Next generation sequencing of viral RNA genomes

BMC Genomics

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

Virus	Sample origin	Extraction method	Concentration after RNA extraction (ng/μl)	Concentration after gDNA depletion (ng/μl)	Concentration RNA using Ribogreen¹(ng/μl)	Total N^oof reads	N^oof 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	Yes^a	Yes
RV61	mouse	TRIzol®	3,362.8	ND	58.9	191,508^#	1,810	0.95	Yes^c	Yes
RV2417	Dog	TRIzol®	5,431.3	1799.21	57.78	96,014	1,488	1.55	Yes^b	Yes
RV2508	African civet^	TRIzol®	6,537.4	66.55	28.18	139,841	626	0.45	Yes^a	Yes
RV2627	Cow	TRIzol®	2,385.8	111.81	26.60	11,470	682	5.95	Yes	Yes
RV2627	Cow	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	Yes^a	Yes
RV2516	Cow	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
RV2772	Dog	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	TCSN 100 ml	TRIzol® LS	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	TCSN 100 ml	TRIzol® LS	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	TCSN 250 μl	TRIzol® LS	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

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.
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.

ISSN: 1471-2164