NON-CODING RNA PREDICTION OF CLINICALLY
IMPORTANT MYCOPLASMA BY COMPARATIVE
GENOMIC ANALYSIS
Dissertation submitted to the Madurai Kamaraj University in partial fulfillment
for the requirement of Masters of Science in Biotechnology
School of Biotechnology
Madurai Kamaraj University
Madurai
Regn. No:A242009
OBJECTIVES:
Mycoplasma.
Past
QRNA - A Blend
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
.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
Genome Size Comparision
[Link]
[Link]
[Link]
[Link]
[Link]
[Link]- Mycoplasma genetalium
[Link] [Link]- Mycoplasma pneumoniae
[Link]- Mycoplasma pulmonis
[Link]- Mycoplasma gallisepticum
0 500000 1000000 1500000 [Link]- Mycoplasma mycoides
[Link]- Mycoplasma penetrans
Genome length
MYCOPLASMA GENOME – INTERGENIC REGION
100%
80%
60%
40%
20%
0%
en
ne
en
al
ul
yc
s
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
Mycoplasma genitalium G37 complete genome - 0..580074
480 proteins
Location Strand Length PID Gene Synonym Code COG Product
Product
735..1829 + 364 3844620 MG001 - - - (dnaN)
1829..2761 + 310 1045670 MG002 - - - dnaJ
2846..4798 + 650 1045671 MG003 - - - (gyrB)
4813..7323 + 836 1045672 MG004 - - - (gyrA)
7295..8548 + 417 1045673 MG005 - - - (serS)
8552..9184 + 210 1045674 MG006 - - - (tmk)
9157..9921 + 254 1045675 MG007 - - - hypothetical
9924..11252 + 442 1045676 MG008 - - - (tdhF)
…… …….. … ….. ……….. ……… .. .. .. …
735 1829 1 734
1829 2761 2762 2845
2846 4798 → 4799 4812
4813 7323 7224 7294
7295 8548 8549 8551
8552 9184 9183 9156
9157 9921 ……. …….
……. …….
Starting Ending Length
Curing of Intergenic Regions 1 734 734
2762 2845 84
4799 4812 14
1200 7324 7294 -29
No. of Intergenic Regions
1000 8549 8551 3
9185 9156 -28
800 9922 9923 2
Before
600 11253 11251 -1
After 12041 12068 28
400
200 12726 12701 -24
13566 13569 4
0 14434 14395 -38
15317 15555 239
n
n
l
l
c
ga
pu
pe
pn
ge
my
M.
M.
M.
M.
M.
M.
Starting Ending Length
1 734 734
2762 2845 84
GRAPH SHOWING THE CULLING OF THE 15317 15555 2390
INTERGENIC SEQUENCES BY THE
C PROGRAMME THAT SELECTS THE REGIONS
WHOSE LENGTH IS GREATER THAN OR EQUAL
TO 50 NUCLEOTIDES ONLY
>L43967_2762_2845 Mycoplasma genitalium G37 intergenic sequence
AAAACCTTTCATTTTTAATGTGTTATAATTATTTGTTATGCCATAAATTTAGTTTGTGGC
AAAAGCTTCTGTACTGTTTATTTA
>L43967_15317_15555 Mycoplasma genitalium G37 intergenic sequence
ACCCTCAACCTCCTGAGTGCAAATCAGGTGCTCTATCAGTTGAGCTACATCCCCATTATT
GGTGGAAGTAAATGGACTTGAACCATCGACCTCACCCTTATCAGGGGTGTGCTCTAACCA
ACTGAGCTATACTTCCAAGCATAATCCTAAGGGTATTTAACTAATTATTATAACAATTTT
AATTTAACCAAAATACCCCTCGAATTTTAACAGTTTTTATAATCAAAACAGCTAATTTT
>L43967_19760_19824 Mycoplasma genitalium G37 intergenic sequence
ATAAATTTAATAGTGTTGAAAGACAAACATTATTAATTTTTGATCAGCTAAATAAAACAA
AGCAA
>L43967_20356_20543 Mycoplasma genitalium G37 intergenic sequence
CTCAAAAAACTAATACATCAAACTTCAACCGTTTACTTTTTTATGAACAAGCACTACAAA
GGTTTTATGAAGAATTATTTCAAATAGATTATTTAAGAAGATTTGAAAACATTCCCATTA
AAGATAAGAATCAAATTGCGCTTTTTAAAACTGTTTTTGATGATTACAAAACCATTGATT
TAGCAGAA
…………………………………………………………………………………………………………..
Similarity Search - WU BLAST 2.0
[Link]
filename.q file
[Link]
QRNA input file
>L43967_15317_15555-1>179-Mycoplasma
ACCCTCAACCTCCTGAGTGCAAATCAGGTGCTCTATCAGTTGAGCTACATCCCCATTATT
GGTGGAAGTAAATGGACTTGAACCATCGACCTCACCCTTATCAGGGGTGTGCTCTAACCA
ACTGAGCTATACTTCCAAGCATAATCCTAAGGGTAT-TTAACTA-ATTATTATAACAATT
T
>gb-U00089--19096>19275-Mycoplasma
ACCCTCAACCTCCTGAGTGCAAATCAGGTGCTCTATCAGTTGAGCTACATCCCCATTATT
GGTGGAAGTAAATGGACTTGAACCATCGACCTCACCCTTATCAGGGGTGTGCTCTAACCA
ACTGAGCTATACTTCCAGGCAAAATCTTC-GTACAGGTTCGCTTCATAATTATATTAATT
T
>L43967_19760_19824-5<65-Mycoplasma
TTGCTTTGTTTTATTTAGCTGATCAA-AAATTAATAATGTTTGTCTTTCAACACTATTAA
AT
>emb-BX293980.1--57200>57261-Mycoplasma
TTGTTTTGTTTTATTTAATTGATCAATAAATTGATTTAGTTTATCTTTATTTATTAATAA
AT
No. of Blast
No. of blastn hits selected hitsinput
for qrna No. of
alignments
[Link] 386 53927
[Link] 565 44433
[Link] 1012 360830
[Link] 850 154026
[Link] 1787 430551
[Link] 1852 560263
GRAPH SHOWING NUMBER OF ALIGNMENTS
SELECTED FOR QRNA INPUT FOR EACH
GENOME THROUGH THE PERLSCRIPT
QRNA – PARAMETERS
• Scanning window approach
– Window =150 nt; Extension = 50 nt
• Maximum length 9999999
• Local viterbi algorithm
• RIBOPROB matrix
• Shuffling the sequence maintaining
the composition
#---------------------------------------------------------------------
# qrna 2.0.1 (Tue Aug 19 11:30:55 CDT 2003) using squid 1.5m
(Sept 1997)
#---------------------------------------------------------------------
# PAM model = BLOSUM62
#---------------------------------------------------------------------
# RNA model = /mix_tied_linux.cfg
# RIBOPROB matrix = /[Link]
#---------------------------------------------------------------------
# seq file = /home/kalyankpy/perlscriptresult/genblast.q
# #seqs: 772 (max_len = 3420)
#---------------------------------------------------------------------
# window version: window = 150 slide = 50 -- length range =
[0,9999999]
#---------------------------------------------------------------------
# 1 [both strands] (sre_shuffled)
>L43967_1_734-90>722-Mycoplasma (664)
>gb-U00089--130>767-Mycoplasma (664)
length of whole alignment after removing common gaps: 664
Divergence time (variable): 0.401
[alignment ID = 61.75 MUT = 29.67 GAP = 8.58
………………………………………………………… ……………….. ( CONTD..)
QRNA OUTPUT
length alignment: 150 (id=61.33) (mut=32.67) (gap=6.00)(sre_shuffled)
posX: 0-149 [0-145](146) -- (0.42 0.08 0.06 0.43)
posY: 0-149 [0-144](145) -- (0.37 0.11 0.06 0.46)
L43967_1_734-90 TTAATTTTATTAAAACTATAACTTATTTTTTATAAACATTCTATGTTTTT
gb-U00089--130> [Link]
………………………………………………………………………………………………………………………………………………………………………………
LOCAL_DIAG_VITERBI -- [Inside SCFG]
OTH ends *(+) = (0..[150]..149)
OTH ends (-) = (0..[150]..149)
COD ends *(+) = (120..[27]..146)
COD ends (-) = (41..[12]..52)
RNA ends *(+) = (0..[21]..20)
RNA ends (-) = (0..[150]..149)
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
Number of ncRNAs
No. of non-coding predicted
predicted
60
50
40
Number
30
20
10
0
[Link] [Link] [Link] [Link] [Link] [Link]
Number of ncRNA predicted for
each organism
Range
Length Range of of Non-coding
Non-coding RNARNA
predicted
350
300
250
Length (nt)
200
150
100
50
0
[Link] [Link] [Link] [Link] [Link] [Link]
PICTURE SHOWING THE LENGTH
RANGE OF NON-CODING RNAs.
•The predicted ncRNa were searched for similarity against the
biochemically characterized ncRNA of Bacteria ( Non-coding RNA
database at [Link] updated 2002)
•Found similar to the Mc_MCS4 ncRNA of Mycoplasma capricolum.
.
•Another motif in one of the putative ncRNA was found to be conserved
across [Link], [Link], [Link] as a part of MicF ncRNA in these
organsims.
•Similarity was also found with OxyS ncRNA of [Link].
- In Eukaryotes
• Similarity was observed with few miRNAs
that were present in the miRNA database
(Rfam miRNA registry)
• Same stretch of sequence was present in Human,
Rat and Mouse miRNA.
• Small stretches of similarity was observed
with various ncRNAs playing role in
regulation of development also.
Sequences producing High-scoring Segment Pairs: Score P(N) N
hsa-mir-190 MI0000486 Homo sapiens miR-190 stem-loop 91 0.26 1
rno-mir-190 MI0000933 Rattus norvegicus miR-190 stem-loop 91 0.26 1
mmu-mir-190 MI0000232 Mus musculus miR-190 stem-loop 86 0.48 1
>hsa-mir-190 MI0000486 Homo sapiens miR-190 stem-loop
Length = 85
Minus Strand HSPs:
Score = 91 (19.7 bits), Expect = 0.31, P = 0.26
Identities = 45/68 (66%), Positives = 45/68 (66%), Strand = Minus / Plus
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
Plus Strand HSPs:
Score = 116 (23.5 bits), Expect = 0.025, P = 0.024
Identities = 60/94 (63%), Positives = 60/94 (63%), Strand = Plus / Plus
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.
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