Meaningful application of the new 454 large scale ... · like QDD also enabled to sort and discard...
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Meaningful application of the new 454 large scale pyrosequencing technology (Roche GS-FLX 454) to the identification of microsatellites for small-scale research projects CLASSICAL METHOD: enriched library & Sanger sequencing For construction of the enriched DNA library, we followed Kijas et al., (1994). 1,5 μg of genomic DNA was digested with Rsa1 restriction enzyme. Digested fragments were ligated to Rsa adaptators and PCR amplified. PCR products were enriched with Streptavidin-coated magnetic beads (Promega) and 3’- biotinylated (TC) 10 or (TG) 10 . Enrichments were PCR amplified and cloned. A total of 509 recombinant clones for the two libraries were picked randomly and tested by PCR for detection of microsatellites. Inserted fragments of 500 to 1,000bp in 96 clones were sequenced using the Sanger ‘s technology. NEW METHOD: 454-GS- FLX Titanium pyrosequencing of enriched library 1 μg of genomic DNA was digested by Rsa1 restriction enzyme. Digested fragments were ligated to Rsa adaptators and PCR amplified. PCR products were enriched with Streptavidin-coated magnetic beads (Promega) and 3’-biotinylated (AG) 10, (AC) 10, (AAC) 8, (AGG) 8, (ACG) 8, (AAG) 8, (ACAT) 6, (ATCT) 6. Subsequent enrichments were PCR amplified. The next steps were specific to the 454 GS-FLX technology and carried out as described by the manufacturer (Roche Applied Science). This implies fragment end polishing, adaptator ligation (A and B), addition of Multiplex identifiers (MIDs), library immobilization, fill-in reaction and isolation of a library of a single strand DNA + adaptors. During an emulsion PCR, each fragment is then mixed with beads carrying on their surfaces oligos complementary to the adaptators. With a PCR amplification occurring within each droplet, each bead is bound with millions of copies of a unique DNA (equivalent to clonal amplification). SstDNA library after addition of DNA polymerase and a cocktail of sulfurylase and luciferase was sequenced by the Genome sequencer FLX. Using extension reaction by DNA polymerase just same as normal PCR, bases will be loaded in a fixed order of T -> A -> G -> C. Sulfurylase generates ATP using pyrophoric acid, which is made in polymerase extension reaction, as a substrate. Luciferase reacts to generate a light signal using this ATP and Luciferin as substrates. This chemiluminescent signal is recorded by the CCD camera. DATA ANALYSIS Sequences obtained in both protocols were analyzed using the bioinformatic QDD pipeline (Meglécz et al., 2010). Sequences shorter than 80bp and containing microsatellite motifs shorter than four repeats for any motif of 2 to 6bp were discarded. Sequences with significant Blasts hits but with flanking region identity levels below 90% were discarded. Primers were designed automatically by Primer3 within QDD. RESULTS We obtained 21,117 sequences for the new technology vs 509 recombinant clones (potentially sequences) for the classical one. In total, 4,183 loci containing microsatellites matching the quality criteria implemented in QDD were detected after the 454 technology (Table 1). For the 454 technology, mean sequence length was of 278 bp (244 bp for classical ones) and maximum was of 548 bp (731 bp for classical ones), 14,3 % of the validated loci corresponded to consensus sequences (28,5 % for the classical ones). Primer design was successful in 6,3 % loci (266 out of 4,183), (7,6 % for classical ones). In both technologies, the primers design targeted more microsatellites with perfect motif than with compound ones. M. Jeanneau 1 , M-C Bon 1 , and J-F Martin 2 . 1. European Biological Control Laboratory USDA-ARS and 2. Centre de Biologie et de Gestion des Populations (CBGP) - SupAgro Campus International de Baillarguet, CS90013 Montferrier sur Lez, 34988 St. Gély du Fesc, France. [email protected] , [email protected] , [email protected]. Cloning : DNA insertion into plasmid vector Transformation : vector insertion into competent bacteria Sreening: Selection of transformed bacteria with SSR Detection of recombinant clones with SSR marker by PCR Sanger’s Sequencing CLASSICAL METHOD This new method was first developed within the framework of a multi-partner pilote project between the French Institut National de la Recherche Agronomique (INRA), AIP Bioressources EcoMicro), the R&D department of Genoscreen, Montpellier SupAgro and the University of Provence. To provide a broadly applicable test during the project, genomic DNA was sourced from Plant, Fungus, Arthropod, Nematod and Stramenopile kingdoms and numerous unrelated taxa within Arthropods including our target. Development of microsatellites has been streamlined by optimizing the numerous steps in microsatellite identification and subsequent sequencing throughput, to make the 454 process even more successful. The flowchart of both the classical microsatellite-library method and this improved 454 technology that has recently been published (Malausa et al., 2010) are presented here. Figure 1 : Microsatellite (CA repetition). INTRODUCTION Microsatellites (simple sequence repeats) are DNA sequences that consist of tandem repeats of 1-6 nucleotides (Figure 1). Because of high levels of polymorphism, ease to use and co-dominance, they are generally seen as the most pertinent markers to study at a fine scale level the genetic structure and demographic history of invasive weed populations. However, their development using the classical microsatellite-library by enrichment remain typically time consuming and labor intensive, impeding their generalization at the level of small-scale research projects. We considered in the present study the extent to which the new generation sequencing such 454 Life Sciences/Roche GS-FLX pyrosequencing based-technology, could lead to a rapid, more efficient and less costly way to identify microsatellites for a small-scale research project than the classical microsatellite-library by enrichment. DNA SOURCE: The DNA was sourced from a non model taxa i.e. a root gall weevil, Ceutorhynchus assimilis (Coleoptera: Curculionidae) that is one natural enemy of the invasive weed, Lepidium draba sp draba (Brassicaceae) (Figure 2). DNA was extracted using the Qiagen DNeasy Tissue DNA extraction kit. Figure 2 : Ceutorhynchus assimilis © JEANNEAU M. Microsatellite polymerase Single strand DNA + adaptators Emulsion PCR : amplification of unique DNA GFLX plating : million copies of DNA per well sequencing Signal processing to determine base sequence 454 flowgram : Y-axis is proportionnal of nucleotides number X-axis corresponds to the succession of nucleotides NEW METHOD polymerase Signal image Anneal primer DNA Capture Bead containing millions of copies of a single clonal fragment APS PPi ATP Light + oxyluciferin Luciferin Sulfurylase Luciferase Expansion of fragments by PCR with adaptator primers Hybridization with biotinylated repeat probes Capture of SSR DNA fragments with streptavidin-coated beads DNA digestion + adaptator ligation Enriched fragments FLOWCHART : COMMON STEPS B B Acknowledgements The autors thank Thibaut Malausa (INRA, UMR 1301 IBSV INRA/UNSA/CNRS, Sophia Antipolis, France), André Gilles, Caroline Costedoat, Vincent Dubut, Nicolas Pech & Emese Meglècz (Aix-Marseille University, CNRS, IRD, UMR 6116-IMEP, France), Stephanie Ferreira, Hélène Blanquart, Stephanie Duthoy (Genoscreen, Genomic Platform and R&D, Lille, France) for their valuable collaboration to this project. References Kijas J.M., Fowler J.C., Garbett C.A., Thomas M.R., 1994. Enrichment of microsatellite from the citrus genome using biotinylated oligonucleotide sequences bound to strepatvidin coated magnetic particles. Biotechniques. 16(4) : 656-60, 662. Malausa T., and al., 2010. High-throughput microsatellite isolation through 454 GS-FLX titanium pyrosequencing of enriched DNA libraries. Molecular Ecology Resources submitted. Meglécz E., Costedoat C., Dubut V., Gilles A., Malausa T., Pech N., Martin J.F., 2010. QDD : a user-friendly program to select microsatellite markers and design primers from large sequencing projects. Bioinformatics . 26,3 : 403-404. CONCLUSIONS Even with a stringent selection of loci imposed by the parameters chosen throughout the analysis, the total number of microsatellites isolated by using the new 454 technology is much higher than the number of SSR obtained following a classical method. The use of multiplex enrichment with 8 probes made the SSR enrichment easier and generalist. Moreover, the analysis of the large dataset obtained, using bioinformatic programs like QDD also enabled to sort and discard loci found at multiples sites in the genome. The data presented here confirmed that the microsatellite isolation through the 454-GS-FLX Titanium pyrosequencing of enriched library can be successfully applied to a non model taxa at much lower cost and more rapidly than by using a classical method. Table 1 : Description of the microsatellite libraries obtained by the two methods. CRITERIA CLASSICAL METHOD NEW METHOD DNA quantity Number of raw sequences Number of sequences with length > 80 bp & SSR motif > 4 repeats Sequences length (Mean-Min-Max) % Loci identified from several sequences Number of sequences with successful Design of primers Ratio of perfect SSR / compound SSR Duration Cost > 1,5 μg 509 91 244-127-731 28,5 7 5-2 1 month > 2500 euros > 2 μg 21117 14,3 266 185-81 2 months (Genoscreen) 1750 euros (Genoscreen) 4183 278-80-548
Transcript of Meaningful application of the new 454 large scale ... · like QDD also enabled to sort and discard...
Meaningful application of the new 454 large scale pyrosequencing technology (Roche GS-FLX 454) to the identification of microsatellites
for small-scale research projects
CLASSICAL METHOD:
enriched library & Sanger sequencingFor construction of theenriched DNA library, wefollowed Kijas et al., (1994).1,5 µg of genomic DNA wasdigested with Rsa1 restrictionenzyme. Digested fragmentswere ligated to Rsa adaptatorsand PCR amplified. PCRproducts were enriched withStreptavidin-coated magneticbeads (Promega) and 3’-biotinylated (TC)10 or (TG)10.Enrichments were PCRamplified and cloned. A totalof 509 recombinant clones forthe two libraries were pickedrandomly and tested by PCRfor detection ofmicrosatellites. Insertedfragments of 500 to 1,000bp in96 clones were sequencedusing the Sanger ‘s technology.
NEW METHOD: 454-GS-FLX Titanium pyrosequencing of enriched library 1 µg of genomic DNA was digested byRsa1 restriction enzyme. Digestedfragments were ligated to Rsaadaptators and PCR amplified. PCRproducts were enriched withStreptavidin-coated magnetic beads(Promega) and 3’-biotinylated (AG)10,
(AC)10, (AAC)8, (AGG)8, (ACG)8,
(AAG)8, (ACAT)6, (ATCT)6. Subsequentenrichments were PCR amplified.The next steps were specific to the454 GS-FLX technology and carried outas described by the manufacturer(Roche Applied Science). This impliesfragment end polishing, adaptatorligation (A and B), addition ofMultiplex identifiers (MIDs), libraryimmobilization, fill-in reaction andisolation of a library of a single strandDNA + adaptors. During an emulsionPCR, each fragment is then mixed withbeads carrying on their surfaces oligoscomplementary to the adaptators.With a PCR amplification occurringwithin each droplet, each bead isbound with millions of copies of aunique DNA (equivalent to clonalamplification). SstDNA library afteraddition of DNA polymerase and acocktail of sulfurylase and luciferasewas sequenced by the Genomesequencer FLX.Using extension reaction by DNApolymerase just same as normal PCR,bases will be loaded in a fixed order ofT -> A -> G -> C. Sulfurylase generatesATP using pyrophoric acid, which ismade in polymerase extensionreaction, as a substrate. Luciferasereacts to generate a light signal usingthis ATP and Luciferin as substrates.This chemiluminescent signal isrecorded by the CCD camera.
DATA ANALYSISSequences obtained in both protocols were analyzed using the bioinformatic QDD pipeline (Meglécz et al., 2010). Sequences shorterthan 80bp and containing microsatellite motifs shorter than four repeats for any motif of 2 to 6bp were discarded. Sequences withsignificant Blasts hits but with flanking region identity levels below 90% were discarded. Primers were designed automatically byPrimer3 within QDD.
RESULTSWe obtained 21,117 sequences for the new technology vs 509recombinant clones (potentially sequences) for the classicalone. In total, 4,183 loci containing microsatellites matching thequality criteria implemented in QDD were detected after the454 technology (Table 1). For the 454 technology, meansequence length was of 278 bp (244 bp for classical ones) andmaximum was of 548 bp (731 bp for classical ones), 14,3 % ofthe validated loci corresponded to consensus sequences (28,5% for the classical ones). Primer design was successful in 6,3 %loci (266 out of 4,183), (7,6 % for classical ones). In bothtechnologies, the primers design targeted more microsatelliteswith perfect motif than with compound ones.
M. Jeanneau1, M-C Bon1, and J-F Martin2.
1. European Biological Control Laboratory USDA-ARS and 2. Centre de Biologie et de Gestion des Populations (CBGP) - SupAgro Campus International de Baillarguet, CS90013 Montferriersur Lez, 34988 St. Gély du Fesc, France.
[email protected], [email protected], [email protected].
Cloning : DNA insertioninto plasmid vector
Transformation : vector insertion into competent
bacteria
Sreening: Selection oftransformed bacteria
with SSR
Detection of recombinant clones with SSR marker by
PCR
Sanger’s Sequencing
CLASSICAL METHOD
This new method was first developed within the framework of a multi-partner pilote project between the French Institut National de la Recherche Agronomique (INRA), AIP BioressourcesEcoMicro), the R&D department of Genoscreen, Montpellier SupAgro and the University of Provence. To provide a broadly applicable test during the project, genomic DNA was sourced fromPlant, Fungus, Arthropod, Nematod and Stramenopile kingdoms and numerous unrelated taxa within Arthropods including our target. Development of microsatellites has been streamlinedby optimizing the numerous steps in microsatellite identification and subsequent sequencing throughput, to make the 454 process even more successful. The flowchart of both the classicalmicrosatellite-library method and this improved 454 technology that has recently been published (Malausa et al., 2010) are presented here.
Figure 1 : Microsatellite (CA repetition).
INTRODUCTIONMicrosatellites (simple sequence repeats) are DNA sequences that consist of tandem repeats of 1-6 nucleotides (Figure 1). Because of high levels of polymorphism, ease to use and co-dominance, they are generally seenas the most pertinent markers to study at a fine scale level the genetic structure and demographic history of invasive weed populations. However, their development using the classical microsatellite-library byenrichment remain typically time consuming and labor intensive, impeding their generalization at the level of small-scale research projects. We considered in the present study the extent to which the new generationsequencing such 454 Life Sciences/Roche GS-FLX pyrosequencing based-technology, could lead to a rapid, more efficient and less costly way to identify microsatellites for a small-scale research project than the classicalmicrosatellite-library by enrichment.
DNA SOURCE:The DNA was sourced from anon model taxa i.e. a root gallweevil, Ceutorhynchusassimilis (Coleoptera:Curculionidae) that is onenatural enemy of the invasiveweed, Lepidium draba spdraba (Brassicaceae) (Figure2). DNA was extracted usingthe Qiagen DNeasy TissueDNA extraction kit.
Figure 2 : Ceutorhynchus assimilis© JEANNEAU M.
Microsatellite
polymerase
Single strand DNA+ adaptators
Emulsion PCR : amplification of unique DNA
GFLX plating : million copiesof DNA per well sequencing
Signal processing to determine base sequence
454 flowgram :
Y-axis is proportionnal of nucleotides number
X-axis corresponds to the succession of nucleotides
NEW METHOD
polymerase
Signal image
Anneal primer
DNA Capture Beadcontaining millions ofcopies of a singleclonal fragment
APS
PPi
ATP
Light + oxyluciferin
LuciferinSulfurylase
Luciferase
Expansion of fragments by PCR
with adaptator primersHybridization with
biotinylated repeat probes
Capture of SSR DNA fragments with
streptavidin-coated beadsDNA digestion + adaptator
ligation
Enriched fragments
FLOWCHART : COMMON STEPS
BB
AcknowledgementsThe autors thank Thibaut Malausa (INRA, UMR 1301 IBSV INRA/UNSA/CNRS, Sophia Antipolis, France), André Gilles, Caroline Costedoat, Vincent Dubut, Nicolas Pech & Emese Meglècz (Aix-Marseille University, CNRS, IRD, UMR 6116-IMEP, France), Stephanie Ferreira, Hélène Blanquart, Stephanie Duthoy (Genoscreen, Genomic Platform and R&D, Lille, France) for their valuable collaboration to this project.ReferencesKijas J.M., Fowler J.C., Garbett C.A., Thomas M.R., 1994. Enrichment of microsatellite from the citrus genome using biotinylated oligonucleotide sequences bound to strepatvidin coated magnetic particles. Biotechniques. 16(4) : 656-60, 662.Malausa T., and al., 2010. High-throughput microsatellite isolation through 454 GS-FLX titanium pyrosequencing of enriched DNA libraries. Molecular Ecology Resources submitted.Meglécz E., Costedoat C., Dubut V., Gilles A., Malausa T., Pech N., Martin J.F., 2010. QDD : a user-friendly program to select microsatellite markers and design primers from large sequencing projects. Bioinformatics . 26,3 : 403-404.
CONCLUSIONSEven with a stringent selection of loci imposed by the parameters chosen throughout the analysis, the total number of microsatellites isolated by using the new 454 technology is much higher than the number of SSRobtained following a classical method. The use of multiplex enrichment with 8 probes made the SSR enrichment easier and generalist. Moreover, the analysis of the large dataset obtained, using bioinformatic programslike QDD also enabled to sort and discard loci found at multiples sites in the genome.The data presented here confirmed that the microsatellite isolation through the 454-GS-FLX Titanium pyrosequencing of enriched library can be successfully applied to a non model taxa at much lower cost and morerapidly than by using a classical method.
Table 1 : Description of the microsatellite libraries obtained by the two methods.
CRITERIA CLASSICAL METHOD NEW METHODDNA quantity
Number of raw sequences
Number of sequences with length > 80 bp& SSR motif > 4 repeats
Sequences length (Mean-Min-Max)
% Loci identified from several sequences
Number of sequences with successfulDesign of primers
Ratio of perfect SSR / compound SSR
Duration
Cost
> 1,5 µg
509
91
244-127-731
28,5
7
5-2
1 month
> 2500 euros
> 2 µg
21117
14,3
266
185-81
2 months (Genoscreen)
1750 euros (Genoscreen)
4183
278-80-548
