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PROTOCOL Europe - Diagenode s.a. / orders@diagenode.com / info@diagenode.com // North America - Diagenode Inc. / orders.na@diagenode.com / info.na@diagenode.com www.diagenode.com Standard protocols DNA shearing for Bioruptor ® Pico Standard operating conditions Sample volume: 100 µl Tubes: 0.65 ml Bioruptor ® Microtubes (Cat. No. WA-005-0500) Tube holder: 0.65 ml tube holder for Bioruptor ® Pico (Cat. No. B01200042) for 12 x 0.65 ml tubes Sonication buffer: TE (10 mM Tris, 1mM EDTA, pH 7.5 - 8.0) DNA concentration: 1-20 ng/µl (10 ng/µl recommended) Samples are vortexed (5-10 sec) and centrifuged (10 sec) before shearing. For optimal results samples should be stored on ice during 5-10 minutes prior to sonication. Temperature: 4°C – Water Cooler (Cat. No. BioAcc-Cool) & Single Cycle Valve (Cat. No. B02020004) Sonication cycle & total sonication time: varies depending on desired DNA size (see table) Note: Recommended protocols are subject to change without notice. Additional protocols are available on demand. 125 bp 135 bp 183 bp 200 bp 234 bp 319 bp 476 bp 696 bp 848 bp 1036 bp 1230 bp Programmable DNA size distributions, excellent reproducibility, and high dsDNA yields with Bioruptor ® Pico Figure shows different DNA size distributions of sheared genomic DNA produced by varying the duration of sonication. The different curves depict a specific Bioruptor ® Pico Next- Generation run, optimized to produce specific mean sizes and size ranges for Next-Generation sequencing. All samples were analyzed on Bioanalyzer 2100 using DNA High Sensitivity chip.

Standard protocols DNA shearing for Bioruptor Pico · DNA concentration: 1-20 ng/µl (10 ng/µl recommended) Samples are vortexed (5-10 sec) and centrifuged (10 sec) before shearing

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Page 1: Standard protocols DNA shearing for Bioruptor Pico · DNA concentration: 1-20 ng/µl (10 ng/µl recommended) Samples are vortexed (5-10 sec) and centrifuged (10 sec) before shearing

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Europe - Diagenode s.a. / [email protected] / [email protected] // North America - Diagenode Inc. / [email protected] / [email protected]

www.diagenode.com

Standard protocols DNA shearing for Bioruptor® Pico

Standard operating conditions

Sample volume: 100µl

Tubes: 0.65mlBioruptor®Microtubes(Cat.No.WA-005-0500)

Tube holder: 0.65mltubeholderforBioruptor®Pico(Cat.No.B01200042)for12x0.65mltubes

Sonication buffer: TE(10mMTris,1mMEDTA,pH7.5-8.0)

DNA concentration:1-20ng/µl(10ng/µlrecommended)

Samplesarevortexed(5-10sec)andcentrifuged(10sec)beforeshearing.For optimal results samples should be stored on ice during 5-10 minutes prior to sonication.

Temperature: 4°C – Water Cooler (Cat. No. BioAcc-Cool) & Single Cycle Valve (Cat. No.B02020004)

Sonication cycle & total sonication time:variesdependingondesiredDNAsize(seetable)

Note: Recommended protocols are subject to change without notice. Additional protocols areavailableondemand.

125 bp

135 bp

183 bp

200 bp

234 bp

319 bp

476 bp

696 bp

848 bp

1036 bp

1230 bp

Programmable DNA size distributions, excellent reproducibility, and high dsDNA yields with Bioruptor® Pico

Figure shows different DNA sizedistributions of sheared genomicDNAproducedbyvaryingthedurationof sonication. The different curvesdepictaspecificBioruptor®PicoNext-Generationrun,optimizedtoproducespecific mean sizes and size rangesforNext-Generationsequencing.All samples were analyzed onBioanalyzer 2100 using DNA HighSensitivitychip.

Page 2: Standard protocols DNA shearing for Bioruptor Pico · DNA concentration: 1-20 ng/µl (10 ng/µl recommended) Samples are vortexed (5-10 sec) and centrifuged (10 sec) before shearing

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Europe - Diagenode s.a. / [email protected] / [email protected] // North America - Diagenode Inc. / [email protected] / [email protected]

www.diagenode.com

Target sizeCycle condition

(On/Off cycle time)Cycle number

150bp 30’’/30’’ 30

200bp 30’’/30’’ 13

300bp* 30’’/90’’ 6

400bp* 15’’/90’’ 7-8

1000bp* 5’’/90’’ 7-8

*Forlongerfragments(300upto1000bp),ashortcentrifugationstepafterhalfofthecyclenumberscansignificantlyimprovetheresults.Protocolsforothersizeranges(incl.longerfragmentsupto1300bp)areavailableonrequest.

Broad DNA size distribution, excellent reproducibility, and high dsDNA yields with Bioruptor® Pico

Figure A shows different DNA sizedistributions of sheared genomic DNAproduced by varying the duration ofsonicationfrom2separateexperiments(run-to-runvariation).Thedifferentlanesdepict4lanesfrom2differentBioruptor®

Picoruns,optimizedtoproducespecificmean sizes corresponding to 150, 200,300 and 400 bp (based on the standardIonTorrentstandardsamplepreparationsizerequirements).

Figure B shows different DNA sizedistributions of sheared genomic DNAproduced by varying the duration ofsonication. The different lanes depict aspecific Bioruptor® Pico run, optimizedto produce specific mean sizes andsize ranges for Next-Generation DNAsequencing.

Panel A: peak electropherogram view.Lanes 1,2: 150 bp; Lanes 3,4: 200 bp;Lanes5,6:300bp;Lanes7,8:400bp.

PanelB:gelvirtualview

AllsampleswereanalyzedonBioanalyzer2100usingDNAHighSensitivitychip.

A.

B.

The protocol settings listed above are recommended guidelines and actual results may vary depending on the type and amount of starting material, purity level, concentration and/or sample viscosity. It is highly recommended that a time course response experiment be carried out (e.g. varying the time of “on” and “off” durations as well as the number of cycles) to determine the appropriate treatment for your specific sample. Starting material with a smaller sample volume and a greater concentration than the recommended range may require a different time course to ensure homogenous shearing results.

Page 3: Standard protocols DNA shearing for Bioruptor Pico · DNA concentration: 1-20 ng/µl (10 ng/µl recommended) Samples are vortexed (5-10 sec) and centrifuged (10 sec) before shearing

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Europe - Diagenode s.a. / [email protected] / [email protected] // North America - Diagenode Inc. / [email protected] / [email protected]

www.diagenode.com

A.TargetSize:150bp

C.TargetSize:300bp

B.TargetSize:200bp

D.TargetSize:400bp

Excellent reproducibility and programmable size distribution with Bioruptor® Pico

PanelsAtoDshowsizedistributionsofsheared genomic DNA from separateexperimentsaimingatproducinghigh-qualityDNAfragmentscompatiblewithIonTorrentstandardsamplepreparationprotocols(150,200,300and400bp).

Panel A: 30 cycles, 30 sec ON/30 secOFFcycles.Expectedfragmentsize:150bp, observed average fragment size is150.6bp(CV%:2.07%).

Panel B: 13 cycles, 30 sec ON/30 secOFFcycles.Expectedfragmentsize:200bp, observed average fragment size is202.2bp(CV%:4.7%).

PanelC:6cycles,15secON/90secOFFcycles.Expectedfragmentsize:300bp,observedaveragefragmentsizeis291.3bp(CV%:4.31%).

PanelD:7cycles,15secON/90secOFFcycles.Expectedfragmentsize:400bp,observedaveragefragmentsizeis406.9bp(CV%:7.96%).

All samples were analyzed onBioanalyzer 2100 DNA High Sensitivitychips.

Page 4: Standard protocols DNA shearing for Bioruptor Pico · DNA concentration: 1-20 ng/µl (10 ng/µl recommended) Samples are vortexed (5-10 sec) and centrifuged (10 sec) before shearing

PR

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OL

Europe - Diagenode s.a. / [email protected] / [email protected] // North America - Diagenode Inc. / [email protected] / [email protected]

www.diagenode.com

Important comments about DNA shearing

The Diagenode ACT (Adaptative Cavitation Transfer technology) process is highly reproducible.However,attentionmustbepaidtothefollowingtreatmentattributestoensurebestresults:

• Tubes:Atpresent,therecommendedtubevesselsarethe0.65mlBioruptor®Microtubes(CatNo.WA-005-0500).Payattentionnottodamagethecapwhenclosingthetubessincethiscouldaltersonicationresults.

• Sample volume:Therecommendedvolumeofthe0.65mlBioruptor®Microtubes(CatNo.WA-005-0500)is100µl.Whenusinglowervolumes(e.g.≤50µl),lessreproducibleresultsmaybeobserved due to an alteration of the ultrasonic waves distribution in the sample fluid; thus,reducing the efficiency of sonication which may result in broader size distribution or largerpeaks.

• Sample concentration:DiagenoderecommendsusingDNAconcentrationrangingbetween1and20ng/µl(10ng/µlrecommended).Usinglargerconcentration(e.g.50-100ng/µl)mayresultinbroaderpeaksorvariablepeakdistribution.

• Sample preparation:Sampleviscositymayhaveamajorimpactonsonicationresults.CarefulresuspensionofDNAsampleisstronglyrecommendedbeforesonicationprocessing.Multiplepipettingandgentlevortexingfollowedbyashortcentrifugationtorecoversamplevolumeatthebottomofthetubeisthereforestronglyrecommended.StoringDNAsamplesoniceduring5-10minutesbeforesonicationhasalsobeenshowntoimprovereproducibility.

• DNA quality: DNA quality and quantity must be considered carefully since bad quality andquantityDNAmayhaveseveralimpactsonsonicationandNext-Gensequencingdownstreamapplications.First,DNAcontamination(e.g.fromsuperfluousnucleicacidssuchasRNA,smallnucleicacidfragments,excessproteins,orothercontaminatingmaterials)mayinterferewithDNA measurement method leading to incorrect DNA quantitation thus. Also contaminatingRNA in genomic DNA preparation might generate a biased fragment distribution profile onmicrofluidics-basedplatform(e.g.AgilentBioanalyzer)oraltersonicationeffiency.

Therefore it is highly recommended to use only high quality DNA when sonicating DNA forNext-Gensequencing librarypreparation.TheDNAsample tobeprocessedshouldbehighlypure,havinganOD260/280ratioofbetween1.8and2.0,andshouldbeas intactaspossible.DNAextractedusingstandardtechniques(e.g.ProteinaseKdigested,doublephenol/chloformextraction, ethanol precipitated, treatment with RNase-DNase free enzymatic digestion toremovecontaminantRNA)orcommercialspin-columnbasedkitsarerecommended.

• Water temperature:PropagationofultrasoundinaliquidunavoidablyproducesheatthatcanultimatelyalterDNAsample(e.g.bythermaldenaturation).Toensurethebestpreservationofthesample,itisrecommendedtostartthesonicationprocesswithcoldwaterinthewaterbath.Duringsonication,especiallywhendoinglongsonicationruns,thetemperaturemustalsobecontrolled.Thisisobtainedbytheautomatictemperaturecontrol.

Note: Thepermanent installationof theBioruptor®Pico inacold room ispossible,althoughnotsufficienttoavoidthetemperatureincreaseduetosonication.

• Automatic temperature control:Arecirculatingwatercoolerisusedtoguaranteetheautomatictemperaturecontrolofthewaterbathduringthewholesonicationprocess.Thiswatercooler(Cat.No.BioAcc-cool)producesaregularwaterflowwithaconstantwater level in thetank.

Page 5: Standard protocols DNA shearing for Bioruptor Pico · DNA concentration: 1-20 ng/µl (10 ng/µl recommended) Samples are vortexed (5-10 sec) and centrifuged (10 sec) before shearing

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Europe - Diagenode s.a. / [email protected] / [email protected] // North America - Diagenode Inc. / [email protected] / [email protected]

www.diagenode.com

Anadditionalregulatingvalve(SingleCycleValve,Cat.No.B02020004)ensuresthatwaterwillonlybereplacedduringtheoffcycletoavoidanyinterferencebetweenthewaterflowandthesonicationprocess.

• Sonication time:Minoradjustmentsincyclenumbermaybemadetooptimizeresultsforvarioussampletypesandconcentrations.Cyclenumberlistedaboveisarecommendedguideline.Actualresultsmayvarydependingontheamountandtypeofstartingmaterial,concentration,viscosityand/orplastictubes.Diagenoderecommendssettingupatimedoseresponseexperimentfordeterminingappropriatecyclenumber.Largerlengthstartingmaterial(e.g.totalgenomicDNA)andhigherconcentrationmayrequirealongerdosetoensureahomogeneousshearingresult.

• Water bath:ThesonicationwaterbathisacriticalcomponentoftheBioruptor®Picosonicationsystem.

1. Water purity: Contaminants such as algae and particules may alter the ultrasonic wavespropagation,resultinginbroadersizedistributionorlargerpeaks.Bathwatershouldbepuredistilled water,changedregularly(atleastonceperweek).

2. Water bath maintenance: The water bath metal surface is fragile and requires a carefulmaintenance.Useonlysoftspongetoremovetraces.Neverusescratchscrubspongesincethiswouldaltertheultrasonicwaveemittersurface.

3. Water type:Distilled water

Supplementary Data:

Pleasenotethattherearethreemainsourcesofvariationinbothpeakbase-pairsizeanddistribution:

1) ThephysicalprocessofDNA fragmentationmightnotbeentirely random inAT-orGC-richregions.

2) The analytical process to determine fragment size has inherent variances (for example, gelelectrophoresisandmicrofluidics-basedplatform).Therefore,fragmentdistributionsandpeakvalues,even fromtechnical replicates,maynotappear identical. If theshearedDNAsamplewillberesinorcolumnpurifiedorconcentratedpriortoanalysis,pleaseremembertotakeoutanaliquot foruseascontrolprior to thatstep.Columnpurificationandconcentrationof theshearedDNAwillgenerateabiasedfragmentdistributionprofileduetotheinherentgreaterlossofthesmallerDNAfragments.

3) RNA contamination in genomic DNA preparation should be carefully removed using RNase-DNasefreeenzymaticdigestionsincetheymightgenerateabiasedfragmentdistributionprofileonmicrofluidics-basedplatform(e.g.AgilentBioanalyzer)oraltersonicationeffiency.

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