A de novo expression profiling of Anopheles funestus, malaria vector in Africa, using 454 pyrosequencing. PLoS One 2011, 6(2):e17418.
- [25]Paris M, Despres L: Identifying insecticide resistance genes in mosquito by combining AFLP genome scans and 454 pyrosequencing. Mol Ecol 2012, 21(7):1672-1686.
- [26]Zheng W, Peng T, He W, Zhang H: High-throughput sequencing to reveal genes involved in reproduction and development in Bactrocera dorsalis (Diptera: Tephritidae). PLoS One 2012, 7(5):e36463.
- [27]Vera JC, Wheat CW, Fescemyer HW, Frilander MJ, Crawford DL, Hanski I, Marden JH: Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing. Mol Ecol 2008, 17(7):1636-1647.
- [28]Zagrobelny M, Scheibye-Alsing K, Jensen N, Moller B, Gorodkin J, Bak S: 454 pyrosequencing based transcriptome analysis of Zygaena filipendulae with focus on genes involved in biosynthesis of cyanogenic glucosides. BMC Genomics 2009, 10(1):574. BioMed Central Full Text
- [29]Pauchet Y, Wilkinson P, van Munster M, Augustin S, Pauron D, ffrench-Constant RH: Pyrosequencing of the midgut transcriptome of the poplar leaf beetle Chrysomela tremulae reveals new gene families in Coleoptera. Insect Mol Biol 2009, 39(5–6):403-413.
- [30]Bai X, Zhang W, Orantes L, Jun TH, Mittapalli O, Mian MA, Michel AP: Combining next-generation sequencing strategies for rapid molecular resource development from an invasive aphid species. Aphis glycines. PLoS One 2010, 5(6):e11370.
- [31]Bai X, Mamidala P, Rajarapu SP, Jones SC, Mittapalli O: Transcriptomics of the Bed Bug (Cimex lectularius). PLoS One 2011, 6(1):e16336.
- [32]Pauchet Y, Wilkinson P, Vogel H, Nelson DR, Reynolds SE, Heckel DG, ffrench-Constant RH: Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence. Insect Mol Biol 2010, 19(1):61-75.
- [33]Zou Z, Najar F, Wang Y, Roe B, Jiang H: Pyrosequence analysis of expressed sequence tags for Manduca sexta hemolymph proteins involved in immune responses. Insect Biochem Mol Biol 2008, 38(6):677-682.
- [34]Zhang F, Guo H, Zheng H, Zhou T, Zhou Y, Wang S, Fang R, Qian W, Chen X: Massively parallel pyrosequencing-based transcriptome analyses of small brown planthopper (Laodelphax striatellus), a vector insect transmitting rice stripe virus (RSV). BMC Genomics 2010, 11(1):303. BioMed Central Full Text
- [35]Olafson PU, Lohmeyer KH, Dowd SE: Analysis of expressed sequence tags from a significant livestock pest, the stable fly (Stomoxys calcitrans), identifies transcripts with a putative role in chemosensation and sex determination. Arch Insect Biochem Physiol 2010, 74(3):179-204.
- [36]O’Neil S, Dzurisin J, Carmichael R, Lobo N, Emrich S, Hellmann J: Population-level transcriptome sequencing of nonmodel organisms Erynnis propertius and Papilio zelicaon. BMC Genomics 2010, 11(1):310. BioMed Central Full Text
- [37]Hull JJ, Geib SM, Fabrick JA, Brent CS: Sequencing and de novo assembly of the western tarnished plant bug (Lygus hesperus) transcriptome. PLoS One 2013, 8(1):e55105.
- [38]Mittapalli O, Bai X, Mamidala P, Rajarapu SP, Bonello P, Herms DA: Tissue-specific transcriptomics of the exotic invasive insect pest emerald ash borer (Agrilus planipennis). PLoS One 2010, 5(10):e13708.
- [39]WHO: Test Procedures for Insecticide Resistance Monitoring in Malaria Vector Mosquitoes. Geneva, Switzerland: World Health Organization; 2013.
- [40]Gao Q, Beebe NW, Cooper RD: Molecular identification of the malaria vectors Anopheles anthropophagus and Anopheles sinensis (Diptera: Culicidae) in central China using polymerase chain reaction and appraisal of their position within the Hyrcanus group. J Med Entomol 2004, 41(1):5-11.
- [41]Bonizzoni M, Afrane Y, Dunn WA, Atieli FK, Zhou G, Zhong D, Li J, Githeko A, Yan G: Comparative transcriptome analyses of deltamethrin-resistant and -susceptible Anopheles gambiae mosquitoes from Kenya by RNA-Seq. PLoS One 2012, 7(9):e44607.
- [42]Bräutigam A, Mullick T, Schliesky S, Weber APM: Critical assessment of assembly strategies for non-model species mRNA-Seq data and application of next-generation sequencing to the comparison of C3 and C4 species. J Exp Bot 2011, 62(9):3093-3102.
- [43]Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M: Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005, 21(18):3674-3676.
- [44]Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L, Wang J: WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 2006, 34(suppl 2):W293-W297.
- [45]Audic S, Claverie JM: The significance of digital gene expression profiles. Genome Res 1997, 7(10):986-995.
- [46]Li H, Chen S, Song A, Wang H, Fang W, Guan Z, Jiang J, Chen F: RNA-Seq derived identification of differential transcription in the chrysanthemum leaf following inoculation with Alternaria tenuissima. BMC Genomics 2014, 15(1):9. BioMed Central Full Text
- [47]Young ND, Jex AR, Li B, Liu S, Yang L, Xiong Z, Li Y, Cantacessi C, Hall RS, Xu X, Chen F, Wu X, Zerlotini A, Oliveira G, Hofmann A, Zhang G, Fang X, Kang Y, Campbell BE, Loukas A, Ranganathan S, Rollinson D, Rinaldi G, Brindley PJ, Yang H, Wang J, Wang J, Gasser RB: Whole-genome sequence of Schistosoma haematobium. Nat Genet 2012, 44(2):221-225.
- [48]Benjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B Stat Methodol 1995, 57(1):289-300.
- [49]Faircloth BC: msatcommander: detection of microsatellite repeat arrays and automated, locus-specific primer design. Mol Ecol Resour 2008, 8(1):92-94.
- [50]Rice P, Longden I, Bleasby A: EMBOSS: the European molecular biology open software suite. Trends Genet 2000, 16(6):276-277.
- [51]Pellino M, Hojsgaard D, Schmutzer T, Scholz U, Hörandl E, Vogel H, Sharbel TF: Asexual genome evolution in the apomictic Ranunculus auricomus complex: examining the effects of hybridization and mutation accumulation. Mol Ecol 2013, 22(23):5908-5921.
- [52]Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008, 3(6):1101-1108.
- [53]Copley SD: Evolution of a metabolic pathway for degradation of a toxic xenobiotic: the patchwork approach. Trends Biochem Sci 2000, 25(6):261-265.
- [54]Copping LG: Metabolic pathways of agrochemicals: part two –insecticides and fungicides. Pest Manag Sci 2000, 56(1):103-104.
- [55]David JP, Strode C, Vontas J, Nikou D, Vaughan A, Pignatelli PM, Louis C, Hemingway J, Ranson H: The Anopheles gambiae detoxification chip: a highly specific microarray to study metabolic-based insecticide resistance in malaria vectors. Proc Natl Acad Sci USA 2005, 102(11):4080-4084.
- [56]Vontas J, Blass C, Koutsos AC, David JP, Kafatos FC, Louis C, Hemingway J, Christophides GK, Ranson H: Gene expression in insecticide resistant and susceptible Anopheles gambiae strains constitutively or after insecticide exposure. Insect Mol Biol 2005, 14(5):509-521.
- [57]Chow C-Y: Malaria vector in China. Chinese J Entomology Special Publ 1991, 6:67-79.
- [58]Ree H-I: Studies on Anopheles sinensis, the vector species of vivax malaria in Korea. Korean J Parasitol 2005, 43(3):75-92.
- [59]Sinka M, Bangs M, Manguin S, Chareonviriyaphap T, Patil A, Temperley W, Gething P, Elyazar I, Kabaria C, Harbach R, Hay S: The dominant Anopheles vectors of human malaria in the Asia-Pacific region: occurrence data, distribution maps and bionomic precis. Parasit Vectors 2011, 4(1):89. BioMed Central Full Text
- [60]Pan J-Y, Zhou S-S, Zheng X, Huang F, Wang D-Q, Shen Y-Z, Su Y-P, Zhou G-C, Liu F, Jiang J-J: Vector capacity of Anopheles sinensis in malaria outbreak areas of central China. Parasit Vectors 2012, 5(1):136. BioMed Central Full Text
- [61]Zhou D, Zhang D, Ding G, Shi L, Hou Q, Ye Y, Xu Y, Zhou H, Xiong C, Li S, Yu J, Hong S, Yu X, Zou P, Chen C, Chang X, Wang W, Lv Y, Sun Y, Ma L, Shen B, Zhu C: Genome sequence of Anopheles sinensis provides insight into genetics basis of mosquito competence for malaria parasites. BMC Genomics 2014, 15(1):42. BioMed Central Full Text
- [62]Price DP, Nagarajan V, Churbanov A, Houde P, Milligan B, Drake LL, Gustafson JE, Hansen IA: The Fat body transcriptomes of the yellow fever mosquito, Aedes aegypti, Pre- and post- blood meal. PLoS One 2011, 6(7):e22573.
- [63]Poelchau M, Reynolds J, Denlinger D, Elsik C, Armbruster P: A de novo transcriptome of the Asian tiger mosquito, Aedes albopictus, to identify candidate transcripts for diapause preparation. BMC Genomics 2011, 12(1):1-19. BioMed Central Full Text
- [64]Pridgeon JW, Liu N: Overexpression of the cytochrome c oxidase subunit I gene associated with a pyrethroid resistant strain of German cockroaches, Blattella germanica (L.). Insect Biochem Mol Biol 2003, 33(10):1043-1048.
- [65]McLaughlin LA, Niazi U, Bibby J, David JP, Vontas J, Hemingway J, Ranson H, Sutcliffe MJ, Paine MJ: Characterization of inhibitors and substrates of Anopheles gambiae CYP6Z2. Insect Mol Biol 2008, 17(2):125-135.
- [66]Muller P, Donnelly M, Ranson H: Transcription profiling of a recently colonised pyrethroid resistant Anopheles gambiae strain from Ghana. BMC Genomics 2007, 8(1):36. BioMed Central Full Text
- [67]Jones C, Haji K, Khatib B, Bagi J, Mcha J, Devine G, Daley M, Kabula B, Ali A, Majambere S, Ranson H: The dynamics of pyrethroid resistance in Anopheles arabiensis from Zanzibar and an assessment of the underlying genetic basis. Parasit Vectors 2013, 6(1):343. BioMed Central Full Text
- [68]Nardini L, Christian R, Coetzer N, Koekemoer L: DDT and pyrethroid resistance in Anopheles arabiensis from South Africa. Parasit Vectors 2013, 6(1):229. BioMed Central Full Text
- [69]Hardstone MC, Komagata O, Kasai S, Tomita T, Scott JG: Use of isogenic strains indicates CYP9M10 is linked to permethrin resistance in Culex pipiens quinquefasciatus. Insect Mol Biol 2010, 19(6):717-726.
- [70]Liu N, Li T, Reid WR, Yang T, Zhang L: Multiple cytochrome P450 genes: their constitutive overexpression and permethrin induction in insecticide resistant mosquitoes. Culex quinquefasciatus. PLoS One 2011, 6(8):e23403.
- [71]Zhu F, Li T, Zhang L, Liu N: Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies. Musca domestica. BMC Physiol 2008, 8(1):18. BioMed Central Full Text
- [72]Zhu F, Liu N: Differential expression of CYP6A5 and CYP6A5v2 in pyrethroid-resistant house flies. Musca domestica. Arch Insect Biochem Physiol 2008, 67(3):107-119.
- [73]Sun Y, Zou P, Yu XY, Chen C, Yu J, Shi LN, Hong SC, Zhou D, Chang XL, Wang WJ, Shen B, Zhang DH, Ma L, Zhu CL: Functional characterization of an arrestin gene on insecticide resistance of Culex pipiens pallens. Parasit Vectors 2012, 5:134. BioMed Central Full Text
- [74]Nardini L, Christian R, Coetzer N, Ranson H, Coetzee M, Koekemoer L: Detoxification enzymes associated with insecticide resistance in laboratory strains of Anopheles arabiensis of different geographic origin. Parasit Vectors 2012, 5(1):113. BioMed Central Full Text
- [75]Yang T, Liu N: Genome analysis of cytochrome p450s and their expression profiles in insecticide resistant mosquitoes. Culex quinquefasciatus. PLoS One 2011, 6(12):e29418.
- [76]Carvalho RA, Azeredo-Espin AM, Torres TT: Deep sequencing of New World screw-worm transcripts to discover genes involved in insecticide resistance. BMC Genomics 2010, 11:695. BioMed Central Full Text
- [77]Cui PH, Lee AC, Zhou F, Murray M: Impaired transactivation of the human CYP2J2 arachidonic acid epoxygenase gene in HepG2 cells subjected to nitrative stress. Br J Pharmacol 2010, 159(7):1440-1449.
- [78]Marinotti O, Nguyen QK, Calvo E, James AA, Ribeiro JM: Microarray analysis of genes showing variable expression following a blood meal in Anopheles gambiae. Insect Mol Biol 2005, 14(4):365-373.
- [79]Bonizzoni M, Britton M, Marinotti O, Dunn W, Fass J, James A: Probing functional polymorphisms in the dengue vector. Aedes aegypti. BMC Genomics 2013, 14(1):739. BioMed Central Full Text
- [80]Morlais I, Severson DW: Intraspecific DNA variation in nuclear genes of the mosquito Aedes aegypti. Insect Mol Biol 2003, 12(6):631-639.
- [81]Holt RA, Subramanian GM, Halpern A, Sutton GG, Charlab R, Nusskern DR, Wincker P, Clark AG, Ribeiro JC, Wides R, Salzberg SL, Loftus B, Yandell M, Majoros WH, Rusch DB, Lai Z, Kraft CL, Abril JF, Anthouard V, Arensburger P, Atkinson PW, Baden H, de Berardinis V, Baldwin D, Benes V, Biedler J, Blass C, Bolanos R, Boscus D, Barnstead M: The genome sequence of the malaria mosquito Anopheles gambiae. Science 2002, 298(5591):129-149.
- [82]Wondji C, Hemingway J, Ranson H: Identification and analysis of Single Nucleotide Polymorphisms (SNPs) in the mosquito Anopheles funestus, malaria vector. BMC Genomics 2007, 8(1):5. BioMed Central Full Text
- [83]Ning J, Wang M, Li C, Sun S: Transcriptome sequencing and De Novo analysis of the copepod Calanus sinicus Using 454 GS FLX. PLoS One 2013, 8(5):e63741.
- [84]Roberts SB, Hauser L, Seeb LW, Seeb JE: Development of genomic resources for pacific herring through targeted transcriptome pyrosequencing. PLoS One 2012, 7(2):e30908.
- [85]Bustamante CD, Fledel-Alon A, Williamson S, Nielsen R, Todd Hubisz M, Glanowski S, Tanenbaum DM, White TJ, Sninsky JJ, Hernandez RD, Civello D, Adams MD, Cargill M: Natural selection on protein-coding genes in the human genome. Nature 2005, 437(7062):1153-1157.