Enzyme Activity analysis from gut bacterial MetaTranscriptome

1. NGS Meta-Transcriptome Analysis and
CAZy Family Annotation
(April, 2015 – September, 2015)
Minesh Jethva, JRF
PI : Dr. Hemant Kushwaha

2. Previous Work
• Development of NGS Data Analysis Pipeline
– QC & Normalization
– Mapping
– Assembly
– Annotation
For Presentation @ SBB Group, ICGEB, India 2
28/9/2015

3. Current Objectives
• To Analyze Meta-Transcriptomics Data
– Rice Stem Borer Gut Bacteria
– Expression Level
• To Annotate using CAZy Family Members
– Classes : GH, AA, CBM, GT, PL, CE
– Diversity Study
– Identifying homology based taxonomical origin
For Presentation @ SBB Group, ICGEB, India 3
28/9/2015

4. Meta-Transcriptome Data Analysis
• Challenges in NGS Data Analysis
– Storage and Analysis problem because of
Large amount of Data
– Resulting Short Reads
– De novo Assembly
• What can we get?
– Transcript Abundance
– Novel Transcripts
– Isoform Information
– Splice Junction Information (In Eukaryotes)
– Gene Function
For Presentation @ SBB Group, ICGEB, India 4
28/9/2015

5. Annotation
• Approaches
– Homology Based
– Model/Profile Based
– Hybrid
• Levels
– Genome (Variation, Regulatory Motif, Gene
Distribution, etc.)
– Transcriptome
– Protein (Domain Arrangement, Active Sites, etc.)
• Resources
– Databases : NR, CDD, RefSeq, Swissprot, Pfam, KEGG,
Motif databases
– Tools : BLAST, HAMAP, Pfamscan, DAVID Server,
Interpro
For Presentation @ SBB Group, ICGEB, India 5
28/9/2015

6. CAZy Annotation
• CAZy Database
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Class Sub-classes Proteins
Glycoside Hydrolases (GH) 135 80,448
Glycosyl Transferases (GT) 98 52,364
Polysaccharide Lyases (PL) 24 6,703
Carbohygrate Esterases (CE) 17 11,031
Auxiliary Activities (AA) 14 7,444
Carhohydrate-Binding Modules (CBM) 72 27,871
Total 360 1,85,861

7. Work Done
• Quality Check
– Paired Ended Reads for Day 4th (14 GB), 7th (12 GB),
13th (12 GB) and 20th (12 GB)
• Protein Prediction
– # of Contigs : 4,68,225
– # of Predicted Proteins
• 5,70,771
• Expression Analysis
• CAZy Annotation
– GH48 : 3 Proteins
– GH6 : 3 Proteins
– GH9 : 11 Proteins
• Diversity and Homology Study
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8. Result of Quality Check
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Position in Reads
Quality
Value

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No.
of
Reads
Mean Sequence Quality
Result of Quality Check

10. Result of Quality Check
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• Nucleotide content showing presence of
adaptor/primer sequence in the beginning of
reads

11. • All reads are having same length as 101
bases
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Result of Quality Check

12. Protein Prediction
• MetaGeneMark
• TransGeneScan
• FragGeneScan
• MetaGeneAnnotator
• Glimmer-MG
• MetaGUN
• Eugene-PP
• GeneMark
• Glimmer
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13. GH48
Expression Analysis
28/9/2015 For Presentation @ SBB Group, ICGEB, India 14
• FPKM (Frag. Per Kb of transcript per Million
mapped reads) value of transcript coded for
GH48 member protein by sample
FPKM
0
5
10
15
D4 D7 D13 D20
Days
Ids

14. GH48
Diversity
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% Identity Matrix

15. GH48
Protein Species Coverage E-Value Identity PID
exoglucanase B Cellulomonas fimi 100% 0.0 81% WP_013772537.1
exoglucanase Cellvibrio gilvus 100% 0.0 70% WP_013884685.1
exoglucanase Cellulomonas sp. FA1 72% 0.0 92% WP_046529316.1
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ID: Gene_140649 Length: 1081 aa
Top Blast Hits

16. GH48
ID: Gene_140654 Length: 692 aa
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Protein Species Coverage E-Value Identity PID
exoglucanase Cellulomonas sp. FA1 100 % 0.0 99 % WP_046529316.1
exoglucanase Cellulomonas flavigena 98 % 0.0 81 % WP_013118318.1
exoglucanase B Cellulomonas fimi 98 % 0.0 74 % WP_013772537.1
Top Blast Hits

17. GH9
Expression Analysis
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D4 D7 D13 D20
Days
FPKM
0
10
20
30
40

18. GH9
Diversity
28/9/2015 For Presentation @ SBB Group, ICGEB, India 19
% Identity Matrix

19. GH9
Domain Arrangement
• gene_108278/gene_108280/gene_108283 (750 aa)
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• gene_110436/gene_11487 (583 aa)
• gene_119033 (864 aa)

20. GH6
Diversity
28/9/2015 For Presentation @ SBB Group, ICGEB, India 21
% Identity Matrix

21. GH6
Expression
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FPKM
0
7
14
D4 D7 D13 D20
Days

22. GH6
Domain Arrangement
• Gene_127481 (280 aa)
• Gene_178854 (450 aa)
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• Gene_263424 (434 aa)

23. Future Plan
• Annotation of other CAZy members
• Differential Expression Analysis
• Correlating Transcriptome with Proteome
data
28/9/2015 For Presentation @ SBB Group, ICGEB, India 24

24. Thank You