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Viral metagenomic analysis of sweet potato using high-throughput deep sequencing

Student: Thulile Faith NhlapoSupervisors: Dr. J. Rees, Prof. M.E.C Rey, Dr. D.A. Odeny

Collaborators: Ms J. Mulabisana, Dr. M. Cloete

Sweet potato viruses and their effects on production

• Sweet potato is highly nutritious and is used as a poverty alleviation crop (food security)

– Good source of carbohydrates, proteins, fiber, iron, vitamin C, B, and Vitamin A (beta carotene)

• Viral diseases can reduce crop quality and yield by up to 100%

• A collection of viruses may infect sweet potato (disease complex)

• In SA 12 viruses have been identified either occurring singly or in combination (viral synergy) decreasing yield by 50-100%

Healthy

Viral Infection/diseased

“Potential infection”

Sweet potato virus families

• Compared to viruses of other agriculturally important crops, sweet potato viruses have been poorly studied but recently more viruses infecting sweet potato are being described

• Over 30 sweet potato viruses have been identified and assigned to 9 families

• 7 RNA virus families have been identified: Bromoviridae, Bunyaviridae, Closteroviridae, Comoviridae, Flexiviridae, Luteoviridae, and Potyviridae

• 2 DNA virus families have been identified: Caulimoviridae, Geminiviridae

Symptoms associated with viral infection

Symptoms observed on sweet potato plants in the field (A&B) chlorotic spots with purple rings, (C) upward curling of leaves, (D) insect damage. Symptoms observed in the glasshouse (A) chlorotic spots with purple rings, (B) chlorotic spots with purple rings, and purple edged vein feathering, (C) upward curling of young leaves, (D) chlorotic spots and vein clearing.

Metagenomics and viral metagenomics?

• Metagenomics- “or community genomics, is an approach aimed at analyzing the genomic content of microbial communities within a particular niche”

• Viral metagenomics- the study of viral communities. Viral metagenomics can be used to analyse viral sequences in any sample type (soil, plant, water, human gut etc.)

• Is a powerful tool for virus discovery, can be applied to the problem of determining etiology in diseases

• Also a metagenomic study or analysis is not biased towards culturable organisms; therefore the total genetic diversity of microorganisms can be studied

Cloning dependent sequencing

Deep sequencing

Expensive Cheaper

Time consuming Faster, accurate

Require large amounts of DNA

Small amounts of DNA (detect low virus titers)

Inserts sometimes unstable No cloning

Produces large contiguous sequences

Short reads

1. Viral metagenomics-viruses small genomes, so assembly not a problem2. Bioinformatic- developed software and algorithm for analysis of short reads

Using next generation sequencing approach for metagenomics

Aims

1. To carry out a metagenomic study of sweet potato viruses in the Western and Eastern Cape provinces of South Africa

2. To undertake genetic characterisation of sweet potato viruses under South African conditions in order to generate a basis for their classification

3. Explore diagnostic strategies using next generation sequencing (NGS)

Overview of metagenomics strategy

OutputData Analysis-CLC Bio

Input Symptomatic & Asymptomatic leaves

DNA IsolationRCA

Sample preparation

Nextera

RNA Isolation

Sample preparationRibo-Zero&TruSeq

Sequencing-MiSeq

Sampling

Overview of bioinformatics strategy

1. Download reference sequences (NCBI)

CLC Bio 6.0.1- Plug-ins for additional alignments-

MUSCLE and ClustalW MEGA 5.05

5. Multiple Sequence Alignment

2. Read map to reference viral genomes (0.8-0.99 stringency)

3. Extract new consensus sequence

BLASTn

4. Retrieve full genomes of most closely related species

Sequence Reads (Raw Data)

2. Trim reads for adaptors

Unmapped reads

BLASTn

Identify contigs

De novo assembly (25-64 k-mer)

7. Phylogenetic tree

6. Pairwise Comparison- Sequence ID 8. Full genomes

9. Design primers

10. Confirm by PCR

Western CapeEastern Cape

Sweet potato sampling sites

Date Location Type of farming

November 2012 P.E. (Eastern Cape) Subsistence

November 2012 P.E. (Eastern Cape) Subsistence

November 2012 P.E. (Eastern Cape) Subsistence

November 2012 Alice (Eastern Cape) Subsistence/Commercial

January 2013 Klawer (Western Cape) Commercial

January 2013 Lutzville (Western Cape) Commercial

January 2013 Paarl (Western Cape) Commercial

January 2013 Franschhoek (Western Cape) Commercial

Eastern Cape

3 Z

3 K

3 A

3 M

Symptomatic

Eastern Cape

2 Z

2 K

2 A

2 M

Asymptomatic

NSample size= 20

RESULTS

Rolling circle amplification (RCA) provides DNA sequencing template

• Genomic DNA (gDNA) isolation - Qiagen DNeasy Plant Mini Kit• Rolling circle amplification (RCA) - IllustraTM TempliPhi 100 Amplification Kit• Nextera DNA sample preparation• Sequencing on the Illumina MiSeq Benchtop Sequencer

DNA isolation of symptomatic and asymptomaticplants collected from the Eastern Cape

10Kb

3Kb

1Kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

RCA products for Eastern Cape samples

Results: DNA data, symptomatic samples

Reference genome Percentage identity

Average coverage

Percentage of genome covered

Consensus length

Sweet potato geminivirus strain SPLCSPV (JQ621844)

94.38% 3 359 X 99.3% 2 769 bp

Sweet potato geminivirus strain SPMaV (JQ621843)

98.10% 2 940 X 99.92% 2 781 bp

Ipomoea batatas mitochondrial plasmid-like DNA (FN421476)

100% 3 713 X 100% 1 027 bp

Western Cape sample (KT10): Sequence identity and percentage genome coverage of DNA circular viruses and mitochondrial DNA

Example of mapping and coverage-KT10

Reads mapped to SPLCSPV-ZA (94 % similarity)

New consensusReference

Neighbour-joining tree of geminiviruses

• Total RNA isolation - Qiagen RNeasy Mini Kit• DNase treatment of samples prior to sequencing• rRNA depletion- Ribo-ZeroTM Magnetic Kit (Plant Leaf)• TruSeq Stranded Total RNA Sample Preparation• Sequencing on the Illumina MiSeq Benchtop Sequencer

RNA isolation of symptomatic and asymptomatic plants collected from the Eastern and Western Cape

Ribo-zeroed total RNA provides sequencing template for RNA sequencing

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 M

10Kb

3Kb

1Kb

Results: RNA data, symptomatic samples

• 1% of data mapped to viral genomes

• Majority of reads mapped to sweet potato chloroplast genome

• Assembled near complete genomes of:

– Sweet potato chlorotic stunt virus (RNA 2 segment) (SPCSV)• Still need to assemble RNA 1 segment (Total genome size = 17 630nt)• 3 593 reads out of 5 432 520, consensus length 4 811bp, 61 X average coverage

– Sweet potato feathery mottle virus (SPFMV)• Ordinary-strain• Common-strain (Sweet potato virus C) (SPVC)

– Sweet potato virus G (SPVG)

Summary of results for RNA viruses

Reference genome Percentage identity

Average coverage

Percentage of genome covered

Consensus length

Sweet potato virus C Peru(GU207957)

94.07% 446 X 99.92% 10 812 bp

Sweet potato feathery mottle virus (AB439206)

93.96 % 255 X 98.83% 10 694 bp

Sweet potato virus G(JQ824374)

97.92% 51 X 99.92% 10 743 bp

Sweet potato chlorotic stunt virus RNA 2 (KC146843)

96.99 % 750 X 99.85 % 8 205 bp

Consensus length= 10 694 bp Average coverage= 255 X

New consensus

Mapping sequence reads to SPFMV reference genome

Reference

New consensus shares 94% similarity with reference (variation)

ZOOM-in

Neighbour-joining tree of criniviruses (SPCSV)

Sweet potato chlorotic stunt virus isolates: WA- West African strain EA-East African strain

EA

WA

Neighbour-joining tree of potyviruses (SPFMV, SPVC, SPVG)

Sweet potato feathery mottle virus isolates: EA-East African strain S-S strain C-Common strain G-Sweet potato virus G 2-Sweet potato virus 2

EA & O

S

C

SPFMV lineage

Sequence data suggests multiple infection

Observed symptoms on sweet potato plants. (A1) Purple ringspots and chlorotic spots on KT10 sample, these symptoms are associated with Sweet potato feathery mottle virus (SPFMV). (A2) Upward curling of leaves associated with Sweet potato leaf curl virus (SPLCV). (B) Upward curling of leaves and chlorotic spots on sample KF1, symptoms associated with SPLCV and SPFMV. (C) Purple ringspots, leaf vein feathering with purple feathering and chlorotic spots on sample F11, these are symptoms associated with SPFMV and Sweet potato virus G (SPVG). (D) Chlorotic spots and vein clearing on sample K17, symptoms associated with Sweet potato virus C (SPVC), the C strain of the potyvirus SPFMV.

Sweet potato virus distribution

SPVC (SPFMV C-strain)

SPVG

SPFMV (O-strain) SPCSV SPLCSPV-ZA SPMaV-ZA

Western Cape

Eastern Cape

Advantages of this sequencing approach?

• Detect viruses by direct sequencing

• Generate complete/near complete viral genomes

• High average sequence depths

• Deep sequencing is efficient diagnostic tool– Detected viral pathogens– Detected mixed infections– Detected diverse viral strains

Acknowledgements• Supervisors

– Dr. J. Rees– Prof. C. Rey– Dr. D. Odeny

• Collaborators– Julia Mulabisana– Dr. M. Cloete

• ARC-VOPI senior researchers, technicians, and staff– Sidwell Tjale– Thakhani Ramathavhatha – Dr. Laurie

• ARC-BTP senior students, researchers and bioinformaticians • Farmers in Western and Eastern Cape • This work is based on the research support in part by the National Research

Foundation of South Africa  (Grant reference number UID 79983)• Other funding sources: ARC-PDP and DAFF

THANK YOU