Genome Wide Transcriptome Profiling of Agave sisalana Leaves with

Next Generation Sequencing under Drought Stress

Muhammad Bilal Sarwar

Supervisor: Dr. Bushra Rashid
Plant Genomics Lab
Centre of Excellence in Molecular Biology
University of the Punjab, Lahore Pakistan
bilal.sarwar@cemb.edu.pk
 Layout…….
1. INTRODUCTION 4. RESULTS
◦ Drought Stress
◦ Drought Responsive Mechanisms
◦ Agave sisalana; A New Model For Drought Mechanism Study
◦ RNA-Seq Advantages

2. OBJECTIVES
5. CONCLUSION

3. EXPERIMENTAL DESIGN
◦ Plant Sowing, Drought Stress, Physiological and Biochemical
Analysis
◦ Bio-replicates and Sequencing
◦ Stable Housekeeping Gene Identification
◦ Data Validation
 The Drought Stress an Emerging Threat

 Up to 16% yield loss risk by the end of 21st Century: Leng, G., & Hall, J. (2019).

Source: World Resources Institute https://www.wri.org/blog/2015/08/ranking-world-s-most-water-stressed-countries-2040

 Plant Response to Drought Stress

Chaves MM, Maroco JP, Pereira JS. Understanding plant responses to drought—from genes to the whole plant. Functional plant biology.
2003;30(3):239-64.
 Agave as a Model CAM Crop System for a Warming and Drying World Classification
Kingdom: Plantae
Clade: Angiosprems
Clade: Monocots
Order: Asparagales
Family: Asparagaceae
Subfamily: Agavoidae
Remarkable Genus: Agave
resistance to Species: Agave sisalana
Xeric Enviro

Application
• wine, ropes, string, yarn, Sisal pulp and paper, Textile,
Plastic and rubber composites

Thermo-tolerance survival range (-16.1c to 61.4c)

All these features related to heat and drought stress make the
Agave species an important model plant for creating the applied Drought-tolerance up to 1 year without Rain Fall
solutions to the Agricultural challenges associated with the
climate change.
 RNA Seq Advantages

 High Throughput
 Cheaper
 Better Dynamic Range
 Better Coverage
 Required Low amount of RNA with low Noise
 Required High Computational Power

https://www.nature.com/scitable/content/microarray-chip-3d-6630068/

Nature Review Genetics 2012 (10) 57-63. BMC Genomics 2009 (10) 347. Nucleic Acid Research (2012) 40. The Plant Cell 2012 (22) 2058
 OBJECTIVES
 Identification of Drought Responsive transcripts from Agave sisalana leaves under water stressed condition.

 Development of Agave sisalana off shoots from the Single mother plant
 Induction of drought stress
 Monitoring of Morphological, physiological and Biochemical response of control and stressed plants.
 Total RNA isolation from leaves, mRNA Library Construction and illumine mRNA Sequencing.
 Development of De novo assembled Transcriptome based and Annotation

 Identification, characterization, and Validation of the Housekeeping genes for quantitative real-time PCR data
normalization under abiotic stress in Agave sisalana

 Induction of abiotic stress (Drought, Rehydration, High Temperature, Low temperature, Salt stress)
 Time Course Leaf Sampling, RNA Isolation And cDNA Synthesis
 Development of the Local Database based on the reported Housekeeping Genes from the Model Plant
 Identification of the Candidate HKGs within the Unigene database based on Homology Study
 Ranking of Candidate Reference Genes Based on Gene Expression Stability Values
 Validation of stable HKGs
 Validation of DEG Expression through Quantitative Real-time PCR (qRT-PCR)
Materials & Methods
/
Work Flow
 Quantity and quality Testing
Plant Propagation
Offshoots

Leaf Sampling
RNA Isolation
RNA Library Preparation
&
Sequencing
High Quality RNA

Mother Plant

Leaf Sampling
Drought Stress

Offshoots

Plant Propagation
 RAW Reads

FastQC
Removal of bad Reads and Adopter Sequence
NGS-QC Tool Kit  BUSCO Score
 Unigenes Statistics
Clean Reads  N50 & N25 Indicators
 AT & GC Ratio
Reads Statistics by BBmap  CDS Prediction
 % reads alignment Back to assembly
Trinity Assemblies Evaluation
Trans-Abbys Contigs Unigenes Parameters

SOAP-de novo
MEGAN 6

de novo assembly
Swiss-Prot NCBI nr

InterProScan Viridiplantae
RSEM Functional
SNPs….SSR edgeR Annotation Pfam Oryza sativa
GO database Arabidopsis

Differentially KEGG Plant TF

Expressed Genes COG HSPIR database
qPCR
 Plant Material, Stress Conditions, and Tissue Sampling

Drought Treated

Mature
Off Shoots
Plants

Control Group
RESULTS
 Monitoring of Physiological and Biochemical response of control and stressed plants
Photosynthetic Attributes Chlorophyll Contents Relative Water Contents
and
Cell Membrane Stability

Biochemical Parameters

Total Proline Contents Lipid Peroxidation

Activity
 Total RNA isolation, mRNA Library Construction and Illumina mRNA
Sequencing
Agilent bioanalyzer 2100 Report for Agilent bioanalyzer 2100 Report for
RNA Quality and Quantity RNA Quality and Quantity
 Total RNA isolation, mRNA Library Construction and Illumina mRNA
Sequencing
 Raw Reads Statistics

size Total reads

File Name read length read with (bp) GC (%) AT (%) Q20 (%)
(GB) (Million)

C1 1.fastq 5.71 48.6 51.4 97.899

43.5 M 4,395,545,048
2.fastq 5.71

C2 1.fastq 7.07 48.2 51.87 97.822

53.8 5,440,548,820
2.fastq 7.07

C3 1.fastq 7.21 48.07 51.93 97.745

55.1 5,571,765,192
2.fastq 7.21

T1 1.fastq 5.8 47.5 52.48 97.78

44.3 4,478,188,702
2.fastq 5.8

T2 1.fastq 5.3 101

40.5 4,097,010,056 48.1 51.9 97.737
2.fastq 5.3

T3 1.fastq 5.15 48.2 51.97 97.299

39.3 3,978,366,972
2.fastq 5.15

Total 72.49 276.8 27,961,424,790 48.11 51.93 97.71

 RNA-Seq Clean Reads Qualitative Indicators

Per Base Sequence Quality B. per sequence quality Score C. Per Base Sequence Contents
https://www.ncbi.nlm.nih.gov/sra

BioProject: SUB2289050
Agave sisalana Genome
sequencing and assembly

NCBI Raw data

Submission ID:
D. Adopter Contents E. Per Sequence GC contents D. Sequence Length
Distribution  SRA5137659
 SRR5137661
 SRA5137662

 SRA5137658
 SRA5137663
 SRA5137660
 Short Clean Reads

Reads alignment
Transcriptome de novo Assembly Over view of
Individual Genome
Contigs

Unigenes

Draft Transcriptome
Type of Assembler used of Draft Transcriptome
De novo assembly-Results….

Boxplot comparisons

Length distribution using Trinity, SOAPdenovo-trans and Trans-ABySS software.

 De novo assembly-Results…. Bioinformatics' Parameters
Parameters Trinity Trans-ABySS / K. 64 Trans-Abyss / K. 51 SOAPdenovo-trans K.

Total contigs 93,141 647,990 950,646 37,731

Total bases 68,048,194 270,466,052 309,981,046 28,640,994

Min contig length 201 100 100 100

Max contig length 9,304 16,240 18,530 3,637,672

Ave. contig length 731 417 326 759

Median contig length 432 228 177 234

N25 length 1,887 1,253 1,024 3,541

N50 length 1,164 676 521 1,882

N75 length 537 330 245 875

N90 length 297 149 115 243

N95 length 248 127 101 152
As 27.44% 27.92 28.18% 24.05%
 Trinity Ts 27.28% 27.81 28.01% 23.90%

 Trans-Abayss Gs
Cs
22.99%
22.29%
22.01
22.24
21.77%
22.05%
19.54%
19.81%

 SOAPdenovo-trans K. (A + T)s 54.72% 55.73 56.19% 47.95%

(G + C)s 45.28% 44.27 43.81% 39.35%

Ns 0% 0% 0% 12.70%

Summary of the results from the de novo assembly with Trinity, Trans-ABySS and SOAP denovo-trans software.
Bioinformatics Parameters….
Trinity assembled Assembly Evaluation (prior-down stream analyses)
• Benchmarking Universal Single-Copy Orthologs (BUSCO Score (%)
• Reads Mapping Back to Transcriptome (RMBT)
De novo assembly-Results….
Contigs and Unigenes Counts

Contigs/Transcripts = 93141
Unigenes= 67327
Functional Annotation
Functional Annotation
Species Specfic Homology
 Functional Annotation KEGG (Kyoto Encyclopedia of Genes and Genomes)

KEGG is a collection of databases

dealing with genomes, biological
pathways, diseases, drugs, and chemical
substances
Plant Transcription Factor and Heat Shock Protein
 SSR and SNP Detection

Statistics of SSRs identified in A. sisalana Statistics of SNPs identified in A. sisalana

 Differential Gene Expression (DEGs)
 RSEM (RNA-Seq by Expectation Maximization) Package

 edgeR (Empirical Analysis of Digital Gene Expression Data in R)

Go Enrichment Hits after Drought Stress
Differentially Expressed Genes -Results….
De novo assembly-Results….
Other Enriched Genes Families

Sarwar, Muhammad Bilal, Zarnab Ahmad, Bushra Rashid, Sameera Hassan, Per L. Gregersen, Maria De la O. Leyva, Istvan Nagy, Torben
Asp, and Tayyab Husnain. "De novo assembly of Agave sisalana transcriptome in response to drought stress provides insight into the
tolerance mechanisms." Scientific reports 9 (2019).
 Stable/Superior Housekeeping Genes Identification from the Agave sisalana
De novo Assembly
Objective 2: Identification, and Validation of the Housekeeping genes for quantitative real-time PCR data normalization
under abiotic stress in Agave sisalana (CAM plant)

 Induction of abiotic stress (Drought, Rehydration, High Temperature, Low temperature, Salt stress)
 Time Course Leaf Sampling, RNA Isolation And cDNA Synthesis
 Development of the Local Database based on the reported Housekeeping Genes from the Model Plant
 Identification of the Candidate HKGs within the Unigene database based on Homology Study
 Ranking of Candidate Reference Genes Based on Gene Expression Stability Values
 Validation of stable HKGs
 Validation of DEG Expression through Quantitative Real-time PCR (qRT-PCR)
 Sorghum bicolor
Sudhakar Reddy et al. 2016
Stable HKG Identification Experimental Workflow
Janská, Anna, et al. 2013
Reddy et al. 2016
Zea mays Hordeum vulgare tblastn
Jain et al. 2006
Protein Translated Nucleotide
Oryza sativa Kumar et al. 2012
Arabidopsis thaliana
SUB4239256
Paolacci et al. 2009
Jain et al. 2006
Pennisetum glaucum
Oryza sativa
Agave sisalana Unigenes Database
Manoli et al. 2012
Li-hua Xie al. 2019
Triticum aestivum
Candidates HKG
Nicotiana tabacum

Setaria italica Prasad, M. (2013) Primer Designing

Local Housekeeping Gene Testing of Reference Genes under various abiotic Stress Condition
Database Ortholog Locus Sanger Sequencing Control Cold Salt
Cellular Ho Study
Homology
Drought Heat
Function time course sampling
NCBI 1
Annotation 2
Accession
3
IDs
4
Gene Symbol
5
Ranking 6
RNA-Seq (DEGs) Validation Validation 7 RefFinder geNorm NormFinder BestKeeper
by Using Stable HKG 8
for qPCR data Normalization 9
10
Stable HKG Identification…… Agave sisalana candidate reference genes with gene symbols, accession numbers,
descriptions, cellular functions and ortholog locus
Details of the candidate genes evaluated for reference genes selection in Agave sisalana under abiotic stress conditions
The primer specificity and amplicon size determination

Control Drought Stress Rehydration Condition

High Temperature Stress Low Temperature Stress Salt Stress Condition

Total pooled samples

The expression level of tested reference genes under various abiotic stress conditions
Software Output
Venn diagram showing the (A) most stable and (B) least stable candidate’s reference genes
The Recommended most stable and least stable combination of reference genes (up to four) as determined by
RefFinder
 Venn diagram of (A) most stable and (B) least stable candidates reference genes in common based on the recommended
comprehensive ranking (RefFinder) under abiotic stress condition

Most Stable Least Stable

Validation of the Selected Housekeeping Genes
 Absolute Quantification
Relative Quantification

Small Heat Shock Protein Gene AsHSP20 (MH555356)

Cloned int the pJET1.2 Cloning Vector

Get the Expression data of AsHSP20 under abiotic stress condition

Data Normalization was Carried out by the Stable and Least Stable Genes individually

Relative Fold Change determination and Comparative Study

 Normalization of AsHSP20 Gene Expression for Validation of Selected Reference Genes

Drought High Temperature Salt

Cold Rehydration
Absolute Quantification

Copy number of the AsHSP20

DEGs Data Validation by qPCR
 Conclusion
Global overview of the Agave sisalana transcriptome under drought stress : De novo
assembly Transcriptome
Provide Comprehensive annotation
Predict the Possible SNPs and SSR Markers
Differentially Expressed Gene Identification (Heat Shock Protein, Transcription Factors )
- 1195 upregulated Transcripts - 1864 Down regulated Transcripts
- Potential Heat Shock Proteins and Transcription Factor Candidates
Identify and Validate the possible stable and least stable housekeeping genes in the Agave
sisalana under abiotic stress condition for accurate expression study
 List of Publications
Batcho, A. A., Sarwar, M. B., Tariq, L., Rashid, B., Hassan, S., & Husnain, T.. "Identification
and characterization of heat shock protein gene (HSP70) family and its expression in Agave
sisalana under heat stress." The Journal of Horticultural Science and Biotechnology (2019): 1-
13.

Sarwar, M.B., Ahmad, Z., Batcho, A., Ahmed, M., Sajid, M., Hassan, S., Rashid, B., Husnain,
T. Identification and Validation of Suitable Housekeeping Gene for Quantitative RT-PCR Data
Normalization under abiotic Stress Conditions in Agave sisalana (a CAM-plant). Accepted.
Plant physiology and Molecular Biology

Sarwar, M.B., Ahmad, Z., Rashid, B., Hassan, S., Gregersen, P.L., Leyva, M.D.L.O., Nagy, I.,
Asp, T. and Husnain, T. "De novo assembly of Agave sisalana transcriptome in response to
drought stress provides insight into the tolerance mechanisms." Scientific Reports 9, no. 1
(2019): 396-396. https://doi.org/10.1038/s41598-018-35891-6.
 Acknowledgements
 Prof. Dr. Tayyab Husnain (Director)
 Dr. Bushra Rashid (Supervisor)
 Prof. Dr. Per L. Gregeson (Aarhus University)
 Dr. Maria De la O. Leyva (Aarhus University)
 Prof. Dr. Ahamd Ali Shahid
 Prof. Dr. Idrees Ahmad Nasir
 Dr. Sameera Hassan
 Dr. Abdul Qayyum Rao
 Mr. Atif and Zulfiqar (IT Department)
 Dr.Mukhtar Ahmad, Dr.Salah ud Din, Fayyaz Ahmad
 Higher Education Commission of Pakistan (IRSIP Fellowship)
 Leipzig University, Germany.
 Plant Genomics Lab
 CEMB Family …..
Any Question