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Improved assembly procedure of viral RNA genomes amplified with Phi29 polymerase from new generation sequencing data

View Article: PubMed Central - PubMed

ABSTRACT

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.

Findings: 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.

Electronic supplementary material: The online version of this article (doi:10.1186/s40659-016-0099-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Phylogenetic tree of mengo and encephalomyocarditis viruses. Phylogenetic analysis was based on nucleic acid sequences of the whole genomes of mengo and encephalomyocarditis viruses from the NCBI database
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Fig2: Phylogenetic tree of mengo and encephalomyocarditis viruses. Phylogenetic analysis was based on nucleic acid sequences of the whole genomes of mengo and encephalomyocarditis viruses from the NCBI database

Mentions: The Mengovirus belongs to the genus Cardiovirus and the Picornaviridae family. It was isolated for the first time in 1948 in Uganda from a rhesus monkey which had developed hind limb paralysis. Genomic analysis of 7717 nucleotides for the strain ArNB-3741 showed the typical organization of the genome of the Mengovirus with one ORF encoding a polyprotein of 2293 amino acid flancked by two UTR in 5′ and 3′. Our genome of the Mengovirus shares 79.7 and 94.5 % at nucleic and amino acids levels with the sequence of the EMC virus (1086C), the closest strain. The phylogenetic tree based on the polyprotein sequence showed that our strain of Mengovirus isolated in the CAR belongs to the group of mengo/EMC viruses isolated from different species of rodents (mice and rats) (Fig. 2).Fig. 2


Improved assembly procedure of viral RNA genomes amplified with Phi29 polymerase from new generation sequencing data
Phylogenetic tree of mengo and encephalomyocarditis viruses. Phylogenetic analysis was based on nucleic acid sequences of the whole genomes of mengo and encephalomyocarditis viruses from the NCBI database
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5015205&req=5

Fig2: Phylogenetic tree of mengo and encephalomyocarditis viruses. Phylogenetic analysis was based on nucleic acid sequences of the whole genomes of mengo and encephalomyocarditis viruses from the NCBI database
Mentions: The Mengovirus belongs to the genus Cardiovirus and the Picornaviridae family. It was isolated for the first time in 1948 in Uganda from a rhesus monkey which had developed hind limb paralysis. Genomic analysis of 7717 nucleotides for the strain ArNB-3741 showed the typical organization of the genome of the Mengovirus with one ORF encoding a polyprotein of 2293 amino acid flancked by two UTR in 5′ and 3′. Our genome of the Mengovirus shares 79.7 and 94.5 % at nucleic and amino acids levels with the sequence of the EMC virus (1086C), the closest strain. The phylogenetic tree based on the polyprotein sequence showed that our strain of Mengovirus isolated in the CAR belongs to the group of mengo/EMC viruses isolated from different species of rodents (mice and rats) (Fig. 2).Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

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.

Findings: 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.

Electronic supplementary material: The online version of this article (doi:10.1186/s40659-016-0099-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus