Improved assembly procedure of viral RNA genomes amplified with Phi29 polymerase from new generation sequencing data.
BACKGROUND
New sequencing technologies have opened the way to the discovery and the characterization of pathogenic viruses in clinical samples. However, the use of these new methods can require an amplification of viral RNA prior to the sequencing. Among all the available methods, the procedure based on the use of Phi29 polymerase produces a huge amount of amplified DNA. However, its major disadvantage is to generate a large number of chimeric sequences which can affect the assembly step. The pre-process method proposed in this study strongly limits the negative impact of chimeric reads in order to obtain the full-length of viral genomes.


RESULTS
Three different assembly softwares (ABySS, Ray and SPAdes) were tested for their ability to correctly assemble the full-length of viral genomes. Although in all cases, our pre-processed method improved genome assembly, only its combination with the use of SPAdes allowed us to obtain the full-length of the viral genomes tested in one contig.


CONCLUSIONS
The proposed pipeline is able to overcome drawbacks due to the generation of chimeric reads during the amplification of viral RNA which considerably improves the assembling of full-length viral genomes.
Institut Pasteur, Epidemiology and Physiopathology of Oncogenic Viruses, 25 Rue Du Docteur Roux, 75724, Paris, France. nicolas.berthet@ird.fr.|Centre National de La Recherche Scientifique, UMR 3569, 25 Rue Du Docteur Roux, 75724, Paris, France. nicolas.berthet@ird.fr.|Département Zoonose Et Maladies Emergentes, Syndromes Cliniques Et Virus Associés, Centre International de Recherches Médicales de Franceville (CIRMF), BP769, Franceville, Gabon. nicolas.berthet@ird.fr.
Nicolas
N
Berthet
Institut Pasteur, Plate-forme de Bioanalyse Génomique, 25 Rue Du Docteur Roux, 75724, Paris, France.
Stéphane
S
Descorps-Declère
Département Zoonose Et Maladies Emergentes, Syndromes Cliniques Et Virus Associés, Centre International de Recherches Médicales de Franceville (CIRMF), BP769, Franceville, Gabon.
Andriniaina Andy
AA
Nkili-Meyong
Département de Virologie, Institut Pasteur de Bangui, BP 923, Bangui, République Centrafricaine.
Emmanuel
E
Nakouné
Institut Pasteur, Epidemiology and Physiopathology of Oncogenic Viruses, 25 Rue Du Docteur Roux, 75724, Paris, France.|Centre National de La Recherche Scientifique, UMR 3569, 25 Rue Du Docteur Roux, 75724, Paris, France.
Antoine
A
Gessain
Unité Environnement Et Risques Infectieux, Institut Pasteur, Cellule D'Intervention Biologique D'Urgence, 25 Rue Du Docteur Roux, 75724, Paris, France.
Jean-Claude
JC
Manuguerra
Département de Virologie, Institut Pasteur de Bangui, BP 923, Bangui, République Centrafricaine.
Mirdad
M
Kazanji
D000524:Alphavirus / Q000235:genetics; D002488:Central African Republic; D019295:Computational Biology; D020451:Contig Mapping; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D016679:Genome, Viral; D008574:Mengovirus / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012367:RNA, Viral; D012016:Reference Values; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA / Q000379:methods*; D012984:Software; D019065:Virus Assembly
D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases
Amplification with phi29 polymerase; Assembling genome; Next generation sequencing; RNA viral genome; SPAdes