Complete Genomics Sample Quality Control Protocol
Contents
1. Complete ip genomics Sample Quality Control Protocol Updated August 2012 DNA Q amtitati oti eae enh nee Materials RE QUire d inaianei aaa a a ra Preparing a PicoGreen Working Solution Preparing the DNA Samples cessssssssessssssseesseecssecsessneecsnsecsecsneesseesseessneesnteessnecsneesneeesaeessuessneeesieesanessneeeateesaeeraneesaeesanes Preparing DNA Standards sscccscciecsssccescscscsicescvsescossusssouscotsececusiciauasttsasece yucceeistteesaseteduintetucetestus seisttsococoubstsusstevaseccousiectsateediie Determining DNA Quantitation sisina niin EEN Expected DNA Quantitation Reproducibility GEL ELE CUO PIVOTS IS eats scics es cscesads aaa aa aaa Eaa a e Materials Re quire a n a EL Nala eS Sec DS aD ates tale at Preparing the Agarose Gel scccssciecsssicesasssrsaceccvsescosssnstoescotsccecsstccousstrss cect icceeisincesasategealedicctestuis snistegocacousstobsstecsseccousiectiatoeaate Loading and Performing Electrophoresis of the DNA Samples eessesssesseesseessseeesstecsteessteesseesateesstersneesateeeanes Inspecting Gell images iio teak atid aaa a E ear area E A E AEA These instructions describe the procedures used by Complete Genomics to quantitate and verify the integrity of DNA samples submitted for complete genome sequencing Sample acceptance is based on results of this QC process DNA is quantitated using the Quant iT PicoGreen dsDNA kit from Invitrogen and integrity is verified using agarose gel e2. 2 6 11 2 26 11 3 18 11 4 7 11 4 27 11 5 17 11 6 6 11 6 26 11 Date Complete Genomics Inc 5 Complete Genomics Sample Quality Control Protocol Gel Electrophoresis Gel Electrophoresis The process of verifying the integrity of your samples using agarose gel electrophoresis includes the following steps Preparing the Agarose Gel Complete Genomics Inc 6 Complete Genomics Sample Quality Control Protocol Gel Electrophoresis Loading and Performing Electrophoresis of the DNA Samples Inspecting Gel Images Materials Required Agarose IBI Scientific Cat 1B70042 or equivalent 1X TBE buffer 96 well full skirted PCR plate VWR Cat 10011 228 or equivalent GeneRuler 1 kb Plus DNA Ladder Fermentas Cat SM1333 or equivalent 5X gel loading dye Amresco Cat E274 or equivalent Lonza GelStar nucleic acid gel stain Fisher Scientific Cat BMA 50535 or equivalent Gel documentation system Alphalmager from Protein Simple or equivalent Gel electrophoresis system Thermo Scientific D2 wide gel system Cat D2 or equivalent Power source Preparing the Agarose Gel The following steps describe preparing a 0 8 agarose gel in a gel mold 1 2 Mix 0 48 g of agarose with 60 mL of 1X TBE buffer in a conical flask Heat the mixture in a microwave until the agarose is completely dissolved approximately one minute Mix well by swirling and allow to cool to 50 55 C Prepare gel casting tray
3. be in the approximate concentration range of 30 to 300 ng uL 1 If frozen thaw DNA samples completely 2 Mix DNA samples thoroughly by vortexing for 20 seconds on a plate mixer at setting 8 Centrifuge briefly to collect contents at the bottom of the plate wells 3 Prepare Dilution Plate 1 o o o o o o o Label a PCR plate as Dilution Plate 1 Add 15 uL 1X TE pH 8 0 to each well positions for which a sample will be quantitated Add 15 uL 1X TE pH 8 0 to one additional well for a control DNA sample Transfer 5 uL from each DNA sample to the corresponding well on Dilution Plate 1 1 4 dilution Add 5 uL of a control DNA sample at a concentration of 100 ng L to the control well Seal the plate and mix by vortexing for 20 seconds at setting 8 Centrifuge briefly to collect contents at the bottom of the plate wells 4 Prepare Dilution Plate 2 o o o o o Label a PCR plate as Dilution Plate 2 Add 42 uL 1X TE pH 8 0 to all well positions for which a sample will be quantitated Transfer 3 uL from each well in Dilution Plate 1 to the corresponding well on Dilution Plate 2 1 15 dilution Seal the plate and mix by vortexing for 20 seconds at setting 8 Centrifuge briefly to collect contents at the bottom of the plate wells 5 Prepare Quantitation Plate o o o Label a UV star 96 well micro plate as Quantitation Plate Add 95 uL 1X TE pH 8 0 to well positions for whic
4. by sealing the ends of the gel chamber with tape or appropriate casting system Place an appropriate number of combs in a gel tray Add 1 uL of GelStar staining reagent to the cooled gel mix well by swirling and pour into the gel tray Allow gel to set for 15 to 30 minutes at room temperature Remove comb s place the gel in electrophoresis chamber and cover with 1X TBE buffer Complete Genomics Inc 7 Complete Genomics Sample Quality Control Protocol Gel Electrophoresis Loading and Performing Electrophoresis of the DNA Samples The following steps describe preparing dye for the electrophoresis putting samples and dye in the agarose gel including a DNA ladder and performing the electrophoresis 1 2 Prepare a 1 67X gel loading dye by mixing 50 uL of 5X gel loading dye with 100 uL TE Prepare the gel plate o Labela PCR plate as Gel Plate o Transfer 4 uL from each well of Dilution Plate 2 to the corresponding well of the Gel Plate Include the control DNA sample o Add 6 uL of 1 67X gel loading dye to each sample in the Gel Plate o Seal the plate and mix by vortexing for 20 seconds at setting 8 o Centrifuge briefly to collect contents at the bottom of the plate wells Prepare the GeneRuler 1 kb Plus DNA Ladder by mixing 1 uL of ladder with 36 uL of the 1 67X gel loading dye prepared in step 1 Load 2 uL of the diluted ladder into the first well of the gel Unseal the Gel Plate and load 4 uL of material
5. d down 10 times to result ina DNA concentration of 125 ng mL 4 Continue in this manner to prepare dilutions at 62 5 ng mL well D12 31 2 ng mL well E12 15 6 ng mL well F12 and 7 8 ng mL well G12 as indicated in Table 2 Table 2 Lambda DNA Serial Dilution Well Final Lambda DNA Concentration ng mL A12 500 B12 250 C12 125 D12 62 5 E12 31 2 F12 15 6 G12 7 8 H12 0 5 Discard 100 uL of the 7 8 ng mL dilution from well G12 Do NOT add it to well H12 Well H12 will contain 100 uL 1X TE pH 8 0 representing 0 ng mL of the Lambda DNA standard Determining DNA Quantitation The following steps describe adding PicoGreen reagent to all samples incubating the Quantitation Plate and determining sample concentrations from standard curve after measuring fluorescence in a plate reader 1 Add the reagent to the samples as follows o Add 100 uL of the PicoGreen working solution to each well of the Quantitation plate that contains a sample control or standard DNA o Seal the plate and mix by pulse vortexing for 30 45 seconds at setting 8 o Centrifuge briefly to collect contents at the bottom of the plate wells o Cover the plate with aluminum foil and incubate at room temperature for 10 minutes Complete Genomics Inc 4 Complete Genomics Sample Quality Control Protocol DNA Quantitation 2 Wipe the bottom of the Quantitation Plate with a KimWipe cleaning tissue 3 Carefully remove the seal and place the plate in t
6. equence data Figure 2 A Gel Image Illustrating High Quality DNA Samples with Single High Molecular Weight Bands B Gel Image Illustrating Degraded Samples Indicated by Arrows C Gel Image Illustrating Samples Containing Single Stranded DNA Indicated by Arrows Complete Genomics Inc 9
7. h a sample will be quantitated Transfer 5 uL from each well in Dilution Plate 2 to the corresponding well on the Quantitation Plate 1 20 dilution Seal the plate and mix by vortexing for 20 seconds at setting 8 Centrifuge briefly to collect contents at the bottom of the plate wells Complete Genomics Inc 3 Complete Genomics Sample Quality Control Protocol DNA Quantitation Preparing DNA Standards The following steps describe preparing a Lambda DNA working solution and serially diluting it in column 12 of the Quantitation Plate to create a standard curve 1 Remove the 100 pg mL Lambda DNA standard stock tube from the PicoGreen kit and mix well by vortexing Spin briefly to collect contents to the bottom of the tube 2 Prepare a Lambda DNA working solution at a concentration of 500 ng mL by adding 5 uL Lambda stock DNA to 995 uL 1X TE pH 8 0 1 200 dilution Mix well by pulse vortexing for 10 to 20 seconds Spin briefly to collect contents to the bottom of the tube 3 Prepare a serial dilution of the Lambda DNA working solution in 100 uL volumes as follows o Add 100 uL 1X TE pH 8 0 to wells B12 H12 of the Quantitation Plate o Add 100 uL of the Lambda DNA working solution 500 ng mL to wells A12 and B12 of the Quantitation Plate o Mix the contents of well B12 by pipetting up and down 10 times to result ina DNA concentration of 250 ng mL o Transfer 100 uL from well B12 to well C12 and mix by pipetting up an
8. he microplate reader following the manufacturer s instructions An excitation wavelength of 480 nm and an emission wavelength of 520 nm should be used 4 Generate a standard curve by plotting the fluorescence values determined for the Lambda DNA standards against the corresponding DNA concentrations The slope of a line fitted to this curve should be between 21 and 27 If the slope is outside this range you need to discard this Quantitation Plate and generate a new Quantitation Plate and repeat the quantitation 5 Calculate the concentrations for each sample DNA and the control DNA from the standard curve using a dilution factor of 1 200 Verify that the concentration determined for the control DNA is in the expected range Expected DNA Quantitation Reproducibility As an estimate of expected day to day reproducibility we have included a run chart of the slopes of standard curves during a six month period Figure 1 Within our hands we expect a 15 range in slope 21 to 27 over time Worst case this should translate to 15 variation in our quantitative measurements and those of our customers who follow this procedure Data are provided below Figure 1 Consistency in Standard Curve Slopes Over the Course of Six Months DNA Quantitation Standard Curve Slopes v Q La O 250 4 s t oe sF wee r gt 7 te re g amp e t A F e y 2 o 7 oe N zg gt f 20 0 T 1 12 28 10 1 17 11
9. kirted PCR plate VWR Cat 10011 228 or equivalent UV star 96 well micro plate VWR Cat 82050 788 or equivalent 1X TE pH 8 0 Plate vortexer Plate centrifuge Adhesive plate seals or plate heat sealer a Aluminum foil Preparing a PicoGreen Working Solution The PicoGreen reagent is diluted in preparation for DNA quantitation Note The PicoGreen working solution must be made fresh on the day of use For best results the solution should be used within a few hours of its preparation Further the PicoGreen solution should be protected from light by covering it with foil or placing it in the dark as the reagent is susceptible to photo degradation Prepare a 1 200 dilution of the PicoGreen reagent in a 15 mL conical tube according to Table 1 Mix well by vortexing Table 1 Preparation of diluted PicoGreen Reagent Number of Samples 1X TE pH 8 0 Volume mL PicoGreen Reagent Volume uL 1 24 4 20 25 48 7 35 48 72 9 45 73 96 11 55 Complete Genomics Inc 2 Complete Genomics Sample Quality Control Protocol DNA Quantitation Preparing the DNA Samples The following steps describe diluting DNA samples by a factor of 1 1 200 in a three step serial dilution and placing the samples into the quantitation plate Samples can be quantitated only in columns 1 to 11 of the 96 well quantitation plate maximum of 88 samples column 12 is reserved for the Lambda DNA standards Note For accurate concentration DNA should
10. lectrophoresis Complete Genomics uses liquid handling robotic systems for all liquid transfer steps in this process but the same procedure can be applied using manual pipettes Complete Genomics data is for Research Use Only and not for use in the treatment or diagnosis of any human subject Information descriptions and specifications in this publication are subject to change without notice Copyright 2011 Complete Genomics Incorporated All rights reserved UG_QC 02 Complete Genomics Sample Quality Control Protocol DNA Quantitation DNA Quantitation Note The quantitation instructions include modifications to those provided by Invitrogen in the Quant iT PicoGreen dsDNA kit user manual probes invitrogen com media pis mp07581 pdf The process of quantitating samples to prepare for submission to Complete Genomics includes the following steps Preparing a PicoGreen Working Solution Preparing the DNA Samples Preparing DNA Standards Determining DNA Quantitation The document also includes a description of the variation in DNA quantitation over time to help you assess the success of your process Expected DNA Quantitation Reproducibility Materials Required Quant iT PicoGreen dsDNA kit Invitrogen Cat P7589 http products invitrogen com ivgn product P7589 Control DNA of known concentration 75 to 150 ng uL Tecan SpectraFluor fluorescent plate reader or equivalent 96 well full s
11. per sample to a well of the gel starting with the well adjacent to the one containing the ladder Load 2 uL of the diluted ladder into the well adjacent to the last sample Place the lid on the gel apparatus connect to the power source and electrophorese for one hour at 80 volts Image the gel using a gel documentation system following the manufacturer s instructions Complete Genomics Inc 8 Complete Genomics Sample Quality Control Protocol Gel Electrophoresis Inspecting Gel Images Use the gel images to verify that the DNA for each sample is of high molecular weight and is double stranded Examine the gel profile for each sample to verify the presence of a single band above the location of the 20 KB band of the GeneRuler 1 KB Plus DNA Ladder see Figure 2A The control DNA should also have a single band of high molecular weight DNA Samples for which a high molecular weight band is not present and a significant amount of the DNA is smeared below the 5 KB band of the ladder are deemed to be degraded see Error Reference source not found These samples will fail the Complete Genomics QC process Samples in which the DNA is partially denatured will have a second high molecular weight band that represents single stranded DNA This single stranded DNA band will run above the double stranded DNA band see Error Reference source not found The presence of single stranded DNA may negatively impact the quality of the resultant s
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