User Bulletin - Guidelines for Preparing 20 kb SMRTbell™ Templates
Contents
1. approximately 200 ng of gDNA on a field inversion gel p Figure 2 FIGE was performed using a CHEF Mapper Bio Rad according to the manufacturer s recommendations with a low range tar get of 1 kb and a high range target of 50 kb 100 200 ng of each sample was loaded per lane e Lane 1 2 5 kb Molecular Ruler Bio Rad P N 170 8205 e Lane 2 starting gDNA sample e Lane 3 20 kb sheared DNA e Lane 4 20 kb SMRTbell template before size selection e Lane 5 BluePippin size selected 20 ko SMRTbell template PN 100 286 700 01 10 07 2013 Because constant voltage gel electrophoresis does not resolve high molecular weight fragments what appears to be high molecular weight DNA may not be accurate Figure 3 below shows the same samples run on both constant voltage electrophoresis and field inversion gel electrophoresis FIGE 4 kb 4 kb Figure 3a Samples run on 1 2 Lonza Gel Lanes 3 8 Figure 3b The same samples run on FIGE using a Pippin Pulse appear to be high molecular weight on agarose gel Power Supply show sample degradation Lane 1 FlashGel DNA marker 100 bp 4 kb Lane 1 8 kb 48 kb DNA Size Standard from Bio Rad Lane 2 Sample 1 Lane 2 2 5 kb Molecular Ruler from Bio Rad Lane 3 Sample 2 Lane 3 Sample 1 Lane 4 Sample 3 Lane 4 Sample 2 Lane 5 Sample 4 Lane 5 Sample 3 Lane 6 Sample 5 Lane 6 Sample 4 Lane 7 Sample 6 Lane 7 Sample 5 Lane 8 Sample 7 Lane 8 Sample 6 Lane 9 FlashGel DNA marker 12. 00 bp 4 kb Lane 9 Sample 7 If the majority of the sample is lt 10 kb and there is no higher quality sample available you may want to prepare a library from the unsheared sample following the standard 10 kb SMRTbell library protocol However this may result in shorter subread lengths If the goal of the project is to generate the longest reads possible using the PacBio P5 C3 chemistry and180 min movies consider preparing higher quality gDNA Figure 4 below shows a comparison of subread lengths from degraded and high quality gDNA N 3 8 of Subreads of Mb gt Read Length of Mb gt Read Length of Subreads re en i 10000 15000 20000 25000 0 Subread Length 0 5000 10000 15000 Subread Length Figure 4a Subread length Distribution from a degraded Figure 4b Subread length Distribution from high quality genomic genomic DNA DNA PN 100 286 700 01 10 07 2013 Once you have assessed the quality of your gDNA you can proceed by following the workflow below ac QC Input gDNA Optional 0 45X AMPure Purification Shearing 0 45X AMPure Purification QC Sheared DNA Library Construction DNA Damage Repair DNA End Repair 0 45X AMPure Purification Ligation and Exo Digest 0 45X AMPure Purification BluePippin System Size Selection 0 40X AMPure Purification 1 0X AMPure Purification Library Size Selection Anneal and Bind SMRTbell Templates Prepare for MagBead Station Loading
3. PACIFIC BIOSCIENCES D Guidelines for Preparing 20 kb SMRTbell Templates User Bulletin This Bulletin provides recommendations and tips for preparing 20 kb SMRTbell templates using the BluePippin size selection method Once you have read this User Bulletin proceed to the Procedure amp Check list 20 kb Template Preparation and Sequencing for preparing your 20 kb SMRTbell templates Before You Begin Materials and Kits needed To perform this procedure you must have the following kits materials and instrumentation Item DNA Template Prep Kit 2 0 3 kb to 10 kb DNA Polymerase Binding Kit polymerase of choice DNA Sequencing Kit or DNA Sequencing Kit XL MagBead Binding Kit SMRT Cell 8Pac v3 BluePippin system with Software v5 90 or later See www sagescience com for ordering information and links to downloadable software and installation instructions The BluePippin User Manual may be found at http www sagescience com support PacBio SMRTbell cassette definition set see http www sagescience com support for downloadable file and installation instructions 0 75 Dye Free Agarose Gel Cassettes 1 10 kb to order go to http www sagescience com products cassettes 0 75 agarose The Quick Guide for this cassette at http www sagescience com wp content uploads 2011 10 Quick Guide BLF 751 0 marker S1 pdf NanoDrop Spectrophotometer Series 2000 2000c 3300 or 8000 Qubit Fluorometer and Qu
4. Sequence PN 100 286 700 01 10 07 2013 Shear and Purify DNA Note that prior to shearing gentle mixing is recommended This recommendation will hold for larger insert sizes however for 20 kb libraries no damage has been observed as a result of vortexing Also typical yields after shearing and AMPure purification are 50 70 depending on the quality and purity of the input gDNA We rec ommend starting with at least 7 5 ug to 10 ug input gDNA in order to produce 5 ug sheared and concentrated DNA for subsequent steps Use a Covaris g TUBE device to shear gt 5 ug DNA sample The most up to date guidance on how to use the g TUBE device along with recommended centrifuges and centrifugation speeds can be found in the g TUBE device user manual available for download from the Covaris website or the Shared Protocols page of SampleNet with the following changes 1 Dilute your DNA concentration to 200 300 ng uL in Elution Buffer EB The sample volume may range from 25 100 uL 2 Shear at 5500 rom for 1 minute in an Eppendorf MiniSpin plus 3 Check for any residual sample remaining in the upper chamber If present add 30 uL of EB flick the tube and spin again at 5500 rpm for another minute in the same orientation If not proceed to step 5 4 Again check for residual sample If present add another 30 uL EB and spin at 6000 rpm Repeat until you no longer see anything in the upper chamber 5 Invert and re spin 6 Recover yo
5. ant iT HS dsDNA reagent g TUBE microcentrifuge tubes AMPure PB Beads Vendor Pacific Biosciences Pacific Biosciences Pacific Biosciences Pacific Biosciences Pacific Biosciences Sage Science PN BLU0001 Sage Science Sage Science PN BLF7510 Thermo Scientific Invitrogen PN Q32857 or Q32851 Covaris Pacific Biosciences Handling High Molecular Weight DNA Proper care of JDNA must be taken to minimize damage e Do not vortex gDNA prior to shearing e Avoid excessive pipetting e Avoid multiple freeze thaw cycles e Avoid excessive heat Assess the Quality of Starting DNA Pure free from RNA or other organic and inorganic contaminants high molecular weight DNA is required to prepare size selected 20 kb SMRTbell templates Measure the gDNA concentration using both a NanoDrop spectrophotometer and a Qubit fluorometer If any of the following is true use 0 45X AMPure PB beads to con centrate and or purify the DNA before shearing following the steps below 1 The gDNA concentration is less than 200 ng uL 2 The OD 260 280 is less than 1 8 or greater than 2 0 If the gDNA concentration determined by the Qubit fluorometer is significantly less than the concentration deter mined by the NanoDrop spectrophotometer this may indicate RNA contamination Performing 0 45X AMPure PB purification removes most RNA contaminations To assess the integrity of gDNA we recommend running both an agarose gel electrophoresis
6. emote to anneal sequencing primer at 0 8333 nM template and bind poly merase at 0 5 nM template to size selected SMRTbell templates Enter 20 000 bases as the average insert size and select Magnetic Beads small scale reaction and no DNA control template For more information about using the Binding Calculator see the Pacific Biosciences Template Preparation and Sequencing Guide On plate Loading Recommendations There are differences in loading requirements between the BluePippin size selected libraries and AMPure selected libraries BluePippin size selected libraries require higher loading concentrations with approximately 25 pM on the SMRT Cell 125 pM on Plate However if your library is size selected using AMPure PB beads a 25 pM on SMRT Cell loading will result in overloading Long insert libraries size selected with 0 40X or 0 375X AMPure PB beads will still contain shorter SMRT bells therefore the required loading concentration is lower approximately 5 10 pM on SMRT Cell 25 50 pM on plate Prior to loading these samples on the PacBio RS system please perform titration runs to maximize the yield per SMRT Cell For Research Use Only Not for use in diagnostic procedures Copyriant 2013 Pacific Biosciences of California Inc All rights reserved Information in this document is sub pa to change without notice Pacific Biosciences assumes no responsibility for any errors or omissions in this document Certain notices term
7. s conditions and or use restric ions may pertain to your use of Pacific Biosciences products and or third party products Please refer to the applicable Pacific Biosciences Terms and Conditions of Sale and to the applicable license terms at http www pacificbiosciences com licenses html Pacific Biosciences the Pacific Biosciences logo PacBio SMRT and SMRTbell are trademarks of Pacific Biosciences in the United States and or certain other countries All other trademarks are the sole property of their respective owners PN 100 286 700 01 10 07 2013
8. size selection system to avoid losing the library PN 100 286 700 01 10 07 2013 Alternative Removal of Short Fragments with 0 40X AMPure PB Beads If the library is lt 0 5 ug or if there is a broad size distribution of lt 4 kb recovery from gel sizing will likely be very low and a lower X AMPure PB bead purification may be preferred This will remove or minimize fragments in the 1 2 kb range Note that it is critical to accurately pipet the desired volume of AMPure PB bead solution there is a steep drop off in recovery for concentrations lt 0 40X To facilitate accurate pipetting carry out 0 40X AMPure PB bead puri fication separations with a minimum 100 uL sample and 40 uL AMPure PB beads dilute sample in EB if neces sary Follow the protocol on page 12 except for the volume of AMPure PB beads in Step 1 Recovery is typically 60 90 from 0 40X AMPure PB Beads If a higher stringency size selection is desired AMPure PB concentration may be reduced to as low as 0 35X For lt 0 40X AMPure purifications recovery is often in the range of 5 25 For lt 0 40X AMPure purifications be sure to save the supernatant from the binding step until after you have verified the eluted library If the final yield is too low add 0 6X AMPure PB beads to the saved supernatant and re bind to recover lost the sample Continue from above Anneal and Bind BluePippin Size Selected SMRTbell Templates Use the Binding Calculator in RS R
9. such as 1 2 Lonza FlashGel with FlashGel DNA marker 100 bp 4 kb and a Field Inversion Gel Electrophoresis FIGE system such as the CHEF Mapper XA System Bio Rad P N 170 3760 or Pippin Pulse power supply Sage Science P N PP10200 Agarose gel electrophoresis allows you to quickly view degraded DNA Depending on the goals of your project you may not want to proceed with library construction if the DNA sample is degraded Use a standard low per cent gel with a DNA ladder that goes to gt 10 kb Genomic DNA samples should run above the largest band in the ladder One such system is E Gel 0 8 gels Ata minimum 1 2 Lonza FlashGel gels with FlashGel DNA marker 100 bp 4 kb can be used to rule out highly degraded samples See figure 1 below Note that small fragments often inflate quantitation Check the Lonza gel for small fragments 1 minute after starting the run to check for small fragments Figure 1 Quality assessment of two samples run on agarose gel electrophoresis 1 2 Lonza Gel e Lane 1 FlashGel DNA marker 100 bp 4 kb e Lane 2 Degraded gDNA e Lane 3 Intact gDNA e Lane 4 FlashGel DNA marker 100 bp 4 kb PN 100 286 700 01 10 07 2013 Consider using an FIGE system to evaluate the quality of gDNA and determine the sizes of sheared DNA and SMRTbell templates see figure 2 below On a field inversion gel high quality gDNA should migrate as a single band of approximately 50 kb Lane 2 Run
10. ur sample into a 1 5 or 2 0 mL LoBind microcentrifuge tube Add EB if necessary to adjust volume to at least 100 uL BluePippin Size Selection With BluePippin size selection the lower and upper limits of the selected range can be adjusted We recom mend keeping the upper size limit BP end at 50000 50 kb The lower size limit BP start may be varied depending on several factors e Size distribution in the library e Amount of SMRTbell DNA in the library e Target size for sequencing reads for the project e Number of Mbases required for the project For size selection of an optimized large insert library we recommend a BP start of 7000 bp If most of the frag ments in the library are gt 15kb and you plan to size select gt 4 ug of template you may consider increasing the BP start to 10000 bp For shorter inserts use a size cut off well below the size of most of the fragments to obtain the typical yield For example if the peak of the library size distribution is around 10 kb 12kb you may consider a BP start of 4000 If a more stringent size selection is desired the yield is likely to be considerably lower Important Note When loading 2 ug or less per well subtract 3000 from the desired BP start as lower amounts tend to run faster on the BluePippin system If the library contains lt 0 5 ug or if there is a broad size distribution to lt 4 kb then a 0 40X AMPure purification step is recommended instead of the BluePippin
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