title: Comparative evaluation of rumen metagenome community using qPCR and MG-RAST.
abstract: Microbial profiling of metagenome communities have been studied extensively using MG-RAST and other related metagenome annotation databases. Although, database based taxonomic profiling provides snapshots of the metagenome architecture, their reliability needs to be validated through more accurate methods. Here, we performed qPCR based absolute quantitation of selected rumen microbes in the liquid and solid fraction of the rumen fluid of river buffalo adapted to varying proportion of concentrate to green or dry roughages and compared with the MG-RAST based annotation of the metagenomes sequences of 16S r-DNA amplicons and high throughput shotgun sequencing. Animals were adapted to roughage-to-concentrate ratio in the proportion of 50:50, 75:25 and 100:00, respectively for six weeks. At the end of each treatment, rumen fluid was collected at 3 h post feeding. qPCR revealed that the relative abundance of Prevotella bryantii was higher, followed by the two cellulolytic bacteria Fibrobacter succinogens and Ruminococcus flavefaciens that accounted up to 1.33% and 0.78% of the total rumen bacteria, respectively. While, Selenomonas ruminantium and archaea Methanomicrobiales were lower in microbial population in the rumen of buffalo. There was no statistically significant difference between the enumerations shown by qPCR and analysis of the shotgun sequencing data by MG-RAST except for Prevotella. These results indicate the variations in abundance of different microbial species in buffalo rumen under varied feeding regimes as well as in different fractions of rumen liquor, i.e. solid and the liquid. The results also present the reliability of shotgun sequencing to describe metagenome and analysis/annotation by MG-RAST.
authors.affiliation: Department of Animal Biotechnology, College of Veterinary Science & Animal Husbandry, Anand Agricultural University, Anand, Gujarat 388 001, India. cgjoshi@rediffmail.com.
authors.forename: Neelam M
authors.initials: NM
authors.lastname: Nathani
authors.forename: Amrutlal K
authors.initials: AK
authors.lastname: Patel
authors.forename: Prakash S
authors.initials: PS
authors.lastname: Dhamannapatil
authors.forename: Ramesh K
authors.initials: RK
authors.lastname: Kothari
authors.forename: Krishna M
authors.initials: KM
authors.lastname: Singh
authors.forename: Chaitanya G
authors.initials: CG
authors.lastname: Joshi
references.citation: Nucleic Acids Res. 2005 Oct 07;33(17):5691-702
references.pmid: 16214803
references.citation: Appl Environ Microbiol. 1998 Oct;64(10):3683-9
references.pmid: 9758785
references.citation: Curr Microbiol. 2009 Apr;58(4):294-9
references.pmid: 19018588
references.citation: Appl Environ Microbiol. 1995 Jul;61(7):2798-801
references.pmid: 7618894
references.citation: Appl Environ Microbiol. 1996 Mar;62(3):1084-8
references.pmid: 8975600
references.citation: J Appl Microbiol. 2003;95(3):621-30
references.pmid: 12911711
references.citation: FEMS Microbiol Lett. 2010 Apr;305(1):49-57
references.pmid: 20158525
references.citation: Appl Environ Microbiol. 2006 Mar;72(3):1719-28
references.pmid: 16517615
references.citation: Gene. 2008 Jun 15;416(1-2):44-7
references.pmid: 18420359
references.citation: Appl Microbiol Biotechnol. 2007 May;75(1):165-74
references.pmid: 17235560
references.citation: Genome Res. 1996 Oct;6(10):986-94
references.pmid: 8908518
references.citation: Appl Environ Microbiol. 1993 Mar;59(3):695-700
references.pmid: 7683183
references.citation: J Anim Physiol Anim Nutr (Berl). 2013 Jun;97(3):439-45
references.pmid: 22356106
references.citation: Appl Environ Microbiol. 2002 Feb;68(2):673-90
references.pmid: 11823207
references.citation: FEMS Microbiol Rev. 1997 Nov;21(3):213-29
references.pmid: 9451814
references.citation: Trends Genet. 1994 Nov;10(11):402-7
references.pmid: 7809946
references.citation: J Clin Microbiol. 2002 Dec;40(12):4423-7
references.pmid: 12454130
references.citation: Appl Environ Microbiol. 1999 Dec;65(12):5372-7
references.pmid: 10583991
references.citation: Nucleic Acids Res. 2007 Jan;35(Database issue):D347-53
references.pmid: 17145713
references.citation: Appl Environ Microbiol. 1998 Feb;64(2):795-9
references.pmid: 9464425
references.citation: Appl Environ Microbiol. 1995 Aug;61(8):3069-75
references.pmid: 7487040
references.citation: Appl Environ Microbiol. 2000 Nov;66(11):4605-14
references.pmid: 11055900
references.citation: J Clin Microbiol. 1999 Feb;37(2):464-6
references.pmid: 9889245
references.citation: Appl Environ Microbiol. 2001 Jun;67(6):2766-74
references.pmid: 11375193
references.citation: J Immunol Methods. 2000 Sep 21;243(1-2):191-210
references.pmid: 10986415
references.citation: Bacteriol Rev. 1959 Sep;23(3):125-53
references.pmid: 13805451
references.citation: FEMS Microbiol Lett. 2001 Nov 13;204(2):361-6
references.pmid: 11731149
references.citation: J Bacteriol. 1998 Sep;180(18):4765-74
references.pmid: 9733676