Centro de Pesquisas René Rachou CPq RR Fundação

Centro de Pesquisas René Rachou (CPq. RR) Fundação Oswaldo Cruz Pós-Graduação em Ciências da Saúde Biologia Celular e Molecular A genomic study of Anopheles aquasalis, a neglected human malaria vector: metagenomic broad survey of associated microbiota and potential virosphere. Luis Eduardo Martínez Villegas PI: Dr. Paulo F. Paolluci Pimenta Medical Entomology Laboratory Col: Dr. Guilherme Corrêa Computational Biology Group August 26, 2011 Belo Horizonte – MG

Materials and methods - DNA extraction ← → Mosquito stage. Tested with 30 - 50 - 90 - 130 adults/pupae (Aedes aegypti) Organic extraction protocol (phenol/chlorofórm): macro-scale / refrigerated. - Mosquito colony Isogenic started in 1993 Training on anopheline rearing at LAFICAVE / Ibex, RJ. (march, 2012) Obtaining enough biomass for extraction took 5 months.

Proposed asembly pipeline Ion Torrent My. Seq Pac. Bio Key concepts: Hybrid assembly / Metagenome-like broad survey

Results SOLi. D sequencing DNA extraction : out of 134 pupae , 170 ug DNA were obtained

Filtering with csfasta perl in house script + SAET tool. 62. 788. 468 7. 149. 898 (11, 39) F 3 62. 787. 134 8. 252. 380 (13, 14) 125. 575. 602 15. 402. 278 (12, 27) R 3 3 -5 kb Initial Nº of reads Total 1 -2 kb Primers R 3 Library Nº de reads after filtering: csfasta_quality_filter * –m + SAET No. (% of initial No. ) 62. 246. 155 5. 205. 086 (8, 36) F 3 58. 121. 290 5. 223. 400 (8, 99) Total 120. 367. 445 10. 428. 486 (8, 66)

Results Ion Torrent sequencing R_2013_04_12_21_10_15_user_PGM-7_Auto_user_PGM -7_15. fastq. gd. html

Results Ion Torrent sequencing R_2013_06_07_18_44_36_user_Genoma_Anopheles 2_user_G enoma_Anopheles 2. fastq. gd. html

Results Maping the SOLi. D generated reads.

In parallel Mapping: Agam. P 3 / DROMEL / Adarc / A. aquasalis ESTś / A. albimanus transcriptome

Materials and methods Shotgun metagenomics: MG-RAST Ion Torrent reads (Glass, 2010)