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Viral genome sequencing by random priming methods.

Djikeng A, Halpin R, Kuzmickas R, Depasse J, Feldblyum J, Sengamalay N, Afonso C, Zhang X, Anderson NG, Ghedin E, Spiro DJ - BMC Genomics (2008)

Bottom Line: For the overwhelming majority, their causative agents are RNA viruses which include but are not limited to HIV, Influenza, SARS, Ebola, Dengue, and Hantavirus.We have adapted the SISPA methodology 123 to genome sequencing of RNA and DNA viruses.We used this technique to generate full viral genome sequence in the presence of host contaminants, using viral preparations from cell culture supernatant, allantoic fluid and fecal matter.

View Article: PubMed Central - HTML - PubMed

Affiliation: Viral Genomics Group, J, Craig Venter Institute, Rockville, MD 20850, USA. adjikeng@jcvi.org

ABSTRACT

Background: Most emerging health threats are of zoonotic origin. For the overwhelming majority, their causative agents are RNA viruses which include but are not limited to HIV, Influenza, SARS, Ebola, Dengue, and Hantavirus. Of increasing importance therefore is a better understanding of global viral diversity to enable better surveillance and prediction of pandemic threats; this will require rapid and flexible methods for complete viral genome sequencing.

Results: We have adapted the SISPA methodology 123 to genome sequencing of RNA and DNA viruses. We have demonstrated the utility of the method on various types and sources of viruses, obtaining near complete genome sequence of viruses ranging in size from 3,000-15,000 kb with a median depth of coverage of 14.33. We used this technique to generate full viral genome sequence in the presence of host contaminants, using viral preparations from cell culture supernatant, allantoic fluid and fecal matter.

Conclusion: The method described is of great utility in generating whole genome assemblies for viruses with little or no available sequence information, viruses from greatly divergent families, previously uncharacterized viruses, or to more fully describe mixed viral infections.

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Related in: MedlinePlus

A. Depth of coverage of viruses. Depth of coverage statistics were generated for each contig (using the output of DNAStar Seqman program). Average coverage is the summed length of all sequence reads in a contig, including gaps divided by the contig length. The average and standard deviation for each virus was determined. B. Correlation of genome coverage with colonies picked. The SISPA method was performed for enterobacteriophage M13 (6407 bp), Newcastle disease virus Lasota (15,186 bp) and enterobacteriophage lambda (48502 bp). One, two or three 96-well blocks of clones were sequenced, trimmed and assembled. The sum of the total lengths of edited contigs for each condition was calculated as percent of the total reference genome length.
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Figure 4: A. Depth of coverage of viruses. Depth of coverage statistics were generated for each contig (using the output of DNAStar Seqman program). Average coverage is the summed length of all sequence reads in a contig, including gaps divided by the contig length. The average and standard deviation for each virus was determined. B. Correlation of genome coverage with colonies picked. The SISPA method was performed for enterobacteriophage M13 (6407 bp), Newcastle disease virus Lasota (15,186 bp) and enterobacteriophage lambda (48502 bp). One, two or three 96-well blocks of clones were sequenced, trimmed and assembled. The sum of the total lengths of edited contigs for each condition was calculated as percent of the total reference genome length.

Mentions: Figure 4A shows an analysis of sequence coverage for the viruses examined in this study. On average, four contigs were generated per experiment, ranging in size from 248 nt to 4495 nt with a median contig size of 1395 nt. The contigs had high sequence redundancy, with a median depth of coverage of 14.33, varying from 11.18 for turkey astrovirus (TA) to a high of 40.29 for MS2.


Viral genome sequencing by random priming methods.

Djikeng A, Halpin R, Kuzmickas R, Depasse J, Feldblyum J, Sengamalay N, Afonso C, Zhang X, Anderson NG, Ghedin E, Spiro DJ - BMC Genomics (2008)

A. Depth of coverage of viruses. Depth of coverage statistics were generated for each contig (using the output of DNAStar Seqman program). Average coverage is the summed length of all sequence reads in a contig, including gaps divided by the contig length. The average and standard deviation for each virus was determined. B. Correlation of genome coverage with colonies picked. The SISPA method was performed for enterobacteriophage M13 (6407 bp), Newcastle disease virus Lasota (15,186 bp) and enterobacteriophage lambda (48502 bp). One, two or three 96-well blocks of clones were sequenced, trimmed and assembled. The sum of the total lengths of edited contigs for each condition was calculated as percent of the total reference genome length.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254600&req=5

Figure 4: A. Depth of coverage of viruses. Depth of coverage statistics were generated for each contig (using the output of DNAStar Seqman program). Average coverage is the summed length of all sequence reads in a contig, including gaps divided by the contig length. The average and standard deviation for each virus was determined. B. Correlation of genome coverage with colonies picked. The SISPA method was performed for enterobacteriophage M13 (6407 bp), Newcastle disease virus Lasota (15,186 bp) and enterobacteriophage lambda (48502 bp). One, two or three 96-well blocks of clones were sequenced, trimmed and assembled. The sum of the total lengths of edited contigs for each condition was calculated as percent of the total reference genome length.
Mentions: Figure 4A shows an analysis of sequence coverage for the viruses examined in this study. On average, four contigs were generated per experiment, ranging in size from 248 nt to 4495 nt with a median contig size of 1395 nt. The contigs had high sequence redundancy, with a median depth of coverage of 14.33, varying from 11.18 for turkey astrovirus (TA) to a high of 40.29 for MS2.

Bottom Line: For the overwhelming majority, their causative agents are RNA viruses which include but are not limited to HIV, Influenza, SARS, Ebola, Dengue, and Hantavirus.We have adapted the SISPA methodology 123 to genome sequencing of RNA and DNA viruses.We used this technique to generate full viral genome sequence in the presence of host contaminants, using viral preparations from cell culture supernatant, allantoic fluid and fecal matter.

View Article: PubMed Central - HTML - PubMed

Affiliation: Viral Genomics Group, J, Craig Venter Institute, Rockville, MD 20850, USA. adjikeng@jcvi.org

ABSTRACT

Background: Most emerging health threats are of zoonotic origin. For the overwhelming majority, their causative agents are RNA viruses which include but are not limited to HIV, Influenza, SARS, Ebola, Dengue, and Hantavirus. Of increasing importance therefore is a better understanding of global viral diversity to enable better surveillance and prediction of pandemic threats; this will require rapid and flexible methods for complete viral genome sequencing.

Results: We have adapted the SISPA methodology 123 to genome sequencing of RNA and DNA viruses. We have demonstrated the utility of the method on various types and sources of viruses, obtaining near complete genome sequence of viruses ranging in size from 3,000-15,000 kb with a median depth of coverage of 14.33. We used this technique to generate full viral genome sequence in the presence of host contaminants, using viral preparations from cell culture supernatant, allantoic fluid and fecal matter.

Conclusion: The method described is of great utility in generating whole genome assemblies for viruses with little or no available sequence information, viruses from greatly divergent families, previously uncharacterized viruses, or to more fully describe mixed viral infections.

Show MeSH
Related in: MedlinePlus