Biology Reference
In-Depth Information
to nearly 60-100% of that present in plants. Furthermore, their contribution is highest in the open
oceans. Thus the newly emerging metagenomic analysis has to its credit more than 100 projects in
the GOLD web site, Genomes OnLine Database [February, 2008; http://www.Genomesonline.org/
gold (gi)]. Of these, 31 projects have already been completed. By now, nearly 120,000 sequences are
deposited in databases such as RDP. Mention may be made of four large environmental sequencing
projects that have been completed from acid mine drainage (Tyson
et al
., 2004), Sargasso Sea (Venter
et al
., 2004), farm soil (Tringe
et al
., 2005) and whale falls (Tringe
et al
., 2005). In terms of minimum
number of species Sargasso Sea is at fi rst position (with 1000 species) wheras in terms of estimated
total number of species farm soil occupied the fi rst place with >3000 species. Likewise, in terms of
total number of ORFs annotated Sargasso Sea showed maximum (>1000,000) followed by farm soil
(>180,000) (Foerstner
et al
., 2006). There are seven techniques identifi ed for analysis of 16S rRNA
gene. These are as follows.
(i)
Oligonucleotide cataloguing:
This is one of the earliest techniques used to identify bacteria. The
16S rRNAs from individual bacteria is digested with ribonuclease T1 (which cleaves specifi cally at
G residues) and produces short oligonucleotides of lengths up to 20 or so. A collection of these from
a given rRNA consists of oligonucleotide catalogue (Fox
et al
., 1977). A specifi c pattern characteristic
of a given species is obtained that enables one to arrange bacteria into phylogenetic groupings. The
data are represented in terms of binary association coeffi cients (called as
S
AB
values) defi ned as the
ratio of twice the sum of bases in oligonucleotides in two catalogues. Because of its failure to identify
bacteria beyond their phyla and branching orders or their subdivisions, sequencing approach received
more attention subsequently (Woese, 1987).
ii)
Sequencing of 16S rRNA gene:
Total community DNA or DNA extracted from identifi ed cultures
is used as a template for polymerase chain reaction (PCR) amplifi cation of 16S rRNA genes with
universal or domain specifi c primers. A clone library is constructed for genes encoding rRNA
(rDNAs) and is screened for fi nding out the sequence differences and phylogenetic relationships
are drawn. By applying this method one can assess the richness and evenness of a community
qualitatively based on the number of unique clones and the relative frequencies of each “ribotype”.
Schmidt
et al
. (1991) described a technique for analysis of marine picoplankton community by 16S
rRNA gene cloning and sequencing bypassing culture of the organisms. In this method, natural
picoplankton populations are collected and fi ltered by tangential fl ow fi ltration. The collected
biomass is subjected to DNA extraction and DNA is purifi ed by CsCl gradient centrifugation. The
DNA obtained is fragmented, size-fractionated and cloned into bacteriophage lambda. Out of 3.2
x 10
4
recombinant phages, 38 clones of lambda phage containing the 16S rDNA were screened by
hybridization with “mixed kingdom” 16S rRNA probes. Ribosomal DNA clones are then sorted, PCR
amplifi ed with r-DNA specifi c primers. Single strand rDNA clones were subjected to sequencing to
obtain nucleotide pattern. Then phylogenetic characterization of the constituents of the population
revealed the identity of 4 cyanobacterial, 11 proteobacterial and 1 eukaryote 16S rDNA sequences. An
alternative method for 16S rDNA sequencing that is quite faster and cheaper is Random Sequence
Reads (RSRs). RSRs are quite suitable for comparative study of the biodiversity of a metagenomic
library. In this method, the sequence analysis of oligonucleotide frequencies represented in the
genomes permits the identifi cation of different species (Karlin and Burge, 1995; Abe
et al
., 2003).
Manichanh
et al
. (2008) compared RSR method against 16S rDNA sequencing for estimating the
biodiversity of a metagenomic library. The sequences of the oligonucleotides are compared with a
database of prokaryotic sequences (GenBank-prok) using BLASTN. They applied this local alignment
programme to fi nd out the closest relative for each of the RSRs out of a total of 516,770 entries. By
