Next Generation Sequencing for virus discovery and large ...€¦ · Monica Carvajal-Yepes1,...
1
Monica Carvajal-Yepes 1 , Maritza Cuervo 2, Cristian Olaya 1+ , Angélica Martínez 2 , Ivan Lozano 1 , Diana Niño 2 , Ericson Aranzales 2 Daniel Debouck 2 , Peter Wenzl 2 , Wilmer Cuellar 1 and Joe Tohme 3 1 Virology Laboratory, 2 Genetic Resource Program, 3 Biotechnology research Unit; International Center for Tropical Agriculture (CIAT), AA 6713, Cali, Colombia. + Current address: Department of Plant Pathology, Washington State University, USA BACKGROUND Analyzing the virome of plants using next generation sequencing (NGS) has accelerated the identification of viruses associated with disease that could not be isolated otherwise. Deep sequencing of small interfering RNAs (siRNAs) is used to diagnose and identify viruses in different crops showing disease symptoms, such as cassava, bean, rice and papaya. By using this approach, novel viruses infecting cassava fields in Colombia, were discovered and reported from plants with Cassava Frogskin disease (CFSD) symptoms (Carvajal-Yepes et al. 2014). CFSD is a quarantine disease that causes significant yield losses and is associated to infection of multiple pathogens (Alvarez et al., 2009; Calvert et al., 2008; Carvajal-Yepes et al., 2014). The Genetic Resource Program (GRP) at the International Center for Tropical Agriculture (CIAT) conserves the world’s largest and most diverse collection of cassava (Manihot esculenta). The health certification of the cassava collection material is done by the Germplasm Health Laboratory (GHL), ensuring the safe distribution of these materials. Following discoveries in cassava with NGS, in addition to screening other viruses such as: Cassava virus X (CsXV), Cassava common mosaic virus (CsCMV) and Cassava vein mosaic virus (CVMV), it was necessary to implement and standardize methods for the detection of Cassava torrado-like virus (CsTLV), Cassava polero-like virus (CsPLV), Cassava frogskin-associated virus (CsFSaV) and Cassava new alphaflexivirus (CsNAV) in the large-scale screening workflow. Up to date, 61% of the in vitro cassava collection has been evaluated. The use of NGS for discovering important quarantinable pathogens will contribute to the safe distribution of germplasm around the world. Next Generation Sequencing for virus discovery and large-scale virus screening of the cassava collection at CIAT’s genebank REFERENCES - Alvarez E; Mejía JF; Llano GA; Loke JB; Calari A; Duduk B and Bertaccini A. 2009. Characterization of a Phytoplasma Associated with Frogskin Disease in Cassava. Plant Disease 93:11, 1139-1145 - Calvert L; Cuervo M; Lozano I; Villareal N; Arroyave J. 2008. Identification of three strains of a virus associated with cassava plants affected by frogskin disease. Journal of Phytopathology 156:647-653. - Carvajal-Yepes M; Olaya C; Lozano I; Cuervo M; Castaño M; Cuellar WJ. 2014. Unraveling complex viral infections in cassava (Manihot esculenta Crantz) from Colombia. Virus Research 186:76–86. - Zerbino, D. R. and Birney, E. (2008). Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome research. 18, 821-829. De novo assembly allowed the identification of three novel viruses infecting cassava in Colombia in plants with CFSD (Carvajal-Yepes et al., 2014). By exploiting the plant immune response system, it is posible to detect viruses infecting plants. During virus replication, host endoribonucleases cleave the viral-derived dsRNAs limiting viral replication. Cleaved small virus-derived RNAs (svRNAs) are used for NGS. VIRUS DISCOVERY BY USING NGS 21% 5% 74% Rice 1'676,078 67% 28% 3% 1% 1% Cassava 1'692,420 i) De novo assembly of 21-24nt reads with VELVET v1.2.03 (Zerbino and Birney, 2008) produces longer assembled sequences (nodes or contigs). BLASTn and BLASTx search are done against the NCBI database using assembled nodes to identify viral sequences. RNA isolation and library preparation Sequencing Data analysis ii) Reads can also be mapped to known viruses (used as reference genomes) by using MAQ 0.7.1 software. This allows multiple detection of viruses simultaneously. Below: Rice hoja blanca virus (RHBV) derived reads mapped to a RHBV reference genome Virus name Classification GenBank Cassava torrado-like virus (CsTLV) Secoviridae / Torradovirus KC 505250, KC 505151 Casava polero-like virus (CsPLV) Luteoviridae / Polerovirus KC 505249 Cassava new alphaflexivurs (CsNAV) Alphaflexiviridae / Potexvirus KC 505252 Fig. 2. Deep sequencing of Small interfering RNAs (siRNAs) using the Miseq platform from Illumina (a) and MiSeq Reagent Kit v2 for 50 cycles. Sequencing produces around 15 M reads of 21-24 nt per run (b). [email protected] [email protected] Fig. 1 a) Source of RNAs for library preparation (yellow dashed circle) b) Representation of library preparation c) Library confirmation/Enrichment PCR. (siRNAs: Small interfering RNAs); svRNAs: small virus-derived RNAs) a. b. c. Cassava germplasm distributed 6,492 accessions since 1979 to 84 countries LARGE-SCALE VIRUS SCREENING IN THE CASSAVA COLLECTION The in vitro collection of the genus Manihot of the GRP at CIAT is currently represented by 6,643 materials; which have been registered in the Multilateral System of Access and Benefit sharing of the International Treaty on Plant Genetic Resources for Food and Agriculture. These materials must be free of quarantine diseases for distribution to users worldwide. Cassava in vitro collection at CIAT Following the discovery of cassava viruses by NGS associated to the quarantine disease CFSD, the large-scale virus screening in the cassava collection updated the number of viruses to be tested and the procedures in the GHL operations workflow (shown within the red rectangle). Up to date, 61% (4087 accessions) of the in vitro cassava collection has been evaluated. The remaining 39% are in this process. Samples with disease symptoms showing the percentage of reads (siRNAs) that were mapped to known viral sequences (shown in red color). Viral sequences used: PRSV: Papaya ring spot virus, RHBV: Rice hoja blanca virus, BGYMV: Bean golden yellow mosaic virus, Cassava viruses (mentioned above). The (%) of unmapped reads are shown in grey. The number of reads that were obtained per sample are shown by the dashed black line. PERSPECTIVES: - Implementation of NGS for seed borne pathogen detection is highly desirable in genebank collections, especially for collections that need to be regenerated in the field, being exposed to emerging pathogens during regeneration crop seasons. - NGS is a reliable and sensitive diagnostic option for the evaluation of germplasm with special country phytosanitary requirements, particularly when certain pathogen detection methods are not yet validated in the facility or positive controls are not available. - The establishment of DNA banks will increase the possibility of optimizing operations for DNA pathogen detection by using NGS. (a) (b) RHBV genome has 4 RNA segments Reads mapped to virus RNA segments Frequency Number of reads and percentage of reads mapped to virus references in different plant species Rice Rice Bean Cassava Cassava Cassava BLASTx search Papaya
Transcript of Next Generation Sequencing for virus discovery and large ...€¦ · Monica Carvajal-Yepes1,...
Monica Carvajal-Yepes1, Maritza Cuervo2, Cristian Olaya1+, Angélica Martínez2, Ivan Lozano1, Diana Niño2, Ericson Aranzales2 Daniel Debouck2, Peter Wenzl2, Wilmer Cuellar1 and Joe Tohme 3
1 Virology Laboratory, 2Genetic Resource Program, 3Biotechnology research Unit; International Center for Tropical Agriculture (CIAT), AA 6713, Cali, Colombia. + Current address: Department of Plant Pathology, Washington State University, USA
BACKGROUNDAnalyzing the virome of plants using next generation sequencing (NGS) has accelerated the identification of viruses associated with disease that could notbe isolated otherwise. Deep sequencing of small interfering RNAs (siRNAs) is used to diagnose and identify viruses in different crops showing diseasesymptoms, such as cassava, bean, rice and papaya. By using this approach, novel viruses infecting cassava fields in Colombia, were discovered and reportedfrom plants with Cassava Frogskin disease (CFSD) symptoms (Carvajal-Yepes et al. 2014). CFSD is a quarantine disease that causes significant yield lossesand is associated to infection of multiple pathogens (Alvarez et al., 2009; Calvert et al., 2008; Carvajal-Yepes et al., 2014). The Genetic Resource Program(GRP) at the International Center for Tropical Agriculture (CIAT) conserves the world’s largest and most diverse collection of cassava (Manihot esculenta).The health certification of the cassava collection material is done by the Germplasm Health Laboratory (GHL), ensuring the safe distribution of thesematerials. Following discoveries in cassava with NGS, in addition to screening other viruses such as: Cassava virus X (CsXV), Cassava common mosaic virus(CsCMV) and Cassava vein mosaic virus (CVMV), it was necessary to implement and standardize methods for the detection of Cassava torrado-like virus(CsTLV), Cassava polero-like virus (CsPLV), Cassava frogskin-associated virus (CsFSaV) and Cassava new alphaflexivirus (CsNAV) in the large-scale screeningworkflow. Up to date, 61% of the in vitro cassava collection has been evaluated. The use of NGS for discovering important quarantinable pathogens willcontribute to the safe distribution of germplasm around the world.
Next Generation Sequencing for virus discovery and large-scale virus screening of the cassava collection at CIAT’s genebank
REFERENCES
- Alvarez E; Mejía JF; Llano GA; Loke JB; Calari A; Duduk B and Bertaccini A. 2009. Characterization of a Phytoplasma Associated with Frogskin Disease in Cassava. Plant Disease 93:11, 1139-1145
- Calvert L; Cuervo M; Lozano I; Villareal N; Arroyave J. 2008. Identification of three strains of a virus associated with cassava plants affected by frogskin disease. Journal of Phytopathology 156:647-653.
- Carvajal-Yepes M; Olaya C; Lozano I; Cuervo M; Castaño M; Cuellar WJ. 2014. Unraveling complex viral infections in cassava (Manihot esculenta Crantz) from Colombia. Virus Research 186:76–86.
- Zerbino, D. R. and Birney, E. (2008). Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome research. 18, 821-829.
De novo assembly allowed the identification ofthree novel viruses infecting cassava in Colombiain plants with CFSD (Carvajal-Yepes et al., 2014).
By exploiting the plant immune response system, it is posible to detect virusesinfecting plants. During virus replication, host endoribonucleases cleave theviral-derived dsRNAs limiting viral replication. Cleaved small virus-derived RNAs(svRNAs) are used for NGS.
VIRUS DISCOVERY BY USING NGS
21%
5%
74%
Rice1'676,078
67%
28%
3%1%
1% Cassava1'692,420
i) De novo assembly of 21-24nt reads with VELVET v1.2.03 (Zerbino and Birney,2008) produces longer assembled sequences (nodes or contigs). BLASTn andBLASTx search are done against the NCBI database using assembled nodes toidentify viral sequences.
RN
A is
ola
tio
n a
nd
lib
rary
pre
par
atio
nSe
qu
enci
ng
Dat
a an
alys
is
ii) Reads can also be mapped to known viruses (used as reference genomes) byusing MAQ 0.7.1 software. This allows multiple detection of virusessimultaneously. Below: Rice hoja blanca virus (RHBV) derived reads mapped to aRHBV reference genome
Virus name Classification GenBank
Cassava torrado-like virus (CsTLV) Secoviridae / TorradovirusKC 505250, KC 505151
Casava polero-like virus (CsPLV) Luteoviridae / Polerovirus KC 505249
Cassava new alphaflexivurs (CsNAV) Alphaflexiviridae / Potexvirus KC 505252
Fig. 2. Deep sequencing ofSmall interfering RNAs(siRNAs) using the Miseqplatform from Illumina (a) andMiSeq Reagent Kit v2 for 50cycles. Sequencing producesaround 15 M reads of 21-24 ntper run (b).
[email protected] [email protected]
Fig. 1 a) Source of RNAs for library preparation (yellow dashed circle) b) Representation oflibrary preparation c) Library confirmation/Enrichment PCR. (siRNAs: Small interfering RNAs);svRNAs: small virus-derived RNAs)
a.
b. c.
Cassava germplasm distributed 6,492accessions since 1979 to 84 countries
LARGE-SCALE VIRUS SCREENING IN THE CASSAVA COLLECTION The in vitro collection of the genus Manihot of the GRP at CIAT is currentlyrepresented by 6,643 materials; which have been registered in the MultilateralSystem of Access and Benefit sharing of the International Treaty on PlantGenetic Resources for Food and Agriculture. These materials must be free ofquarantine diseases for distribution to users worldwide.
Cassava in vitro collection at CIAT
Following the discovery of cassava viruses by NGS associated to the quarantinedisease CFSD, the large-scale virus screening in the cassava collection updatedthe number of viruses to be tested and the procedures in the GHL operationsworkflow (shown within the red rectangle).
Up to date, 61% (4087 accessions) of the in vitro cassava collection has been evaluated. The remaining 39% are in this process.
Samples with disease symptomsshowing the percentage of reads(siRNAs) that were mapped to knownviral sequences (shown in red color).Viral sequences used: PRSV: Papayaring spot virus, RHBV: Rice hojablanca virus, BGYMV: Bean goldenyellow mosaic virus, Cassava viruses(mentioned above). The (%) ofunmapped reads are shown in grey.The number of reads that wereobtained per sample are shown bythe dashed black line.
PERSPECTIVES:- Implementation of NGS for seed borne pathogen detection is
highly desirable in genebank collections, especially for collectionsthat need to be regenerated in the field, being exposed toemerging pathogens during regeneration crop seasons.
- NGS is a reliable and sensitive diagnostic option for the evaluationof germplasm with special country phytosanitary requirements,particularly when certain pathogen detection methods are not yetvalidated in the facility or positive controls are not available.
- The establishment of DNA banks will increase the possibility ofoptimizing operations for DNA pathogen detection by using NGS.
(a) (b)
RHBV genome has 4 RNA segments
Reads mapped to virus RNA segmentsFr
equ
ency
Number of reads and percentage of reads mapped to virus references in different plant species
Rice Rice Bean Cassava Cassava Cassava
BLASTx search
Papaya
