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School of Biotechnology
Madurai Kamaraj University
Madurai
Regn. No:A242009
OBJECTIVES:
• To choose the best possible approach to predict the
ncRNA
QRNA - A Blend
• Secondary structure alone is not statistically significant for the detection of
ncRNAs.
• Important sequences that code for proteins and performing important functions
are conserved across the related organisms.
QRNA was developed to screen the conserved RNA secondary structures from the
background of the other conserved sequences.
OUTLINE
INTERGENIC REGIONS OF ORGANISM OF INTEREST
↓ blastn
SEARCH FOR HOMOLOGY ACROSS RELEATED ORGANISMS
↓ Perl scripts
PARSE THE ALIGNMENTS WITH CERTAIN CUTOFFS
↓
THE ALIGNMENTS WERE GIVEN AS INPUT FOR THE QRNA
↓
PUTATIVE ncRNA
PROTEIN CODING REGION→INTERGENIC REGION
.ptt file
↓
Co-ordinates of protein coding regions
↓
Intergenic region co-ordinates
↓
Intergenic region co-ordinates
difference > 50 nucleotides
↓
Range file
↓
Intergenic sequence extraction by EMBOSS application
extractseq –regions @rangefile -separate
GENOME LENGTH COMPARISION OF
THE MYCOPLASMA
Organism Genome
Genome Size Comparision size
M.gallisepticum 9,96,422
M.myc
M.gen- Mycoplasma genetalium
M.pen M.pne- Mycoplasma pneumoniae
M.pul- Mycoplasma pulmonis
M.gal- Mycoplasma gallisepticum
0 500000 1000000 1500000 M.myc- Mycoplasma mycoides
M.pen- Mycoplasma penetrans
Genome length
MYCOPLASMA GENOME – INTERGENIC REGION
100%
80%
60%
40%
20%
0%
en
ne
en
al
ul
s
yc
en
.g
.p
.p
.p
.g
.m
pi
M
M
M
sa
o
m
Ho
BAR GRAPH SHOWING THE PERCENTAGE OF INTERGENIC
REGION IN THE GENOME OF MYCOPLASMA
PROTEIN TABLE OF THE GENOME
4799 4812 14
1200 7324 7294 -29
1000 8549 8551 3
9185 9156 -28
800 9922 9923 2
Before
600 11253 11251 -1
After 12041 12068 28
400
12726 12701 -24
200
13566 13569 4
0 14434 14395 -38
15317 15555 239
n
n
l
l
yc
u
e
e
.g
.p
.p
.p
.g
.m
M
M
M
M
L43967_1_734-90 TTAATTTTATTAAAACTATAACTTATTTTTTATAAACATTCTATGTTTTT
gb-U00089--130> TTTATTTTATTAAAATTATAATGTATTTTTGTTAAATTTT.TAATTCTTT
………………………………………………………………………………………………………………………………………………………………………………
winner = OTH
OTH = 184.281 COD = 166.408 RNA = 179.710
logoddspostOTH = 0.000 logoddspostCOD = -17.873 logoddspostRNA = -4.571
sigmoidalOTH = 4.571 sigmoidalCOD = -17.932 sigmoidalRNA = -4.571
60
Number of ncRNAs
No. of non-coding predicted
predicted
50
40
Number
30
20
10
0
M.pen M.myc M.gal M.pul M.pne M.gen
350
300
Length (nt) 250
200
150
100
50
0
M.pen M.myc M.gal M.pul M.pne M.gen
Query: 77 AGGTTTAGGTGTTCT-TATTT-ATTTATTAGGTTGTTTAGTT--TC-AATTATTTTTGGA 23
||| | |||| | | ||| || |||||||||||| | || || || ||| | | |
Sbjct: 4 AGGCCTCTGTGTGATATGTTTGATATATTAGGTTGTT-ATTTAATCCAACTATATATCAA 62
Query: 22 ATACTAGT 15
| | || |
Sbjct: 63 ACA-TATT 69
>Hs_NTT
Length = 17,572
Query: 11 TATTTAATATTTATAATTGCTATTTAGCATCTTAAAA-AAGA-CG-TCTTT-AAA-TATA 65
|| |||| | || ||| | | || | |||| | ||| | |||| ||| ||||
Sbjct: 5336 TACATAAT-TAGATCATTTATTCTAAGTAAATTAAGAGAAGCTCTATCTTCCAAAATATA 5394
Query: 66 GATAGTTATACTAATTAGAAAATAGTTAAT-AAG 98
|||| | || ||| |||| | ||||| |||
Sbjct: 5395 GATATCTCTAGCAAT-AGAAGAGTTTTAATTAAG 5427
CONCLUSIONS
• Comparative genomic analysis was selected for
the ncRNA prediction.
• Procedure for the prediction was standardized.
• One of the putative ncRNA was found to be
similar to the already characterized ncRNA from
the same genus.
• Conserved region of MicF was found to be
present in the putative ncRNA also.
• Identification of the eukaryotic miRNA
counterpart in Mycoplasma.
Future Plans
• To develop programmes for getting the intergenic
region co-ordinates given the protein table file as
input.
• To verify the genuinity of the predictions beyond
the homologous regions found in bacteria.
• To extend the prediction procedure for Eukaryotes.
• To develop the procedure required for classification
of the predicted ncRNAs into subclasses.
• To identify the functions of the putative ncRNAs by
searching their effector targets.
• To automize the whole procedure.
ACKNOWLEDGMENTS
Dr. Z. A. Rafi
Dr. S. Krishnaswamy
The Whole SBT family
Ministry of Human Recourses Development
Department of Education
Department of Science and Technology
Department of Biotechnology
All my classmates