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Table 2 Details of brain and BHK cultured samples, relating to the extraction method used and RNA concentration during preparation stages resulting in number of reads obtained and outcome of obtaining full viral genome sequence

From: Next generation sequencing of viral RNA genomes

Virus Sample origin Extraction method Concentration after RNA extraction (ng/μl) Concentration after gDNA depletion (ng/μl) Concentration RNA using Ribogreen1(ng/μl) Total Noof reads Noof vial reads % viral specific reads Genome as single contig De Novo detection
Brain tissue samples
RV437 mouse TRIzol® 5,404.4 410.77 121.77 282,370 1,489 0.53 Yesa Yes
RV61 mouse TRIzol® 3,362.8 ND 58.9 191,508# 1,810 0.95 Yesc Yes
RV2417 Dog TRIzol® 5,431.3 1799.21 57.78 96,014 1,488 1.55 Yesb Yes
RV2508 African civet^ TRIzol® 6,537.4 66.55 28.18 139,841 626 0.45 Yesa Yes
RV2627 Cow TRIzol® 2,385.8 111.81 26.60 11,470 682 5.95 Yes Yes
RNeasy mini N/A 4.99 0.95 239 2 0.84 No No
RV2516 Cow TRIzol® 1,891.2 45.84 11.09 154,068# 550 0.36 Yesa Yes
RNeasy mini N/A 11.12 2.14 7,991 17 0.21 No No
RV50 mouse TRIzol® 2,186.2 18.76 7.64 76,568 293 0.38 Yes Yes
RV2772 Dog TRIzol® 1,833.9 3.27 1.50 305,110 391 0.13 Yes Yes
RNeasy mini N/A 7.19 1.08 48,849 180 0.37 No Yes
BHK cultured virus samples
RV2324 cell pellet TRIzol® 10,232.9 870.72 75.63 101,599 2,027 1.99 Yes Yes
RV20 depleted TCSN 100 ml TRIzol® LS 3,732.6 2.8 0.52 2,537 1,054 41.55 Yes Yes
RV20 Diluted to 1,000 ND 23.47 10,332 4,369 42.29 Yes Yes
RV1787 depleted TCSN 100 ml TRIzol® LS 4,603 37.87 11.56 8,162 53 0.65 No Yes
RV1787 Diluted to 1,000 ND 119.47 6,861 87 1.27 No Yes
RV2508 cells TCSN 250 μl TRIzol® LS 129.44 ND 11.45 14,587# 223 1.529 No* Yes
RV2508 no cells 127.05 ND 0.46 326 42 12.88 No* Yes
RV2508 high titre    112.5 ND 1.31 1,132 12 1.06 No* Yes
  1. TCSN tissue culture supernatant; N/A – Not Applicable as DNAse treatment is incorporated in the RNA extraction protocol, therefore no concentration before gDNA depletion is available; ND - Not Done as gDNA was not depleted from these samples; 1This column contains the RNA concentration after rRNA depletion for all samples, except RV20, RV1787 and RV2508 TCSN where no rRNA depletion was undertaken. brain either from host, or from one passage in a mouse, *combining these reads resulted in a single genome contig; # combined results from two 454 runs of same library; ^ Stored in RNAlater; a 3’UTR not represented; b 5’UTR not represented; c 3’ and 5’ UTR not represented.
  2. Brain tissue samples ordered by concentration after the depletion process. Where rRNA depletion was not undertaken (RV20, RV1787 and RV2508 TCSN), the concentration values in the Ribogreen column are directly comparable to the RNA extraction concentration.