Circular RNA Profiling by Illumina Sequencing via Template ...downloads.hindawi.com/journals/bmri/2019/2756516.pdf · ResearchArticle Circular RNA Profiling by Illumina Sequencing

Research ArticleCircular RNA Profiling by Illumina Sequencing viaTemplate-Dependent Multiple Displacement Amplification

Ashirbad Guria 1 Kavitha Velayudha Vimala Kumar 1 Nagesh Srikakulam 1

Anakha Krishnamma 1 Saibal Chanda 1 Satyam Sharma 1 Xiaofeng Fan 2

and Gopal Pandi 1

1Department of Plant Biotechnology School of Biotechnology Madurai Kamaraj University Madurai 625021 Tamil Nadu India2Division of Gastroenterology and Hepatology Saint Louis University Liver Center Saint Louis University St Louis MO 63110 USA

Correspondence should be addressed to Xiaofeng Fan xiaofengfanhealthslueduand Gopal Pandi pgopalbiotechmkuniversityorg

Received 26 October 2018 Revised 10 December 2018 Accepted 31 December 2018 Published 28 January 2019

Academic Editor Yujiang Fang

Copyright copy 2019 Ashirbad Guria et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Circular RNAs (circRNAs) are newly discovered incipient non-coding RNAs with potential roles in disease progression in livingorganisms Significant reports since their inception highlight the abundance and putative functional roles of circRNAs in everyorganism checked for like O sativa Arabidopsis human andmouse CircRNA expression is generally less than their linear mRNAcounterparts which fairly explains the competitive edge of canonical splicing over non-canonical splicing However existingmethods may not be sensitive enough for the discovery of low-level expressed circRNAs By combining template-dependentmultiple displacement amplification (tdMDA) Illumina sequencing and bioinformatics tools we have developed an experimentalprotocol that is able to detect 1875 novel and known circRNAs from O sativa The same method also revealed 9242 putativecircRNAs in less than 40million reads for the first time from theNicotiana benthamianawhose genomehasnot been fully annotatedSupported by the PCR-based validation and Sanger sequencing of selective circRNAs our method represents a valuable tool inprofiling circRNAs from the organisms with or without genome annotation

1 Introduction

Circular RNA (circRNAs) is an emerging class of non-coding RNA that attracts significant attention in scientificcommunity They are covalently closed without the 51015840 capand polyadenylation in the 31015840 end CircRNAs were initiallydescribed from plant viroids [1] and subsequently identifiedin human and visualised by electronmicroscopy over 35 yearsago [2] Consequent work in sex-determining region Y (SRY)genewas recognised as splicing errors [3 4] However studieson antisense non-coding RNA in INK4 locus (ANRIL) [5]and cerebellar degeneration-related protein 1 (CDR1) [6]have provided compelling evidence for circRNA in human[7] mammals [8] archaea [9] C elegans and mice [10]CircRNAs are synthesized by backsplicing of downstreamdonor site with the upstream acceptor site using the canonicalspliceosomal signals and machinery [11 12] Recently evergrowing reports on circRNAs reveal their pivotal roles in

fundamental biological pathways by their multiple functionalaspects such as microRNAs (miRNAs) sponges [10 13ndash17]cap-independent translation [18ndash21] modulation of cellularproliferation [19 22ndash24] scaffolding the protein activity [1225 26] and competition with linear mRNAs [19]

With the assistance from computational algorithms [7 827ndash32] numerous approaches have been developed to detectand validate the presence of circRNAs in different speciesacross the kingdoms [12 15 33 34] A major challenge incircRNA discovery might be attributed to its extremely lowabundance in samples The cutting-edge method involvesthe enrichment of circRNA by enzymatic digestion RNA-Seq and bioinformatics identification followed by PCR-based validation [13 35 36] A limitation in this approach isthe sensitivity because library preparation in next-generationsequencing (NGS) is often associated with the loss of low-abundant molecules [12 37] Thereby significant sequencingdepth is required in order to identify putative circRNAs [12]

HindawiBioMed Research InternationalVolume 2019 Article ID 2756516 12 pageshttpsdoiorg10115520192756516

2 BioMed Research International

In the present study we have introduced a step of template-dependent multiple displacement amplification (tdMDA)prior to library preparation Together with a newly developedcomputer program we have built an experimental pipelinethat shows an enhanced sensitivity to identify circRNAs fromthe plants

2 Materials and Methods

21 Plant Materials O sativa plants were grown in greenhouse maintained at 32∘C and (70-80) humidity for 2months Similarly N benthamiana were grown in plantgrowth chamber with 16 hrs8hrs lightdark condition and85 humidity for 2 months Plant leaves at 30- and 45-dayold were collected for RNA isolation from O sativa and Nbenthamiana respectively

22 RNA Extraction Reverse Transcription (RT) andTemplate-Dependent Multiple Displacement Amplification(tdMDA) Total RNA was extracted from approximately100mg of leaves of N benthamiana and O sativa usingTri Reagent (Sigma St Louis MO USA) according to themanufacturerrsquos instruction (TRI Reagent (T9424)-Technicalbulletin) Extracted RNA was treated with 4U of TurboDNase (2U120583L Ambion Austin TX USA) at 37∘C for30 minutes and then inactivated at 72∘C for 10 minutesfollowed by phenolchloroform purification Ten microgramof DNase-treated RNA was subjected to 10U of RNaseR (20U120583L Epicentre Madison WI USA) digestion at37∘C for 15 minutes Both integrity and concentration weredetermined respectively on 12 agarose gel and NanodropND-1000 (Thermo Scientific Waltham MA USA)

RNA amplification was achieved by RT-tdMDA protocol[38] In that protocol background amplification in MDAwas eliminated by using exo-resistant random pentamerprimers with their 51015840 ends blocked by C18 spacer [38] Itsefficiency was first evaluated by using extracted RNA andplasmid APTR9 [39 40] with the final primer concentrationranging from 50 to 200 120583M About 1 120583g of RNase R-treated RNA was converted into cDNA using RevertAid orRevertAidHminus first strand cDNA synthesis kit accordingto manufacturerrsquos instructions (Thermo Scientific WalthamMA USA) Approximately 50 ng of converted cDNA wasdirectly used for tdMDA in a 20-120583L reaction consisting of2120583l of 10mM dNTP mix 2 120583l of 10X reaction buffer 2120583lof 500 120583M 51015840end-blocked exo-resistant random pentamerprimers 06120583l of Phi29 DNA polymerase (10U120583l ThermoScientific Waltham MA USA) and 2120583l of pyrophosphatase(001U120583l) (Thermo Scientific Waltham MA USA) Thereaction mixture was incubated at 28∘C for 18 hours andterminated by heating at 65∘C for 10 minutes An aliquot of3120583l reaction was loaded on 1 TAE agarose gel to check fortdMDA performance

23 Identification of circRNA from the tdMDA Ampli-cons by Cloning and Sequencing The tdMDA ampliconwas randomly digested with the restriction enzymes SacIHindIII SspI BamHI EcoRV and EcoRI (10U120583l ThermoScientific Waltham MA USA) for 3 hours at 37∘C The

HindIII SacI digested fragments were purified by GeNeiPCR purification kit (Bangalore Karnataka India) andcloned in pOK12 or pBluescript II KS (+) vector at thecorresponding site at 16∘C for 12 hours After the con-firmation by restriction digestion a total of nine cloneswere sequenced seven for N benthamiana and two for Osativa The mapped clone sequences such as HindIII 10HindIII 33 HindIII 38 and SacI 11 for N benthamiana andHindIII 1 and HindIII 2 for O sativa were subjected toprediction for their possibility of forming circRNA in Plant-circBase [41] (httpibizjueducnplantcircbaseindexphp)Predicted putative circRNA sequences were validated by RT-PCR with divergent primers (Table 1) [42]

24 Identification of circRNA from the tdMDA Ampliconsby Illumina Sequencing About 200 ng of tdMDA prod-ucts was used for library construction using Illumina-compatible NEXTflex Rapid DNA sequencing kit (BIOOScientific Austin Texas USA) according to manufacturerrsquosinstructions followed by sequencing at the Illumina NextSeq500 platform (150-nt paired end) at Genotypic TechnologyBangalore as previously described [40 43] Under genomicannotations from Ensembl plant release 29 [44] trimmedreads at Phred 23 were aligned with O sativa Indica genomeand N benthamiana draft genome for subsequent circRNAidentification using DCC software (v 044) [45] In additionto the consideration of non-canonical splice junction otherparameters were also included for circRNA identification aspostulated in the DCC [45] All the analysis was carried outusing Biolinux 8 OS [46]

25 Validation of circRNAs Derived from tdMDA-IlluminaSequencing Divergent primers were designed from the cir-cRNA derived from NGS-tdMDA containing the junctionsite (Table 1) Most primers designed for O sativa and Nbenthamiana were tested for the validation of correspondingcircRNAs using genomic DNA and cDNA by the standard-ised annealing temperature (TA) Divergent PCR productswere subjected to sequencing or digestion with restrictionenzymes

26 Northern Hybridization Non-radioactive northern hy-bridization was performed with the purified PCR fragment(gt200 nt) as the probe which spanned the corresponding cir-cRNA junction site Probe preparation was followed with theDIG DNA labelling kit (Roche Basel Switzerland) accordingto manufacturerrsquos instructions

27 Analysis on circRNA Conservation and miRNA BindingSites NCBI-BLASTN was used to examine the conserva-tion of circRNAs with other reported plant species suchas S bicolour [47] S italica [47] B distachyon [47] andthose included in plant circular RNA database [41] ThepsRNATarget a plant small RNA target analysis server (2017release) [48] was applied to annotate the possible role ofreported O sativa and N tabacum miRNAs on predictedcircRNAs

BioMed Research International 3

Table 1 List of divergent primers designed for use in confirmation of putative circRNA

Divergent primer Sequence

HindIII 10 Forward 51015840-CTATAGTTGAAGCACCTGATGGTGT-31015840

Reverse 51015840-GAGCCATAAAGATAGGCAGTAACTACA-31015840

HindIII 33 Forward 51015840-TGGTTCACCACAACCCGT-31015840

Reverse 51015840-TGTGTGACTCAAGTTCTCAGTTTGTAA-31015840

osi circ1(136416264-36418547)

Forward 51015840-TGGTAGCAACCGCACAAA-31015840

Reverse 51015840-ATGCTTCCAGGCACATCA-31015840

osi circ2(219273316-20009087)

Forward 51015840-GGGAGCTCAAGGTGAAGAT-31015840

Reverse 51015840-GTTGAACAAACAACACACAAC-31015840

osi circ3(824552647-24573025)

Forward 51015840-ACGTTGAGAGTAAGTTTCCG-31015840

Reverse 51015840-CCCTTTACGATACCACTAGCC-31015840

osi circ4(1216650523-17328210)

Forward 51015840-TAGGCTCACGATGTGTTGC-31015840

Reverse 51015840-CGATGAGGGCTGCGAAC-31015840

osi circ5(915720676-15721227)

Forward 51015840-ATCCTTGGAGCTGGCTATGA-31015840

Reverse 51015840-ATCTCGGTTGACCACACACT-31015840

osi circ6(715534138-15534682)

Forward 51015840-TCAAGTCCGCCGTCAAATC-31015840

Reverse 51015840-CCCAAGGGCAGGTTCTTAC-31015840

osi circ7(627117575-27118530)

Forward 51015840-TGCAGAAACAGCATGGTCA-31015840

Reverse 51015840-ATAGGGTGCAAACCTGTGAG-31015840

osi circ8(84494958-4495647)

Forward 51015840-AGAGTCTCTGGCAGTCTCC-31015840

Reverse 51015840-AACCAGTGACTAGCAACTAAGAA-31015840

osi circ9(141518651-41519075)

Forward 51015840-GCGACCTTACTGCACGAATA-31015840

Reverse 51015840-TTGCAAGCGCAACACAAC-31015840

osi circ10(815854661-15861841)

Forward 51015840-GCTAGCAGGGACAGGTTATC-31015840

Reverse 51015840-CAGAAGACGTGTGTGCCTAT-31015840

Nb circ1(Niben101Scf01334583095ndash583645)

Forward 51015840-CTGGGTCAGTCCTCCATTT-31015840

Reverse 51015840-AGATACGCATGCCTCCAA-31015840

Nb circ2(Niben101Scf01481214685-215144)

Forward 51015840-TCAACGTGCTTCCTGAACT-31015840

Reverse 51015840-AAATGCTTGGGTCCTACTCC-31015840

Nb circ3(Niben101Scf01671738307-738555)

Forward 51015840-TCTTGTCCCAGTCCAGAGA-31015840

Reverse 51015840-TGTCTCCGCGTGTTAATGT-31015840

Nb circ4(Niben101Scf0182033613-33924)

Forward 51015840-GTTGTGCTCATTCCATTGGG-31015840

Reverse 51015840-TGCTTCCTGAGCAAGTTCTG-31015840

Nb circ5(Niben101Scf01505317983-318653)

Forward 51015840-CCCAATCCACCTTGATCCTT-31015840

Reverse 51015840-CACGACTGGATTTGGCGATA-31015840

Nb circ6(Niben101Scf3227610732-11201)

Forward 51015840-TGGGTACCGAAGTGTACTGT-31015840

Reverse 51015840-AAACCTTGGACCGAGATCAAAT-31015840


Forward 51015840-TGAGCCATTCGCAGTTTCA-31015840

Reverse 51015840-GGTCGTCTCGTCCCTTCT-31015840


Forward 51015840-TGGCTAGAATGCGGGTTTC-31015840

Reverse 51015840-ATCTTGAAAGTCGTGGTTTCCT-31015840


Forward 51015840-GCAGTTGGAGACTTTGAGGT-31015840

Reverse 51015840-TGCCGCAAGGGTGATATG-31015840


Forward 51015840-ACAGGTAGTCTGTTCCGACA-31015840

Reverse 51015840-AGATGCCGAGGAGTTGGA-31015840


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Figure 1Amplification of cDNAby Phi29 DNApolymerase Total RNA fromN benthamiana (a) andO sativa (b) was treatedwith DNaseand RNase R to enrich circRNAsThe enriched circRNAs were converted into cDNA using random hexamer and subjected to amplificationby Phi29 DNA polymerase

3 Results

31 e Elimination of Background Amplification by tdMDATemplate independent amplification (TIA) inMDA is amajorconcern owing to high concentrations of random hexamersand an extended incubation period [38] To eliminate TIAwe followed the protocol proposed by Wang et al [38]Total RNA extracted from O sativa was mixed with theplasmid pAPTR9 that harboured Bhendi yellow vein mosaicvirus (BYVMV) After DNase treatment BYVMV-specificPCR yielded no amplification suggesting a complete DNAdigestion in the template (Figure S1a)This template was sub-sequently used to test the efficiency of RT-tdMDA protocol[38] In four primer concentrations (50 100 150 and 200120583M) no amplicon was found in the controls (no template)(Figure S1b) In contrast 50 ng of template along with 50 120583Mfinal primer concentration showed an apparent amplification(Figure S1b) Therefore the use of exo-resistant randompentamer primers with blocked 51015840 ends efficiently eliminatesTIA

32 Novel circRNAs Identified by RT-tdMDA Cloning andSanger Sequencing After DNase and RNase R treatment(Figure S2) total RNA from N benthamiana and O sativaplants was successfully amplified (Figure 1) Again noamplification was observed from the negative controls (notemplate) (Figure 1) Of seven sequenced clones derived fromN benthamiana four sequences named SacI 11 HindIII 10HindIII 33 and HindIII 38 showed 100 sequence identityin BLAST analysis against N benthamiana genome [49](httpssolgenomicsnetorganismNicotiana benthamianagenome) Two clones from O sativa HindIII 1 and HindIII2 were aligned onto the intron region in chromosomes 7and 1 of O sativa with 100 and 99 identity respectivelyThese sequences were then analyzed in PlantcircBase forthe potential of circRNA formation As a result threesequences from N benthamiana HindIII 10 HindIII33 and SacI 11 were predicted to be putative circRNAsThe HindIII 10 sequence was partially mapped onto theintron domain of N3 disease resistance protein gene ofNicotiana paniculata with 96 sequence identity (Figure S3


O Sativa IndicaTotal number of reads 21818956

circRNA obtained 1875

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Figure 2 Identification of rice circRNAs from NGS data CircRNAs identified from total number of reads obtained in rice (a) theirchromosome wise distribution (b) types (c) size distribution (d)

Figure S5 and Figure S6) suggesting its intronic natureThe HindIII 33 sequence was aligned to multiple domainsincluding the unannotated region of the retrotransposonof Nicotiana tabacum (1-156 bp 88 identity) Frigida likeprotein gene of N benthamiana (156-259bp 87 identity)and 40S ribosomal protein gene (227-282 bp 94 identity)Therefore HindIII 33 sequence might be an intronic-exoniccircRNA (Figure S3 Figure S5 and Figure S6) The SacI 11sequence (Acc No MF066173) was predicted as a circRNAHowever this sequence was mapped onto Nicotiana sylvestrismitochondrial genome and thus not included for furtherexperimentation Analysis in PlantcircBase also suggestedthat two clones from O sativa HindIII 1 (osa circ 032545)and HindIII 2 (osa circ 000547) were existing intronic andexon-intronic circRNAs respectively For putative circRNAsequences HindIII 10 andHindIII 33 PCR amplification withdivergent primers provided a positive result when cDNAwas

used as template whereas no amplification was observed atthe same size when various concentration of genomic DNAwas used as template (Figure S4)

33 Complete circRNA Profiles Revealed by Illumina Sequenc-ing Encouraged by the positive outcome from the cloningand Sanger sequencing the amplicons from RT-tdMDAwere subjected to Illumina sequencing for possible captureof entire circRNA repertoire The total number of 150-ntpaired end reads obtained fromO sativa andN benthamianawere 21818956 and 38060238 respectively Using the rawreads from the O sativa the DCC computational pipelinediscovered thousands of circular splicing events that yielded1875 circRNAs (Figure 2(a)) These putative circRNAs arepredominantly distributed on the chromosomes 1 and 5(Figure 2(b)) Perhaps due to the unannotated genome ofIndian cultivar (Pusa Basmati 1) around 200-300 circRNAs


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Figure 3 Rice circRNAs analysis Number of rice genes giving circRNA(s) (a) number of overexpressed circRNAs than their linearcounterparts across chromosomes (b)

came from the genes without any particular chromosomeassignment (Figure 2(b)) With respect to circRNA types theintergenic-intergenic type (n=1359) was the most abundanttype followed by the intronic-intronic type (n=182) and theexonic-exonic type (n=123) (Figure 2(c)) Furthermore 79of putative circRNAs had the length between 100 and 999nt whereas sim1 and sim20 had the size below 100 nt andlarger than 1000 nt respectively (Figure 2(d)) The smallestcircRNA was found to be of 32 nt between positions 5187-5219 in the genome This putative circRNA is surprisinglyan intergenic-intergenic type with CTAC splice junction onscaffold ID AAAA020401371 On the other hand the largestsize of circRNA identified was 737782 nt on the chromosome11 between positions 18852424 and19590206 which harboursmany functional genes like MIR genes tRNA genes andthe genes encoding for hypothetical protein The largestcircRNA is assumingly formed in a non-canonical mannerand categorised as an intron-intergenic type Finally allputative circRNAswere associated with a total of 578 genes inwhich sim72had the translation of hypothetical proteinsThegene ID BGIOSGA000405 on the chromosome 1 contributedthe maximum number of circRNAs (n=35) while most genesgave only one or two circRNAs (Figure 3(a)) Individuallyless than 10 of the genes could produce more than twocircRNAs

Similar analysis in N benthamiana yielded 9242 cir-cRNAs including 6080 intergenic-intergenic 1257 intron-intron 1009 intron-intergenic and 896 intergenic-introniccircRNAs (Figures 4(a) and 4(b)) Interestingly no exonic cir-cRNAs were identified probably because of unavailability ofcomplete genome annotation In comparison with O sativacircRNAs from N benthamiana were larger in size with 69of total identified circRNAs above 1000 nt (Figure 4(c)) TheNiben101Scf02816 an intergenic-intergenic circRNA formedby the non-canonical splice junction had the smallest sizewith about 35 nt located between positions 85132 and 85167on the genome The longest circRNA was Niben101Scf03154with 299801 nt also an intergenic-intergenic type between589 and 300390 genome positions

34 Validation of circRNAs Identified by RT-tdMDA andIllumina Sequencing PCR and northern hybridization wereused for the validation of selective circRNAs For cir-cRNA Niben101Scf27324 (Nb circ7 primer Table 1) PCRproduced two distinct DNA bands with the sizes at sim150 and sim250 bp There was no DNA amplification atsimilar sizes upon the use of the genomic DNA as tem-plate (Figures 5(a) and 5(b)) Sanger sequencing of PCRproduct mapped the larger fragment to the circRNA withextra sequence suggesting a potential alternative splicingevent involved for the biogenesis of this particular circRNA(Figure S7) Further evidence came from the northernhybridization that signalled an apparent presence of cir-cRNA Niben101Scf27324 (Figure S8) Divergent PCR alsogave positive amplification for the osi circ10 (Figure 5(c))as well as other putative circRNAs including Nb circ3Nb circ6 osi circ2 osi circ4 osi circ6 and osi circ8 (datanot shown) Their authenticity was supported by restrictiondigestion of purified DNA bands from the gel (data notshown)

35 CircRNAs Are Conserved across the Species Several re-ports claimed conservative nature of circRNA across species[8]Therefore we compared our circRNAs with all circRNAseither reported [15] or deposited in the plant circular RNAdatabase [41] Of 1875 cicrRNAs from the O sativa signif-icant similarity was found for 1120 (60) to O Sativa sspNipponbare 549 (292) to A thaliana and 145 (775) toT aestivum (Figure 6(a)) For N benthamiana the sequencesimilarity was also shared for 55 circRNAs with S tuberosum60 circRNAswithA thaliana and 44 circRNAswithO Sativa(Figure 7(a)) There was no or little conservation betweenour putative circRNAs and the circRNAs discovered in theplants like S bicolour S italica B distachyon H vulgareand P trifoliataThis is probably due to a rare number of thecircRNAs identified from these plants that could not providea full scenario to explore the conservation (Table 2)


N benthamianaTotal number of reads 38060238

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Figure 4 Identification of N benthamiana circRNA from NGS data CircRNAs identified from total number of reads obtained in Nbenthamiana (a) their types (b) size distribution (c)

4 Discussion

CircRNAs encompass a transcript family with distinctivestructures Various methods are used to detect the circRNAsin both plants and animals [10 13] The difficulty in circRNAidentification lies in the inability to separate the circRNAsfromother RNA species based on their size or electrophoreticmobility Molecular techniques that involve amplificationor fragmentation may destroy their circular nature sincecircRNAs lack a free 51015840 or 31015840 end Likewise methods thatuse polyadenylation ends such as rapid amplification ofcDNA ends (RACE) or poly (A) enrichment cannot beemployed for circRNA identification These hindrances havebeen overcome by the emergence of exonuclease basedenrichment procedures and high throughput sequencingtechniques [12] For instance RNA sequencing has beenused for the identification of circRNAs in Arabidopsis and Osativa [14] However owing to an extremely low expressionof circRNAs comparing to their linear mRNA counterparts ahigh sequencing depth is demanded for productive capture ofcircRNAs In order to improve the sensitivity we exploited theuse of tdMDA to identify circRNAs from the N benthamianaand O sativa plants

Our data have demonstrated the feasibility of tdMDA inthe discovery of circRNAs from small amount of RNA sam-ples First both novel and known circRNAs were identifiedfrom RT-tdMDA product by random enzymatic digestioncloning and Sanger sequencing Two novel circRNAs fromthe N benthamiana were validated by divergent PCR andhad no significant similarity with Arabidopsis and otherplant candidates The function of newly identified circRNAsfromN benthamiana has to be deciphered Second Illuminasequencing of the RT-tdMDA product and bioinformaticsanalysis captured 1875 and 9242 putative circRNAs fromO sativa and N benthamiana respectively The authenticityof selective cirRNAs was confirmed by PCR and northernhybridization Using RNA sequencing Jakobi et al [50]reported the prediction of 575 circRNAs from 335 millionreads in adult mice heart Assuming a similar abundanceamong the samples we could be able to identify much highernumber of circRNAs from almost equal number of readsusing the same computational pipeline Earlier Jeck andSharpless (2013) stressed on the need of having 300000-300000000 reads using traditional sequencing to get a singlecircRNA event whereas exonic circRNA is thought to present


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(c)

Figure 5DivergentPCR for validationofNGS-tdMDAderived circRNA Two products (gt100bp andgt200bp)were amplifiedwith nb circ7primer upon divergent PCR (lane 456) with three N benthamiana cDNA (a) With same primer it did not give same size amplicon withgenomic DNA (lane 123) as template (b) Two amplicons at sim100bp and 270 bp formed from rice cDNA (lane3) with osi circ10 divergentprimer whereas non-specific amplicon also formed when taking genomic DNA as template (lane 2) (c) Non-template PCR was taken asnegative control (lane 2 in (a) lane 5 in (b) and lane 4 in (c)) and 100 bp amplicon formed from 58s as positive control (lane 1 in (a) lane 4in (b) and lane 5 in (c)) Generuler 100 bp plus ladder in lane 3 (a) lane 6 (b) and Fermentas 100 bp ladder in lane 1 (c)

roughly 1 of poly(A) RNA [12 31] Again Wang et al in2017 analyzed over 90 million raw reads and could obtainonly 88 circRNAsAnalyzing saymore than 500million readswill surely increase the chances of getting low abundancecircRNAs but it will spike up the overall cost tremendouslyOur method reduces the cost significantly without compro-mising on findings of lowly expressed circRNAs Finally theconservative nature of most predicted circRNAs across theplants further suggests the methodological reliability

Besides tdMDA our experimental pipeline also takesthe power of the bioinformatics tool We have applied theDCC that gives the expression count of putative circRNAsas well as the linear RNAs expected from the same genomepositions Interestingly 19 of O sativa circRNAs showedoverexpression with respect to their linear counterparts(Figure 3(b)) Functional aspects of circRNAs have not beenfully understood in spite of the reports for their roles inmiRNA sponging transcriptional inhibition and protein

formation [12] Our analysis revealed there are 33 circRNAsthat bind to 156 miRNAs in O sativa Nipponbare (Table S1)This number is translated into 473 miRNA binding sites asa single circRNA can bind to more than one miRNA orvice versa (Table S2) Of the 473 miRNA binding sites 391sites (sim83) are cleavage specific while the remaining 82sites (sim17) are possibly getting sponged by their targets(Figure 6(b)) InN benthamiana 2099 circRNAs could have8149 miRNA binding sites on 163 published N tabacummiRNAs (Table S3 and S4) Approximately 85 (n=6916) ofmiRNA binding sites are cleavage specific and 15 (n=1233)are target inhibitory in action that need to be deciphered(Figure 7(b))

5 Conclusion

In summary through the combination of tdMDA and bioin-formatics tools we have established an experimental protocol


0

200

400

600

800

1000

1200

O sativa A thaliana T aestivumPlants

0

10

20

30

40

50

60

Plants

No

of c

ircRN

A(s

)

Z m

ays

S tu

bero

sum

G m

ax

P tr

ifolia

ta

G h

irsut

um

S ly

cope

rsicu

m

G a

rbor

eum

G ra

imon

dii

H v

ulga

re

S b

icolo

r

S it

alica

Bdi

stach

yon

No

of c

ircRN

A(s

)

(a)

050

100150200250300350400450

cleavage putative spongingPredicted mode of action of miRNAs

No

of m

iRN

A b

indi

ng si

tes

(b)

Figure 6 CircRNA conservation and miRNA action in rice Conservation of 1875 predicted Indica circRNAs against reported circRNAfrom fifteen plants (a) mode of action of reported Japonica miRNAs on predicted circRNAs (b)

010203040506070

Plants

A th

alia

naS

tube

rosu

mO

sat

iva

S ly

cope

rsicu

mT

aes

tivum

P tr

ifolia

taG

arb

oreu

mG

hirs

utum

G ra

imon

dii

G m

axS

bico

lor

Z m

ays

H v

ulga

reS

ital

icaB

dista

chyo

n

No

of c

ircRN

A(s

)

(a)

0

1000

2000

3000

4000

5000

6000

7000

8000


No

of m

iRN

A b

indi

ng si

tes

(b)

Figure 7 CircRNA conservation and miRNA action in N benthamiana Conservation of predicted 9242 circRNAs against reportedcircRNAs from fifteen plants (a) mode of action of reportedN tabacummiRNAs on predicted circRNAs (b)


Table 2 List of circRNAs reported from different plants

Plants Total no of circRNAs ReferenceO sativa Japonica 40311 Chu et al 2017A thaliana 38938 Chu et al 2017T aestivum 88 Chu et al 2017Z mays 3238 Chu et al 2017H vulgare 39 Chu et al 2017G max 5323 Chu et al 2017S tuberosum 1728 Chu et al 2017S lycopersicum 1904 Chu et al 2017G arboreum 1041 Chu et al 2017G raimondii 1478 Chu et al 2017G hirsutum 499 Chu et al 2017P trifoliata 556 Chu et al 2017S bicolor 73 Lu et al 2015S italica 113 Lu et al 2015B distachyon 26 Lu et al 2015

to detect circRNAs from plant samples Currently efficientcircRNA discovery requires the treatment of RNA sampleby DNase and RNase R which are often associated with theabundance loss of RNA species including circRNAs Ourmethod is thus particularly useful in working with limitedamount of biological samples A comprehensive profiling ofcircRNAs as illustrated in O sativa and N benthamianarepresents an essential step toward biological understating ofcircRNAs in plants as well as other organisms This is thefirst report of circRNA identification from the model plantN benthamiana

Data Availability

Three cloned sequences that denote putative circRNAs aredeposited in the GenBank of NCBI under accession numberMF066173 through MF066175 Raw Illumina sequencedata were placed under Bioproject ID PRJNA422356 (SRAaccession nos SRX4502417 SRX4502416 SRX4502415SRX3470454 and SRX3470453)

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Gopal Pandi Ashirbad Guria and Kavitha Velayudha VimalaKumar conceived and designed the experiments AshirbadGuria Kavitha Velayudha Vimala Kumar Anakha Krish-namma Saibal Chanda and Satyam Sharma performed theexperiments Data Analysis was done by Nagesh SrikakulamAshirbad Guria Kavitha Velayudha Vimala Kumar AnakhaKrishnamma Saibal Chanda Satyam Sharma and GopalPandi Paper was written by Ashirbad Guria Xiaofeng FanGopal Pandi and Kavitha Velayudha Vimala Kumar andreviewed by Xiaofeng Fan Gopal Pandi and Ashirbad Guria

Acknowledgments

This work is partially supported by grants from Departmentof Biotechnology (DBT) (Ref no BTPR2061AGR367072011 dated 25-06-2011 and BTPR6466COE34162012dated 28-10-2014) and Science and Engineering ResearchBoard (SERB) (Ref no SBEMEQ-0702013 dated 05-07-2013and SRFTLS-482011 dated 01-05-2012) We are gratefulto Department of Science and Technology (DST) for theINSPIRE Fellowship (Ref no IF110736) to V KavithaThe equipment grants from Department of Science andTechnology-Promotion of University Research and ScientificExcellence (DST-PURSE) University Grant Commission-Special Assistance Programme (UGC-SAP) are gratefullyacknowledged

Supplementary Materials

Table S1 list of 156 miRNAs binding with 33 predicted IndicacircRNAs Table S2 number of miRNA binding sites for eachof the 33 Indica circRNAs with their position in the genomeTable S3 number of miRNA binding sites for each of the2099 N benthamiana circRNAs with their position in thegenome Table S4 list of N tabacum miRNAs with theirnumber of binding sites on 2099 N benthamiana circRNAsFigure S1 absence of TIA inMDA Figure S2 RNA extractionfrom N benthamiana and O sativa plants Figure S3linear and predicted circular maps of the putative circRNAsof N benthamiana Figure S4 divergent PCR for circRNAconfirmation Figure S5 BLAST analysis of N benthamianacloned sequences in Sol Genomics Network Figure S6BLAST analysis of N benthamiana cloned sequences inNCBI Figure S7 mapping and validation of N benthamianacircRNA Figure S8 northern blotting for confirmation ofcircRNA (Supplementary Materials)


References

[1] H L Sanger G Klotz D Riesner H J Gross and A KKleinschmidt ldquoViroids are single stranded covalently closedcircular RNA molecules existing as highly base paired rod likestructuresrdquo Proceedings of the National Acadamy of Sciences ofthe United States of America vol 73 no 11 pp 3852ndash3856 1976

[2] M-T Hsu and M Coca-Prados ldquoElectron microscopic evi-dence for the circular form of RNA in the cytoplasm ofeukaryotic cellsrdquo Nature vol 280 no 5720 pp 339-340 1979

[3] B Capel A Swain S Nicolis et al ldquoCircular transcripts of thetestis-determining gene Sry in adult mouse testisrdquo Cell vol 73no 5 pp 1019ndash1030 1993

[4] I Chen C-Y Chen and T-J Chuang ldquoBiogenesis identifica-tion and function of exonic circular RNAsrdquo Wiley Interdisci-plinary Reviews RNA vol 6 no 5 pp 563ndash579 2015

[5] C E Burd W R Jeck Y Liu H K Sanoff Z Wang and NE Sharpless ldquoExpression of linear and novel circular formsof an INK4ARF-associated non-coding RNA correlates withatherosclerosis riskrdquo PLoS Genetics vol 6 no 12 Article IDe1001233 2010

[6] T B Hansen E D Wiklund J B Bramsen et al ldquomiRNA-dependent gene silencing involving Ago2-mediated cleavage ofa circular antisense RNArdquo EMBO Journal vol 30 no 21 pp4414ndash4422 2011

[7] J Salzman C Gawad P L Wang N Lacayo and P O BrownldquoCircular RNAs are the predominant transcript isoform fromhundreds of human genes in diverse cell typesrdquo PLoS ONE vol7 no 2 Article ID e30733 2012

[8] W R Jeck J A Sorrentino K Wang et al ldquoCircular RNAs areabundant conserved and associated with ALU repeatsrdquo RNAvol 19 no 2 pp 141ndash157 2013

[9] M Danan S Schwartz S Edelheit and R Sorek ldquoTranscripto-me-wide discovery of circular RNAs in ArchaeardquoNucleic AcidsResearch vol 40 no 7 pp 3131ndash3142 2012

[10] S Memczak M Jens A Elefsinioti et al ldquoCircular RNAs area large class of animal RNAs with regulatory potencyrdquo Naturevol 495 no 7441 pp 333ndash338 2013

[11] L-L Chen and L Yang ldquoRegulation of circRNA biogenesisrdquoRNA Biology vol 12 no 4 pp 381ndash388 2015

[12] W R Jeck and N E Sharpless ldquoDetecting and characterizingcircular RNAsrdquoNatureBiotechnology vol 32 no 5 pp 453ndash4612014

[13] Y Wang M Yang S Wei F Qin H Zhao and B SuoldquoIdentification of circular RNAs and their targets in leaves ofTriticum aestivum L under dehydration stressrdquo Frontiers inPlant Science vol 7 no 2024 2017

[14] C-Y Ye L Chen C Liu Q-H Zhu and L Fan ldquoWidespreadnoncoding circular RNAs in plantsrdquo New Phytologist vol 208no 1 pp 88ndash95 2015

[15] J Liu T Liu X Wang and A He ldquoCircles reshaping the RNAworld From waste to treasurerdquoMolecular Cancer vol 16 no 1p 58 2017

[16] C Huang and G Shan ldquoWhat happens at or after transcriptionInsights into circRNA biogenesis and functionrdquo Transcriptionvol 6 no 4 pp 61ndash64 2015

[17] Q Chu E Shen C-Y Ye et al ldquoEmerging roles of plant circularRNAsrdquo Journal of Plant Cell vol 1 no 1 pp 1ndash14 2018

[18] N R Pamudurti O Bartok M Jens et al ldquoTranslation ofCircRNAsrdquoMolecular Cell vol 66 no 1 pp 9ndash21 2017

[19] I Legnini G Di Timoteo F Rossi et al ldquoCirc-ZNF609 Isa Circular RNA that Can Be Translated and Functions inMyogenesisrdquoMolecular Cell vol 66 no 1 pp 22ndash37 2017

[20] Y Wang and Z Wang ldquoEfficient backsplicing produces trans-latable circular mRNAsrdquo RNA vol 21 no 2 pp 172ndash179 2015

[21] Y Yang X FanM Mao et al ldquoExtensive translation of circularRNAs driven by N 6 -methyladenosinerdquo Cell Research vol 27no 5 pp 626ndash641 2017

[22] X Ma X Yang W Bao et al ldquoCircular RNA circMAN2B2facilitates lung cancer cell proliferation and invasion via miR-1275FOXK1 axisrdquo Biochemical and Biophysical Research Com-munications vol 498 no 4 pp 1009ndash1015 2018

[23] J Si-Tu Y Cai T Feng et al ldquoUpregulated circular RNAcirc-102004 that promotes cell proliferation in prostate cancerrdquoInternational Journal of Biological Macromolecules vol 122 pp1235ndash1243 2019

[24] J Zhou HWang J Chu et al ldquoCircular RNA hsa circ 0008344regulates glioblastoma cell proliferation migration invasionand apoptosisrdquo Journal of Clinical Laboratory Analysis vol 32no 7 p e22454 2018

[25] C P Gomes A Salgado-Somoza E E Creemers C DieterichM Lustrek and Y Devaux ldquoCircular RNAs in the cardiovas-cular systemrdquo Non-coding RNA Research vol 3 no 1 pp 1ndash112018

[26] L S Kristensen T B Hansen M T Venoslash and J KjemsldquoCircular RNAs in cancer Opportunities and challenges in thefieldrdquo Oncogene vol 37 no 5 pp 555ndash565 2018

[27] N A Fonseca J Rung A Brazma and J C Marioni ldquoToolsfor mapping high-throughput sequencing datardquo Bioinformaticsvol 28 no 24 pp 3169ndash3177 2012

[28] M G Grabherr B J Haas M Yassour et al ldquoFull-lengthtranscriptome assembly fromRNA-Seq datawithout a referencegenomerdquoNature Biotechnology vol 29 no 7 pp 644ndash652 2011

[29] J E Hooper ldquoA survey of software for genome-wide discoveryof differential splicing in RNA-Seq datardquoHuman Genomics vol8 no 1 article no 3 2014

[30] Z Peng C Yuan L Zellmer S Liu N Xu and D J LiaoldquoHypothesis Artifacts including spurious chimeric RNAs witha short homologous sequence caused by consecutive reversetranscriptions and endogenous random primersrdquo Journal ofCancer vol 6 no 6 pp 555ndash567 2015

[31] J Salzman R E ChenMNOlsen P LWang andPO BrownldquoCell-type specific features of circular RNA expressionrdquo PLoSGenetics vol 9 no 9 Article ID e1003777 2013

[32] KWangD Singh Z Zeng et al ldquoMapSplice accuratemappingof RNA-seq reads for splice junction discoveryrdquo Nucleic AcidsResearch vol 38 no 18 article e178 2010

[33] P L Wang Y Bao M-C Yee et al ldquoCircular RNA is expressedacross the eukaryotic tree of liferdquoPLoSONE vol 9 no 6 ArticleID e90859 2014

[34] H A Vincent and M P Deutscher ldquoSubstrate recognitionand catalysis by the exoribonuclease RNase Rrdquo e Journal ofBiological Chemistry vol 281 no 40 pp 29769ndash29775 2006

[35] A A Alhasan O G Izuogu H H Al-Balool et al ldquoCircularRNA enrichment in platelets is a signature of transcriptomedegradationrdquo Blood vol 127 no 9 pp e1ndashe11 2016

[36] BDarbani S Noeparvar and S Borg ldquoIdentification of circularRNAs from the parental genes involved in multiple aspects ofcellular metabolism in barleyrdquo Frontiers in Plant Science vol 7no 776 2016


[37] L Szabo and J Salzman ldquoDetecting circular RNAs Bioinfor-matic and experimental challengesrdquo Nature Reviews Geneticsvol 17 no 11 pp 679ndash692 2016

[38] W Wang Y Ren Y Lu et al ldquoTemplate-dependent multipledisplacement amplification for profiling human circulatingRNArdquo BioTechniques vol 63 no 1 pp 21ndash27 2017

[39] J Jose and R Usha ldquoBhendi yellow vein mosaic disease inindia is caused by association of a DNA 120573 satellite with abegomovirusrdquo Virology vol 305 no 2 pp 310ndash317 2003

[40] K Babu A Guria J Karanthamalai et al ldquoDNA MethylationSuppression by Bhendi Yellow VeinMosaic Virusrdquo Epigenomesvol 2 no 2 p 7 2018

[41] Q Chu X Zhang X Zhu et al ldquoPlantcircBase A Database forPlant Circular RNAsrdquo Molecular Plant vol 10 no 8 pp 1126ndash1128 2017

[42] S L Mehta G Pandi and R Vemuganti ldquoCircular RNAExpression Profiles Alter Significantly in Mouse Brain afterTransient Focal Ischemiardquo Stroke vol 48 no 9 pp 2541ndash25482017

[43] K Velayudha Vimala Kumar N Srikakulam P Padbhanabhanand G Pandi ldquoDeciphering microRNAs and Their AssociatedHairpin Precursors in a Non-Model Plant Abelmoschus escu-lentusrdquo Non-Coding RNA vol 3 no 2 p 19 2017

[44] B L Aken P Achuthan W Akanni et al ldquoEnsembl 2017rdquoNucleic Acids Research vol 45 no D1 pp D635ndashD642 2017

[45] J Cheng F Metge and C Dieterich ldquoSpecific identificationand quantification of circular RNAs from sequencing datardquoBioinformatics vol 32 no 7 pp 1094ndash1096 2016

[46] D Field B Tiwari T Booth et al ldquoOpen software for biologistsFrom famine to feastrdquo Nature Biotechnology vol 24 no 7 pp801ndash803 2006

[47] T Lu L Cui Y Zhou et al ldquoTranscriptome-wide investigationof circular RNAs in ricerdquo RNA vol 21 no 12 pp 2076ndash20872015

[48] X Dai Z Zhuang and P X Zhao ldquopsRNATarget a plantsmall RNA target analysis server (2017 release)rdquo Nucleic AcidsResearch vol 46 no W1 pp W49ndashW54 2018

[49] A Bombarely H G Rosli J Vrebalov P Moffett L A Muellerand G B Martin ldquoA draft genome sequence of Nicotianabenthamiana to enhance molecular plant-microbe biologyresearchrdquo Molecular Plant-Microbe Interactions vol 25 no 12pp 1523ndash1530 2012

[50] T Jakobi L F Czaja-Hasse R Reinhardt and C DieterichldquoProfiling and Validation of the Circular RNA Repertoire inAdult Murine Heartsrdquo Genomics Proteomics amp Bioinformaticsvol 14 no 4 pp 216ndash223 2016

Hindawiwwwhindawicom

International Journal of

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Anatomy Research International

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Journal of Parasitology Research

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The Scientific World Journal

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BioinformaticsAdvances in

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MicrobiologyHindawiwwwhindawicom

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Submit your manuscripts atwwwhindawicom


In the present study we have introduced a step of template-dependent multiple displacement amplification (tdMDA)prior to library preparation Together with a newly developedcomputer program we have built an experimental pipelinethat shows an enhanced sensitivity to identify circRNAs fromthe plants

2 Materials and Methods

21 Plant Materials O sativa plants were grown in greenhouse maintained at 32∘C and (70-80) humidity for 2months Similarly N benthamiana were grown in plantgrowth chamber with 16 hrs8hrs lightdark condition and85 humidity for 2 months Plant leaves at 30- and 45-dayold were collected for RNA isolation from O sativa and Nbenthamiana respectively

22 RNA Extraction Reverse Transcription (RT) andTemplate-Dependent Multiple Displacement Amplification(tdMDA) Total RNA was extracted from approximately100mg of leaves of N benthamiana and O sativa usingTri Reagent (Sigma St Louis MO USA) according to themanufacturerrsquos instruction (TRI Reagent (T9424)-Technicalbulletin) Extracted RNA was treated with 4U of TurboDNase (2U120583L Ambion Austin TX USA) at 37∘C for30 minutes and then inactivated at 72∘C for 10 minutesfollowed by phenolchloroform purification Ten microgramof DNase-treated RNA was subjected to 10U of RNaseR (20U120583L Epicentre Madison WI USA) digestion at37∘C for 15 minutes Both integrity and concentration weredetermined respectively on 12 agarose gel and NanodropND-1000 (Thermo Scientific Waltham MA USA)

RNA amplification was achieved by RT-tdMDA protocol[38] In that protocol background amplification in MDAwas eliminated by using exo-resistant random pentamerprimers with their 51015840 ends blocked by C18 spacer [38] Itsefficiency was first evaluated by using extracted RNA andplasmid APTR9 [39 40] with the final primer concentrationranging from 50 to 200 120583M About 1 120583g of RNase R-treated RNA was converted into cDNA using RevertAid orRevertAidHminus first strand cDNA synthesis kit accordingto manufacturerrsquos instructions (Thermo Scientific WalthamMA USA) Approximately 50 ng of converted cDNA wasdirectly used for tdMDA in a 20-120583L reaction consisting of2120583l of 10mM dNTP mix 2 120583l of 10X reaction buffer 2120583lof 500 120583M 51015840end-blocked exo-resistant random pentamerprimers 06120583l of Phi29 DNA polymerase (10U120583l ThermoScientific Waltham MA USA) and 2120583l of pyrophosphatase(001U120583l) (Thermo Scientific Waltham MA USA) Thereaction mixture was incubated at 28∘C for 18 hours andterminated by heating at 65∘C for 10 minutes An aliquot of3120583l reaction was loaded on 1 TAE agarose gel to check fortdMDA performance

23 Identification of circRNA from the tdMDA Ampli-cons by Cloning and Sequencing The tdMDA ampliconwas randomly digested with the restriction enzymes SacIHindIII SspI BamHI EcoRV and EcoRI (10U120583l ThermoScientific Waltham MA USA) for 3 hours at 37∘C The

HindIII SacI digested fragments were purified by GeNeiPCR purification kit (Bangalore Karnataka India) andcloned in pOK12 or pBluescript II KS (+) vector at thecorresponding site at 16∘C for 12 hours After the con-firmation by restriction digestion a total of nine cloneswere sequenced seven for N benthamiana and two for Osativa The mapped clone sequences such as HindIII 10HindIII 33 HindIII 38 and SacI 11 for N benthamiana andHindIII 1 and HindIII 2 for O sativa were subjected toprediction for their possibility of forming circRNA in Plant-circBase [41] (httpibizjueducnplantcircbaseindexphp)Predicted putative circRNA sequences were validated by RT-PCR with divergent primers (Table 1) [42]

24 Identification of circRNA from the tdMDA Ampliconsby Illumina Sequencing About 200 ng of tdMDA prod-ucts was used for library construction using Illumina-compatible NEXTflex Rapid DNA sequencing kit (BIOOScientific Austin Texas USA) according to manufacturerrsquosinstructions followed by sequencing at the Illumina NextSeq500 platform (150-nt paired end) at Genotypic TechnologyBangalore as previously described [40 43] Under genomicannotations from Ensembl plant release 29 [44] trimmedreads at Phred 23 were aligned with O sativa Indica genomeand N benthamiana draft genome for subsequent circRNAidentification using DCC software (v 044) [45] In additionto the consideration of non-canonical splice junction otherparameters were also included for circRNA identification aspostulated in the DCC [45] All the analysis was carried outusing Biolinux 8 OS [46]

25 Validation of circRNAs Derived from tdMDA-IlluminaSequencing Divergent primers were designed from the cir-cRNA derived from NGS-tdMDA containing the junctionsite (Table 1) Most primers designed for O sativa and Nbenthamiana were tested for the validation of correspondingcircRNAs using genomic DNA and cDNA by the standard-ised annealing temperature (TA) Divergent PCR productswere subjected to sequencing or digestion with restrictionenzymes

26 Northern Hybridization Non-radioactive northern hy-bridization was performed with the purified PCR fragment(gt200 nt) as the probe which spanned the corresponding cir-cRNA junction site Probe preparation was followed with theDIG DNA labelling kit (Roche Basel Switzerland) accordingto manufacturerrsquos instructions

27 Analysis on circRNA Conservation and miRNA BindingSites NCBI-BLASTN was used to examine the conserva-tion of circRNAs with other reported plant species suchas S bicolour [47] S italica [47] B distachyon [47] andthose included in plant circular RNA database [41] ThepsRNATarget a plant small RNA target analysis server (2017release) [48] was applied to annotate the possible role ofreported O sativa and N tabacum miRNAs on predictedcircRNAs








osi circ1(136416264-36418547)



osi circ2(219273316-20009087)



osi circ3(824552647-24573025)



osi circ4(1216650523-17328210)



osi circ5(915720676-15721227)



osi circ6(715534138-15534682)



osi circ7(627117575-27118530)



osi circ8(84494958-4495647)



osi circ9(141518651-41519075)



osi circ10(815854661-15861841)






Nb circ2(Niben101Scf01481214685-215144)



Nb circ3(Niben101Scf01671738307-738555)



Nb circ4(Niben101Scf0182033613-33924)



Nb circ5(Niben101Scf01505317983-318653)



Nb circ6(Niben101Scf3227610732-11201)
















MD

A w

ith cD

NA

MD

A w

ith R

NA

MD

A w

ithou

t tem

plat

e

231Kb

43Kb

23Kb

MD

A w

ith p

Blue

scrip

t II K

S(+)

pla

smid

H

indI

II la

dder

1 2 3 4 5

(a)

MD

A w

ith cD

NA

MD

A w

ith R

NA

MD

A w

ithou

t tem

plat

e

H

indI

II la

dder

231Kb

43Kb

23Kb

MD

A w

ith p

Blue

scrip

t II K

S(+)

pla

smid

1 2 3 4 5

(b)


3 Results






(a)


chr 10chr 11chr 12

unassigned

Rice 2Rice 1

No of circRNAs

Chro

mos

ome

(b)

1

10

100

1000

10000

circRNAs types

intr

on-in

tron

intr

on-in

terg

enic

intr

on-e

xon

inte

rgen

ic-in

tron

inte

rgen

ic-e

xon

exon

-intr

on

exon

-inte

rgen

ic

exon

-exo

n

inte

rgen

ic-in

terg

enic

No

of c

ircRN

As

(c)

0

50

100

150

200

250

300

350


No

of c

ircRN

As

lt100

100-

199

200-

299

300-

399

400-

499

500-

599

600-

699

700-

799

800-

899

900-

999

1000

-149

915

00-1

999

2000

-999

910

000-

9999

9gt1

0000

0

(d)






0

50

100

150

200

250

300

350

400


No

of g

ene(

s)

(a)

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10 11 12Chromosome

No

of c

ircRN

As

(b)









(a)

0

1000

2000

3000

4000

5000

6000

7000

Types of circRNAs

No

of c

ircRN

As

inte

rgen

ic-in

terg

enic

inte

rgen

ic-in

troni

c

intr

on-in

terg

enic

intr

on-in

tron

(b)

0200400600800

100012001400

lt100

100-

199

200-

299

300-

399

400-

499

500-

599

600-

699

700-

799

800-

899

900-

999

1000

-149

915

00-1

999

2000

-299

930

00-3

999

4000

-499

950

00-9

999

1000

0-99

999

gt100

000


No

of c

ircRN

As

(c)


4 Discussion




PCR

of cD

NA

with

58

s

Div

erge

nt P

CR o

f Nb

3 cD

NA

with

nb_

circ

7

100b

p pl

us D

NA

ladd

er

Div

erge

nt P

CR o

f Nb

1 cD

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

2 cD

NA

with

nb_

circ

7

Neg

ativ

e con

trol

02kb

01kb

1 2 3 4 5 6

(a)

PCR

of D

NA

with

58

s

Div

erge

nt P

CR o

f Nb

3 D

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

2 D

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

1 D

NA

with

nb_

circ

7

Neg

ativ

e con

trol

100b

p pl

us D

NA

ladd

er

01kb

05kb

1kb

1 2 3 4 5 6

(b)Fe

rmen

tas 1

00 b

p pl

us la

dder

Div

erge

nt P

CR o

f ric

e Gen

omic

DN

A

Div

erge

nt P

CR o

f ric

e cD

NA

Div

erge

nt P

CR w

ith W

ater

cont

rol

PCR

of ri

ce cD

NA

with

58

s

01kb

02kb

03kb

1kb

1 2 3 4 5

(c)





5 Conclusion



0

200

400

600

800

1000

1200


0

10

20

30

40

50

60

Plants

No

of c

ircRN

A(s

)

Z m

ays

S tu

bero

sum

G m

ax

P tr

ifolia

ta

G h

irsut

um

S ly

cope

rsicu

m

G a

rbor

eum

G ra

imon

dii

H v

ulga

re

S b

icolo

r

S it

alica

Bdi

stach

yon

No

of c

ircRN

A(s

)

(a)

050

100150200250300350400450


No

of m

iRN

A b

indi

ng si

tes

(b)


010203040506070

Plants

A th

alia

naS

tube

rosu

mO

sat

iva

S ly

cope

rsicu

mT

aes

tivum

P tr

ifolia

taG

arb

oreu

mG

hirs

utum

G ra

imon

dii

G m

axS

bico

lor

Z m

ays

H v

ulga

reS

ital

icaB

dista

chyo

n

No

of c

ircRN

A(s

)

(a)

0

1000

2000

3000

4000

5000

6000

7000

8000


No

of m

iRN

A b

indi

ng si

tes

(b)






Data Availability






Acknowledgments





References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018









osi circ1(136416264-36418547)



osi circ2(219273316-20009087)



osi circ3(824552647-24573025)



osi circ4(1216650523-17328210)



osi circ5(915720676-15721227)



osi circ6(715534138-15534682)



osi circ7(627117575-27118530)



osi circ8(84494958-4495647)



osi circ9(141518651-41519075)



osi circ10(815854661-15861841)






Nb circ2(Niben101Scf01481214685-215144)



Nb circ3(Niben101Scf01671738307-738555)



Nb circ4(Niben101Scf0182033613-33924)



Nb circ5(Niben101Scf01505317983-318653)



Nb circ6(Niben101Scf3227610732-11201)
















MD

A w

ith cD

NA

MD

A w

ith R

NA

MD

A w

ithou

t tem

plat

e

231Kb

43Kb

23Kb

MD

A w

ith p

Blue

scrip

t II K

S(+)

pla

smid

H

indI

II la

dder

1 2 3 4 5

(a)

MD

A w

ith cD

NA

MD

A w

ith R

NA

MD

A w

ithou

t tem

plat

e

H

indI

II la

dder

231Kb

43Kb

23Kb

MD

A w

ith p

Blue

scrip

t II K

S(+)

pla

smid

1 2 3 4 5

(b)


3 Results






(a)


chr 10chr 11chr 12

unassigned

Rice 2Rice 1

No of circRNAs

Chro

mos

ome

(b)

1

10

100

1000

10000

circRNAs types

intr

on-in

tron

intr

on-in

terg

enic

intr

on-e

xon

inte

rgen

ic-in

tron

inte

rgen

ic-e

xon

exon

-intr

on

exon

-inte

rgen

ic

exon

-exo

n

inte

rgen

ic-in

terg

enic

No

of c

ircRN

As

(c)

0

50

100

150

200

250

300

350


No

of c

ircRN

As

lt100

100-

199

200-

299

300-

399

400-

499

500-

599

600-

699

700-

799

800-

899

900-

999

1000

-149

915

00-1

999

2000

-999

910

000-

9999

9gt1

0000

0

(d)






0

50

100

150

200

250

300

350

400


No

of g

ene(

s)

(a)

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10 11 12Chromosome

No

of c

ircRN

As

(b)









(a)

0

1000

2000

3000

4000

5000

6000

7000

Types of circRNAs

No

of c

ircRN

As

inte

rgen

ic-in

terg

enic

inte

rgen

ic-in

troni

c

intr

on-in

terg

enic

intr

on-in

tron

(b)

0200400600800

100012001400

lt100

100-

199

200-

299

300-

399

400-

499

500-

599

600-

699

700-

799

800-

899

900-

999

1000

-149

915

00-1

999

2000

-299

930

00-3

999

4000

-499

950

00-9

999

1000

0-99

999

gt100

000


No

of c

ircRN

As

(c)


4 Discussion




PCR

of cD

NA

with

58

s

Div

erge

nt P

CR o

f Nb

3 cD

NA

with

nb_

circ

7

100b

p pl

us D

NA

ladd

er

Div

erge

nt P

CR o

f Nb

1 cD

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

2 cD

NA

with

nb_

circ

7

Neg

ativ

e con

trol

02kb

01kb

1 2 3 4 5 6

(a)

PCR

of D

NA

with

58

s

Div

erge

nt P

CR o

f Nb

3 D

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

2 D

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

1 D

NA

with

nb_

circ

7

Neg

ativ

e con

trol

100b

p pl

us D

NA

ladd

er

01kb

05kb

1kb

1 2 3 4 5 6

(b)Fe

rmen

tas 1

00 b

p pl

us la

dder

Div

erge

nt P

CR o

f ric

e Gen

omic

DN

A

Div

erge

nt P

CR o

f ric

e cD

NA

Div

erge

nt P

CR w

ith W

ater

cont

rol

PCR

of ri

ce cD

NA

with

58

s

01kb

02kb

03kb

1kb

1 2 3 4 5

(c)





5 Conclusion



0

200

400

600

800

1000

1200


0

10

20

30

40

50

60

Plants

No

of c

ircRN

A(s

)

Z m

ays

S tu

bero

sum

G m

ax

P tr

ifolia

ta

G h

irsut

um

S ly

cope

rsicu

m

G a

rbor

eum

G ra

imon

dii

H v

ulga

re

S b

icolo

r

S it

alica

Bdi

stach

yon

No

of c

ircRN

A(s

)

(a)

050

100150200250300350400450


No

of m

iRN

A b

indi

ng si

tes

(b)


010203040506070

Plants

A th

alia

naS

tube

rosu

mO

sat

iva

S ly

cope

rsicu

mT

aes

tivum

P tr

ifolia

taG

arb

oreu

mG

hirs

utum

G ra

imon

dii

G m

axS

bico

lor

Z m

ays

H v

ulga

reS

ital

icaB

dista

chyo

n

No

of c

ircRN

A(s

)

(a)

0

1000

2000

3000

4000

5000

6000

7000

8000


No

of m

iRN

A b

indi

ng si

tes

(b)






Data Availability






Acknowledgments





References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



MD

A w

ith cD

NA

MD

A w

ith R

NA

MD

A w

ithou

t tem

plat

e

231Kb

43Kb

23Kb

MD

A w

ith p

Blue

scrip

t II K

S(+)

pla

smid

H

indI

II la

dder

1 2 3 4 5

(a)

MD

A w

ith cD

NA

MD

A w

ith R

NA

MD

A w

ithou

t tem

plat

e

H

indI

II la

dder

231Kb

43Kb

23Kb

MD

A w

ith p

Blue

scrip

t II K

S(+)

pla

smid

1 2 3 4 5

(b)


3 Results






(a)


chr 10chr 11chr 12

unassigned

Rice 2Rice 1

No of circRNAs

Chro

mos

ome

(b)

1

10

100

1000

10000

circRNAs types

intr

on-in

tron

intr

on-in

terg

enic

intr

on-e

xon

inte

rgen

ic-in

tron

inte

rgen

ic-e

xon

exon

-intr

on

exon

-inte

rgen

ic

exon

-exo

n

inte

rgen

ic-in

terg

enic

No

of c

ircRN

As

(c)

0

50

100

150

200

250

300

350


No

of c

ircRN

As

lt100

100-

199

200-

299

300-

399

400-

499

500-

599

600-

699

700-

799

800-

899

900-

999

1000

-149

915

00-1

999

2000

-999

910

000-

9999

9gt1

0000

0

(d)






0

50

100

150

200

250

300

350

400


No

of g

ene(

s)

(a)

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10 11 12Chromosome

No

of c

ircRN

As

(b)









(a)

0

1000

2000

3000

4000

5000

6000

7000

Types of circRNAs

No

of c

ircRN

As

inte

rgen

ic-in

terg

enic

inte

rgen

ic-in

troni

c

intr

on-in

terg

enic

intr

on-in

tron

(b)

0200400600800

100012001400

lt100

100-

199

200-

299

300-

399

400-

499

500-

599

600-

699

700-

799

800-

899

900-

999

1000

-149

915

00-1

999

2000

-299

930

00-3

999

4000

-499

950

00-9

999

1000

0-99

999

gt100

000


No

of c

ircRN

As

(c)


4 Discussion




PCR

of cD

NA

with

58

s

Div

erge

nt P

CR o

f Nb

3 cD

NA

with

nb_

circ

7

100b

p pl

us D

NA

ladd

er

Div

erge

nt P

CR o

f Nb

1 cD

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

2 cD

NA

with

nb_

circ

7

Neg

ativ

e con

trol

02kb

01kb

1 2 3 4 5 6

(a)

PCR

of D

NA

with

58

s

Div

erge

nt P

CR o

f Nb

3 D

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

2 D

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

1 D

NA

with

nb_

circ

7

Neg

ativ

e con

trol

100b

p pl

us D

NA

ladd

er

01kb

05kb

1kb

1 2 3 4 5 6

(b)Fe

rmen

tas 1

00 b

p pl

us la

dder

Div

erge

nt P

CR o

f ric

e Gen

omic

DN

A

Div

erge

nt P

CR o

f ric

e cD

NA

Div

erge

nt P

CR w

ith W

ater

cont

rol

PCR

of ri

ce cD

NA

with

58

s

01kb

02kb

03kb

1kb

1 2 3 4 5

(c)





5 Conclusion



0

200

400

600

800

1000

1200


0

10

20

30

40

50

60

Plants

No

of c

ircRN

A(s

)

Z m

ays

S tu

bero

sum

G m

ax

P tr

ifolia

ta

G h

irsut

um

S ly

cope

rsicu

m

G a

rbor

eum

G ra

imon

dii

H v

ulga

re

S b

icolo

r

S it

alica

Bdi

stach

yon

No

of c

ircRN

A(s

)

(a)

050

100150200250300350400450


No

of m

iRN

A b

indi

ng si

tes

(b)


010203040506070

Plants

A th

alia

naS

tube

rosu

mO

sat

iva

S ly

cope

rsicu

mT

aes

tivum

P tr

ifolia

taG

arb

oreu

mG

hirs

utum

G ra

imon

dii

G m

axS

bico

lor

Z m

ays

H v

ulga

reS

ital

icaB

dista

chyo

n

No

of c

ircRN

A(s

)

(a)

0

1000

2000

3000

4000

5000

6000

7000

8000


No

of m

iRN

A b

indi

ng si

tes

(b)






Data Availability






Acknowledgments





References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





(a)


chr 10chr 11chr 12

unassigned

Rice 2Rice 1

No of circRNAs

Chro

mos

ome

(b)

1

10

100

1000

10000

circRNAs types

intr

on-in

tron

intr

on-in

terg

enic

intr

on-e

xon

inte

rgen

ic-in

tron

inte

rgen

ic-e

xon

exon

-intr

on

exon

-inte

rgen

ic

exon

-exo

n

inte

rgen

ic-in

terg

enic

No

of c

ircRN

As

(c)

0

50

100

150

200

250

300

350


No

of c

ircRN

As

lt100

100-

199

200-

299

300-

399

400-

499

500-

599

600-

699

700-

799

800-

899

900-

999

1000

-149

915

00-1

999

2000

-999

910

000-

9999

9gt1

0000

0

(d)






0

50

100

150

200

250

300

350

400


No

of g

ene(

s)

(a)

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10 11 12Chromosome

No

of c

ircRN

As

(b)









(a)

0

1000

2000

3000

4000

5000

6000

7000

Types of circRNAs

No

of c

ircRN

As

inte

rgen

ic-in

terg

enic

inte

rgen

ic-in

troni

c

intr

on-in

terg

enic

intr

on-in

tron

(b)

0200400600800

100012001400

lt100

100-

199

200-

299

300-

399

400-

499

500-

599

600-

699

700-

799

800-

899

900-

999

1000

-149

915

00-1

999

2000

-299

930

00-3

999

4000

-499

950

00-9

999

1000

0-99

999

gt100

000


No

of c

ircRN

As

(c)


4 Discussion




PCR

of cD

NA

with

58

s

Div

erge

nt P

CR o

f Nb

3 cD

NA

with

nb_

circ

7

100b

p pl

us D

NA

ladd

er

Div

erge

nt P

CR o

f Nb

1 cD

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

2 cD

NA

with

nb_

circ

7

Neg

ativ

e con

trol

02kb

01kb

1 2 3 4 5 6

(a)

PCR

of D

NA

with

58

s

Div

erge

nt P

CR o

f Nb

3 D

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

2 D

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

1 D

NA

with

nb_

circ

7

Neg

ativ

e con

trol

100b

p pl

us D

NA

ladd

er

01kb

05kb

1kb

1 2 3 4 5 6

(b)Fe

rmen

tas 1

00 b

p pl

us la

dder

Div

erge

nt P

CR o

f ric

e Gen

omic

DN

A

Div

erge

nt P

CR o

f ric

e cD

NA

Div

erge

nt P

CR w

ith W

ater

cont

rol

PCR

of ri

ce cD

NA

with

58

s

01kb

02kb

03kb

1kb

1 2 3 4 5

(c)





5 Conclusion



0

200

400

600

800

1000

1200


0

10

20

30

40

50

60

Plants

No

of c

ircRN

A(s

)

Z m

ays

S tu

bero

sum

G m

ax

P tr

ifolia

ta

G h

irsut

um

S ly

cope

rsicu

m

G a

rbor

eum

G ra

imon

dii

H v

ulga

re

S b

icolo

r

S it

alica

Bdi

stach

yon

No

of c

ircRN

A(s

)

(a)

050

100150200250300350400450


No

of m

iRN

A b

indi

ng si

tes

(b)


010203040506070

Plants

A th

alia

naS

tube

rosu

mO

sat

iva

S ly

cope

rsicu

mT

aes

tivum

P tr

ifolia

taG

arb

oreu

mG

hirs

utum

G ra

imon

dii

G m

axS

bico

lor

Z m

ays

H v

ulga

reS

ital

icaB

dista

chyo

n

No

of c

ircRN

A(s

)

(a)

0

1000

2000

3000

4000

5000

6000

7000

8000


No

of m

iRN

A b

indi

ng si

tes

(b)






Data Availability






Acknowledgments





References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0

50

100

150

200

250

300

350

400


No

of g

ene(

s)

(a)

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10 11 12Chromosome

No

of c

ircRN

As

(b)









(a)

0

1000

2000

3000

4000

5000

6000

7000

Types of circRNAs

No

of c

ircRN

As

inte

rgen

ic-in

terg

enic

inte

rgen

ic-in

troni

c

intr

on-in

terg

enic

intr

on-in

tron

(b)

0200400600800

100012001400

lt100

100-

199

200-

299

300-

399

400-

499

500-

599

600-

699

700-

799

800-

899

900-

999

1000

-149

915

00-1

999

2000

-299

930

00-3

999

4000

-499

950

00-9

999

1000

0-99

999

gt100

000


No

of c

ircRN

As

(c)


4 Discussion




PCR

of cD

NA

with

58

s

Div

erge

nt P

CR o

f Nb

3 cD

NA

with

nb_

circ

7

100b

p pl

us D

NA

ladd

er

Div

erge

nt P

CR o

f Nb

1 cD

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

2 cD

NA

with

nb_

circ

7

Neg

ativ

e con

trol

02kb

01kb

1 2 3 4 5 6

(a)

PCR

of D

NA

with

58

s

Div

erge

nt P

CR o

f Nb

3 D

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

2 D

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

1 D

NA

with

nb_

circ

7

Neg

ativ

e con

trol

100b

p pl

us D

NA

ladd

er

01kb

05kb

1kb

1 2 3 4 5 6

(b)Fe

rmen

tas 1

00 b

p pl

us la

dder

Div

erge

nt P

CR o

f ric

e Gen

omic

DN

A

Div

erge

nt P

CR o

f ric

e cD

NA

Div

erge

nt P

CR w

ith W

ater

cont

rol

PCR

of ri

ce cD

NA

with

58

s

01kb

02kb

03kb

1kb

1 2 3 4 5

(c)





5 Conclusion



0

200

400

600

800

1000

1200


0

10

20

30

40

50

60

Plants

No

of c

ircRN

A(s

)

Z m

ays

S tu

bero

sum

G m

ax

P tr

ifolia

ta

G h

irsut

um

S ly

cope

rsicu

m

G a

rbor

eum

G ra

imon

dii

H v

ulga

re

S b

icolo

r

S it

alica

Bdi

stach

yon

No

of c

ircRN

A(s

)

(a)

050

100150200250300350400450


No

of m

iRN

A b

indi

ng si

tes

(b)


010203040506070

Plants

A th

alia

naS

tube

rosu

mO

sat

iva

S ly

cope

rsicu

mT

aes

tivum

P tr

ifolia

taG

arb

oreu

mG

hirs

utum

G ra

imon

dii

G m

axS

bico

lor

Z m

ays

H v

ulga

reS

ital

icaB

dista

chyo

n

No

of c

ircRN

A(s

)

(a)

0

1000

2000

3000

4000

5000

6000

7000

8000


No

of m

iRN

A b

indi

ng si

tes

(b)






Data Availability






Acknowledgments





References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018





(a)

0

1000

2000

3000

4000

5000

6000

7000

Types of circRNAs

No

of c

ircRN

As

inte

rgen

ic-in

terg

enic

inte

rgen

ic-in

troni

c

intr

on-in

terg

enic

intr

on-in

tron

(b)

0200400600800

100012001400

lt100

100-

199

200-

299

300-

399

400-

499

500-

599

600-

699

700-

799

800-

899

900-

999

1000

-149

915

00-1

999

2000

-299

930

00-3

999

4000

-499

950

00-9

999

1000

0-99

999

gt100

000


No

of c

ircRN

As

(c)


4 Discussion




PCR

of cD

NA

with

58

s

Div

erge

nt P

CR o

f Nb

3 cD

NA

with

nb_

circ

7

100b

p pl

us D

NA

ladd

er

Div

erge

nt P

CR o

f Nb

1 cD

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

2 cD

NA

with

nb_

circ

7

Neg

ativ

e con

trol

02kb

01kb

1 2 3 4 5 6

(a)

PCR

of D

NA

with

58

s

Div

erge

nt P

CR o

f Nb

3 D

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

2 D

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

1 D

NA

with

nb_

circ

7

Neg

ativ

e con

trol

100b

p pl

us D

NA

ladd

er

01kb

05kb

1kb

1 2 3 4 5 6

(b)Fe

rmen

tas 1

00 b

p pl

us la

dder

Div

erge

nt P

CR o

f ric

e Gen

omic

DN

A

Div

erge

nt P

CR o

f ric

e cD

NA

Div

erge

nt P

CR w

ith W

ater

cont

rol

PCR

of ri

ce cD

NA

with

58

s

01kb

02kb

03kb

1kb

1 2 3 4 5

(c)





5 Conclusion



0

200

400

600

800

1000

1200


0

10

20

30

40

50

60

Plants

No

of c

ircRN

A(s

)

Z m

ays

S tu

bero

sum

G m

ax

P tr

ifolia

ta

G h

irsut

um

S ly

cope

rsicu

m

G a

rbor

eum

G ra

imon

dii

H v

ulga

re

S b

icolo

r

S it

alica

Bdi

stach

yon

No

of c

ircRN

A(s

)

(a)

050

100150200250300350400450


No

of m

iRN

A b

indi

ng si

tes

(b)


010203040506070

Plants

A th

alia

naS

tube

rosu

mO

sat

iva

S ly

cope

rsicu

mT

aes

tivum

P tr

ifolia

taG

arb

oreu

mG

hirs

utum

G ra

imon

dii

G m

axS

bico

lor

Z m

ays

H v

ulga

reS

ital

icaB

dista

chyo

n

No

of c

ircRN

A(s

)

(a)

0

1000

2000

3000

4000

5000

6000

7000

8000


No

of m

iRN

A b

indi

ng si

tes

(b)






Data Availability






Acknowledgments





References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



PCR

of cD

NA

with

58

s

Div

erge

nt P

CR o

f Nb

3 cD

NA

with

nb_

circ

7

100b

p pl

us D

NA

ladd

er

Div

erge

nt P

CR o

f Nb

1 cD

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

2 cD

NA

with

nb_

circ

7

Neg

ativ

e con

trol

02kb

01kb

1 2 3 4 5 6

(a)

PCR

of D

NA

with

58

s

Div

erge

nt P

CR o

f Nb

3 D

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

2 D

NA

with

nb_

circ

7

Div

erge

nt P

CR o

f Nb

1 D

NA

with

nb_

circ

7

Neg

ativ

e con

trol

100b

p pl

us D

NA

ladd

er

01kb

05kb

1kb

1 2 3 4 5 6

(b)Fe

rmen

tas 1

00 b

p pl

us la

dder

Div

erge

nt P

CR o

f ric

e Gen

omic

DN

A

Div

erge

nt P

CR o

f ric

e cD

NA

Div

erge

nt P

CR w

ith W

ater

cont

rol

PCR

of ri

ce cD

NA

with

58

s

01kb

02kb

03kb

1kb

1 2 3 4 5

(c)





5 Conclusion



0

200

400

600

800

1000

1200


0

10

20

30

40

50

60

Plants

No

of c

ircRN

A(s

)

Z m

ays

S tu

bero

sum

G m

ax

P tr

ifolia

ta

G h

irsut

um

S ly

cope

rsicu

m

G a

rbor

eum

G ra

imon

dii

H v

ulga

re

S b

icolo

r

S it

alica

Bdi

stach

yon

No

of c

ircRN

A(s

)

(a)

050

100150200250300350400450


No

of m

iRN

A b

indi

ng si

tes

(b)


010203040506070

Plants

A th

alia

naS

tube

rosu

mO

sat

iva

S ly

cope

rsicu

mT

aes

tivum

P tr

ifolia

taG

arb

oreu

mG

hirs

utum

G ra

imon

dii

G m

axS

bico

lor

Z m

ays

H v

ulga

reS

ital

icaB

dista

chyo

n

No

of c

ircRN

A(s

)

(a)

0

1000

2000

3000

4000

5000

6000

7000

8000


No

of m

iRN

A b

indi

ng si

tes

(b)






Data Availability






Acknowledgments





References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



0

200

400

600

800

1000

1200


0

10

20

30

40

50

60

Plants

No

of c

ircRN

A(s

)

Z m

ays

S tu

bero

sum

G m

ax

P tr

ifolia

ta

G h

irsut

um

S ly

cope

rsicu

m

G a

rbor

eum

G ra

imon

dii

H v

ulga

re

S b

icolo

r

S it

alica

Bdi

stach

yon

No

of c

ircRN

A(s

)

(a)

050

100150200250300350400450


No

of m

iRN

A b

indi

ng si

tes

(b)


010203040506070

Plants

A th

alia

naS

tube

rosu

mO

sat

iva

S ly

cope

rsicu

mT

aes

tivum

P tr

ifolia

taG

arb

oreu

mG

hirs

utum

G ra

imon

dii

G m

axS

bico

lor

Z m

ays

H v

ulga

reS

ital

icaB

dista

chyo

n

No

of c

ircRN

A(s

)

(a)

0

1000

2000

3000

4000

5000

6000

7000

8000


No

of m

iRN

A b

indi

ng si

tes

(b)






Data Availability






Acknowledgments





References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018






Data Availability






Acknowledgments





References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



References






















































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Documents

Circular RNA Profiling by Illumina Sequencing via Template ...downloads.hindawi.com/journals/bmri/2019/2756516.pdf · ResearchArticle Circular RNA Profiling by Illumina Sequencing