Limits...
Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples.

Matranga CB, Andersen KG, Winnicki S, Busby M, Gladden AD, Tewhey R, Stremlau M, Berlin A, Gire SK, England E, Moses LM, Mikkelsen TS, Odia I, Ehiane PE, Folarin O, Goba A, Kahn SH, Grant DS, Honko A, Hensley L, Happi C, Garry RF, Malboeuf CM, Birren BW, Gnirke A, Levin JZ, Sabeti PC - Genome Biol. (2014)

Bottom Line: Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA.This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries.We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries.

View Article: PubMed Central - PubMed

ABSTRACT
We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.

Show MeSH

Related in: MedlinePlus

Depletion of rRNA from rodent and macaque LASV isolates. (A) Depletion of rRNA (top) and unique LASV (bottom) enrichment from Mastomys natalensis spleen and (B) various tissues from cynomolgous macaque (day 12 post LASV infection). Numbers over fraction unique reads represent fold-enrichment in LASV content after rRNA depletion.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4262991&req=5

Fig3: Depletion of rRNA from rodent and macaque LASV isolates. (A) Depletion of rRNA (top) and unique LASV (bottom) enrichment from Mastomys natalensis spleen and (B) various tissues from cynomolgous macaque (day 12 post LASV infection). Numbers over fraction unique reads represent fold-enrichment in LASV content after rRNA depletion.

Mentions: We demonstrated the utility of host rRNA depletion on tissue samples collected from LASV-infected rodents and non-human primate disease models. These tissue samples contain higher levels of 18S rRNA than human plasma or serum (on average 5 times more - data not shown). Using the same human rRNA probes, we depleted rRNA and enriched unique LASV reads approximately five-fold in a Mastomys natalensis spleen sample (Figure 3A). Most of the remaining 10% (approximately) rRNA reads aligned to 28S rRNA sequences which are divergent between humans and rodents [29]. Similarly, our protocol reduced the rRNA content in six different tissue samples from cynomolgous macaques to approximately 10% (Figure 3B). Depletion of rRNA led to an increase in LASV content in all macaque samples, reaching the highest levels in adrenal gland and spleen, two tissues known to accumulate LASV during infection [30].Figure 3


Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples.

Matranga CB, Andersen KG, Winnicki S, Busby M, Gladden AD, Tewhey R, Stremlau M, Berlin A, Gire SK, England E, Moses LM, Mikkelsen TS, Odia I, Ehiane PE, Folarin O, Goba A, Kahn SH, Grant DS, Honko A, Hensley L, Happi C, Garry RF, Malboeuf CM, Birren BW, Gnirke A, Levin JZ, Sabeti PC - Genome Biol. (2014)

Depletion of rRNA from rodent and macaque LASV isolates. (A) Depletion of rRNA (top) and unique LASV (bottom) enrichment from Mastomys natalensis spleen and (B) various tissues from cynomolgous macaque (day 12 post LASV infection). Numbers over fraction unique reads represent fold-enrichment in LASV content after rRNA depletion.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4262991&req=5

Fig3: Depletion of rRNA from rodent and macaque LASV isolates. (A) Depletion of rRNA (top) and unique LASV (bottom) enrichment from Mastomys natalensis spleen and (B) various tissues from cynomolgous macaque (day 12 post LASV infection). Numbers over fraction unique reads represent fold-enrichment in LASV content after rRNA depletion.
Mentions: We demonstrated the utility of host rRNA depletion on tissue samples collected from LASV-infected rodents and non-human primate disease models. These tissue samples contain higher levels of 18S rRNA than human plasma or serum (on average 5 times more - data not shown). Using the same human rRNA probes, we depleted rRNA and enriched unique LASV reads approximately five-fold in a Mastomys natalensis spleen sample (Figure 3A). Most of the remaining 10% (approximately) rRNA reads aligned to 28S rRNA sequences which are divergent between humans and rodents [29]. Similarly, our protocol reduced the rRNA content in six different tissue samples from cynomolgous macaques to approximately 10% (Figure 3B). Depletion of rRNA led to an increase in LASV content in all macaque samples, reaching the highest levels in adrenal gland and spleen, two tissues known to accumulate LASV during infection [30].Figure 3

Bottom Line: Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA.This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries.We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries.

View Article: PubMed Central - PubMed

ABSTRACT
We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.

Show MeSH
Related in: MedlinePlus