dna shearing for bioruptor ngs · dnase free enzymatic digestion since they might generate a biased...
TRANSCRIPT
PR
OT
OC
OL
Europe - Diagenode s.a. / [email protected] / [email protected] // North America - Diagenode Inc. / [email protected] / [email protected]
www.diagenode.com
DNA Shearing for Bioruptor® NGS
Standard operating conditions
Sample volume: 100µl
Tubes: Bioruptor®NGS0.65mlMicrotubesforDNAShearing(Cat.No.WA-005-0500)
Tube holder: 0.5/0.65mltubeholder(Cat.No.UCD-pack0.5)for12x0.65mltubes
Sonication buffer: TE(10mMTris,1mMEDTA,pH7.5-8.0)
DNA concentration:1-20ng/µl(10ng/µlrecommended)
Samplesarevortexed(10-15sec)andcentrifuged(10sec)beforeshearing.For optimal results samples should be stored on ice during 10-15 minutes prior to sonication.
Temperature: 4°C–Bioruptor®WaterCooler(Cat.No.BioAcc-Cool)&SinglecyclevalveforWaterCooler(Cat.No.VB-100-0001)
Power setting: Hposition(High)
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® NGS
Figure shows different DNA size distributionsofshearedgenomicDNAproducedbyvaryingthedurationofsonicationusingpowersettinghigh(H).ThedifferentcurvesdepictaspecificBioruptor® NGS run, optimized to producespecificmeansizesandsizerangesforNext-Generationsequencing.AllsampleswereanalyzedonBioanalyzer2100usingDNAHighSensitivitychip.
PR
OT
OC
OL
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
400bp* 15’’/90’’ 7-8
1000bp* 5’’/90’’ 7-8
*Forlongerfragments(400upto1000bp),ashortcentrifugationstepafterhalfofthecyclenumberscansignificantlyimprovetheresults.Protocolsforothersizeranges(incl.longerfragmentsupto1300bp)areavailableonrequest.
Programmable DNA size distribution and excellent reproducibility with Bioruptor® NGSPanelAandBshowDNAsizedistributionsof200bpofshearedhumangenomicDNAafter13cycles(30secON/OFF)ofsonication.Bioruptor®NGS0.65mlmicrotubesforDNAShearing(WA-005-0500)wereused.AllsampleswereanalyzedonBioanalyzer2100usingDNAHighSensitivitychip.
PanelA:peakelectropherogramview
PanelB:gelvirtualview
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.
PR
OT
OC
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,therecommendedtubevesselsaretheDiagenode’sBioruptor®NGS0.65mlMicrotubesforDNAShearing(CatNo.WA-005-0500).Payattentionnottodamagethecapwhenclosingthetubessincethiscouldaltersonicationresults.
• Sample volume: The recommended volume of the Diagenode’s Bioruptor® NGS 0.65 mlMicrotubesforDNAShearing(CatNo.WA-005-0500)is100µl.Whenusinglowervolumes(e.g.≤50µl),lessreproducibleresultsmaybeobservedduetoanalterationoftheultrasonicwavesdistributioninthesamplefluid;thus,reducingtheefficiencyofsonicationwhichmayresultinbroadersizedistributionorlargerpeaks.
• Sample concentration:DiagenoderecommendsusingDNAconcentrationrangingbetween1and20ng/µl(10ng/µlrecommended).Usinglargerconcentration(e.g.50-100ng/µl)mayresultinbroaderpeaksorvariablepeakdistribution.
• Sample preparation:Sampleviscositymayhaveamajorimpactonsonicationresults.CarefulresuspensionofDNAsampleisstronglyrecommendedbeforesonicationprocessing.Multiplepipettingandgentlevortexingfollowedbyashortcentrifugationtorecoversamplevolumeatthebottomofthetubeisthereforestronglyrecommended.StoringDNAsamplesoniceduring10-15minutesbeforesonicationhasalsobeenshowntoimprovereproducibility.
• DNA quality: DNA quality and quantity must be considered carefully since bad quality andquantityDNAmayhaveseveral impactsonsonicationandnext-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,ethanolprecipitated,treatmentwithRNase-DNasefreeenzymaticdigestiontoremovecontaminantRNA)orcommercialspin-columnbasedkitsarerecommended.
• Water temperature:PropagationofultrasoundinaliquidunavoidablyproducesheatthatcanultimatelyalterDNAsample(e.g.bythermaldenaturation).Toensurethebestpreservationofthesample,itisrecommendedtostartthesonicationprocesswithcoldwaterinthewaterbath.Duringsonication,especiallywhendoinglongsonicationruns,thetemperaturemustalsobecontrolled.Thisisobtainedbytheautomatictemperaturecontrol.
Note:ThepermanentinstallationoftheBioruptor®inacoldroomispossible,althoughnotsufficienttoavoidthetemperatureincreaseduetosonication.
• Automatic temperature control:Arecirculatingwatercoolerisusedtoguaranteetheautomatictemperaturecontrolofthewaterbathduringthewholesonicationprocess.Thiswatercooler(CatNo.BioAcc-cool)producesaregularwaterflowwithaconstantwaterlevelinthetank.Anadditionalregulatingvalve(SinglecyclevalveforWaterCooler,Cat.No.VB-100-0001)ensures
PR
OT
OC
OL
Europe - Diagenode s.a. / [email protected] / [email protected] // North America - Diagenode Inc. / [email protected] / [email protected]
www.diagenode.com
thatwaterwillonlybereplacedduringtheoffcycletoavoidanyinterferencebetweenthewaterflowandthesonicationprocess.
• Sonication time:Minoradjustmentsincyclenumbermaybemadetooptimizeresultsforvarioussampletypesandconcentrations.Cyclenumberlistedaboveisarecommendedguideline.Actualresultsmayvarydependingontheamountandtypeofstartingmaterial,concentration,viscosityand/orplastictubes.Diagenoderecommendssettingupatimedoseresponseexperimentfordeterminingappropriatecyclenumber.Largerlengthstartingmaterial(e.g.totalgenomicDNA)andhigherconcentrationmayrequirealongerdosetoensureahomogeneousshearingresult.
• Water bath: The sonication water bath is a critical component of the Bioruptor® sonicationsystem.
1. Water purity: Contaminants such as algae and particules may alter the ultrasonic wavespropagation,resultinginbroadersizedistributionorlargerpeaks.Bathwatershouldbepuredistilledwater,changedregularly.
2. Water bath maintenance: The water bath metal surface is fragile and requires a carefulmaintenance.Useonlysoftspongetoremovetraces.Neverusescratchscrubspongesincethiswouldaltertheultrasonicwaveemittersurface.
3. Water type:Distilledwater
Supplementary Data:
Please note that there are three main sources of variation in both peak base-pair size anddistribution:
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.
PR
O-D
NAS
h-N
GS_
20_0
4_12