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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.

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Related in: MedlinePlus

Depletion of rRNA from EBOV-Sierra Leone clinical samples. (A) Percentage rRNA (left) and unique EBOV content (right) with (gray) and without (blue) rRNA depletion in four individual clinical serum isolates (G3676-2, G3677-1, G3677-2, G3682-1). (B) Average EBOV genome coverage with (gray) and without (blue) rRNA depletion from four individual isolates with standard deviation (black). N, VP35, VP40, GP, VP30, VP24, L: boundary for each gene in the EBOV genome. Positions and variant allele of two iSNVs (in G3676-2 only) observed after rRNA depletion are depicted.
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Fig5: Depletion of rRNA from EBOV-Sierra Leone clinical samples. (A) Percentage rRNA (left) and unique EBOV content (right) with (gray) and without (blue) rRNA depletion in four individual clinical serum isolates (G3676-2, G3677-1, G3677-2, G3682-1). (B) Average EBOV genome coverage with (gray) and without (blue) rRNA depletion from four individual isolates with standard deviation (black). N, VP35, VP40, GP, VP30, VP24, L: boundary for each gene in the EBOV genome. Positions and variant allele of two iSNVs (in G3676-2 only) observed after rRNA depletion are depicted.

Mentions: Using our approach, we were able to lower the rRNA contamination in all four samples from >80% to <0.5% (Figure 5A). The concomitant increase of EBOV content was approximately 13- to 24-fold, with unique content reaching approximately 35% of total reads in one of the rRNA depleted libraries. Although we sequenced eight libraries on a single MiSeq run, we achieved >50× average coverage for 99% of the EBOV genome (Figure 5B).Figure 5


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 EBOV-Sierra Leone clinical samples. (A) Percentage rRNA (left) and unique EBOV content (right) with (gray) and without (blue) rRNA depletion in four individual clinical serum isolates (G3676-2, G3677-1, G3677-2, G3682-1). (B) Average EBOV genome coverage with (gray) and without (blue) rRNA depletion from four individual isolates with standard deviation (black). N, VP35, VP40, GP, VP30, VP24, L: boundary for each gene in the EBOV genome. Positions and variant allele of two iSNVs (in G3676-2 only) observed after rRNA depletion are depicted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Depletion of rRNA from EBOV-Sierra Leone clinical samples. (A) Percentage rRNA (left) and unique EBOV content (right) with (gray) and without (blue) rRNA depletion in four individual clinical serum isolates (G3676-2, G3677-1, G3677-2, G3682-1). (B) Average EBOV genome coverage with (gray) and without (blue) rRNA depletion from four individual isolates with standard deviation (black). N, VP35, VP40, GP, VP30, VP24, L: boundary for each gene in the EBOV genome. Positions and variant allele of two iSNVs (in G3676-2 only) observed after rRNA depletion are depicted.
Mentions: Using our approach, we were able to lower the rRNA contamination in all four samples from >80% to <0.5% (Figure 5A). The concomitant increase of EBOV content was approximately 13- to 24-fold, with unique content reaching approximately 35% of total reads in one of the rRNA depleted libraries. Although we sequenced eight libraries on a single MiSeq run, we achieved >50× average coverage for 99% of the EBOV genome (Figure 5B).Figure 5

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