FAST MicroSEQ S rdna Bacterial Identification Kit. Protocol

Transcription

1 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

2 For Research Use Use Only. Not intended for any animal or human therapeutic or diagnostic use. Information in this document is subject to change without notice. APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF, WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. NOTICE TO PURCHASER: LIMITED LICENSE for Fast MicroSEQ PCR Kits Use of this product is covered byus patent claims and corresponding patent claims outside the US. 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3 Contents Preface and Safety Safety Information How to use this guide How to Obtain More Information How to Obtain General Support FAST MicroSEQ S rdna Bacterial Identification Kit Protocol Introduction Materials and Equipment Preparing DNA Performing PCR to Amplify the 16S rdna Region Performing Cycle Sequencing Performing Electrophoresis of Extension Products Analyzing Data Troubleshooting Frequently Asked Questions Appendix A Additional Supported Applied Biosystems Instruments Appendix B Preventing Evaporation During Electrophoresis Preparing a Diluted Sample Drying-Down and Resuspending the Sample Appendix C Good PCR Practices FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 3

4 Contents Appendix D Safety Chemical safety General chemical safety SDSs Chemical waste safety Biological hazard safety Safety alerts Chemical alerts Index FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

5 Preface and Safety Safety Information Note: For general safety information, see this Preface and Appendix D, Safety on page 37. When a hazard symbol and hazard type appear by a chemical name or instrument hazard, see the Safety Appendix for the complete alert on the chemical or instrument. Safety Alert Words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards. Each alert word IMPORTANT, CAUTION, WARNING, DANGER implies a particular level of observation or action, as defined below. IMPORTANT! Indicates information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical. CAUTION! Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. WARNING! Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. DANGER! Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations. SDSs The SDSs for any chemicals supplied by Applied Biosystems or Ambion are available to you free 24 hours a day. For instructions on obtaining SDSs, see SDSs on page 39. IMPORTANT! For the SDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer. FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 5

6 Preface and Safety How to use this guide Text conventions This guide uses the following conventions: Bold text indicates user action. For example: Type 0, then press Enter for each of the remaining fields. Italic text indicates new or important words and is also used for emphasis. For example: Before analyzing, always prepare fresh matrix. A right arrow symbol ( ) separates successive commands you select from a drop-down or shortcut menu. For example: Select File Open Spot Set. Right-click the sample row, then select View Filter View All Runs. User attention words Two user attention words appear in Applied Biosystems user documentation. Each word implies a particular level of observation or action as described below: Note: Provides information that may be of interest or help but is not critical to the use of the product. IMPORTANT! Provides information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical. 6 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

7 How to Obtain More Information How to Obtain More Information Related Documentation Information about the MicroSEQ ID Analysis Software is available in the: Documentation for MicroSEQ ID Analysis Software version 2.0 or later: MicroSEQ ID Analysis Software Quick Reference Card MicroSEQ ID Analysis Software Getting Started Guide MicroSEQ ID Analysis Software Online Help Applied Biosystems MicroSEQ web site at Note: For additional documentation, see How to Obtain General Support on page 8. Obtaining Information from the Help System The MicroSEQ ID Analysis Software has a Help system that describes how to use each feature of the user interface. Access the Help system by doing one of the following: Click in the toolbar of the MicroSEQ ID Analysis Software window Select Help F1 You can use the Help system to find topics of interest by: Reviewing the table of contents Searching for a specific topic Searching an alphabetized index Send Us Your Comments Applied Biosystems welcomes your comments and suggestions for improving its user documents. You can your comments to: techpubs@appliedbiosystems.com IMPORTANT! The address above is only for submitting comments and suggestions relating to documentation. To order documents, download PDF files, or for help with a technical question, go to then click the link for Support. (See How to Obtain General Support on page 8). FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 7

8 Preface and Safety How to Obtain General Support For the latest services and support information for all locations, go to then click the link for Support. At the Support page, you can: Search through frequently asked questions (FAQs) Submit a question directly to Technical Support Order Applied Biosystems user documents, SDSs, certificates of analysis, and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition, the Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities. 8 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

9 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol Introduction MicroSEQ System Overview FAST MicroSEQ Product Description The MicroSEQ Microbial Identification System combines all of the instrumentation, reagents, sequence libraries, and software required for automated microbial identification using DNA sequencing. The MicroSEQ system is easy to use and suitable for the routine identification of all bacterial and fungal isolates, including organisms that are difficult to grow, nonviable, or unidentifiable using phenotypic methods. The MicroSEQ system identifies bacterial and fungal isolates from a small sample of pure culture without preliminary testing or growth on selective media. The FAST MicroSEQ S rdna Bacterial Identification Kit comprises two kits: the FAST MicroSEQ S rdna PCR Kit and the MicroSEQ S rdna Sequencing Kit. These kits provide all of the reagents necessary for the amplification and sequencing of the first 500 base pairs of the 16S ribosomal RNA gene (rdna). The DNA sequence of the unknown is deciphered by capillary electrophoresis on an Applied Biosystems genetic analyzer. The Applied Biosystems FAST technology allows identification in five hours. MicroSEQ ID Analysis Software compares the sequence to the validated MicroSEQ ID 16S rdna 500 Library, then generates an identification report. Variations found within the first 500 base pairs of the 16S region are sufficient to identify most bacteria to the species level. Note: The MicroSEQ Full Gene 16S rdna Bacterial Identification Kit is recommended if you need a full 16S rdna sequence to identify a bacterial species. Refer to for more information. About Dyelabeled Terminator Chemistry The MicroSEQ S rdna Sequencing Kit uses Applied Biosystems BigDye Terminator v1.1 chemistry. Forward and Reverse Sequence Mixes contain sequenceterminating 3 -dideoxynucleotide triphosphates (ddntps). Each of the four ddntps is tagged with a different fluorescent dye. When the ddntps are incorporated into extension products during cycle sequencing, the extension products are simultaneously terminated and labeled with the dye that corresponds to the incorporated base, as shown in the following figure. FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 9

10 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol For more information about dye-labeled terminators and other sequencing chemistries, refer to the Automated DNA Sequencing Chemistry Guide (PN ). (See How to Obtain General Support on page 8.) Instrument Platforms For optimum performance of the FAST MicroSEQ S rdna Bacterial Identification Kit, use the: Applied Biosystems Veriti 96-Well or 9800 Fast Thermal Cyclers Applied Biosystems 3500 or 3100 series Genetic Analyzer Note: The FAST MicroSEQ PCR chemistry reduces amplification time when used on the GeneAmp PCR System 9700 in Maximum ramp mode, but the time time is further reduced when the FAST chemistry is used with the recommended thermal cyclers. For information on older instruments that can also be used, see Appendix A, Additional Supported Applied Biosystems Instruments, on page 31. Protocol Overview This protocol provides: Instructions for performing amplification, cycle sequencing, and electrophoresis Information on preparing samples, purifying amplification and extension products, and data analysis 10 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

11 Introduction Procedure Overview Sample Collection Harvest bacterial colony Sample Preparation Isolate DNA, then prepare dilution for PCR Amplification Prepare reactions, perform PCR, analyze PCR products, then purify PCR products Applied Biosystems Veriti or 9800 Fast Thermal Cycler Cycle Sequencing Prepare reactions, perform cycle sequencing, then purify extension products Applied Biosystems Veriti or 9800 Fast Thermal Cycler Electrophoresis Configure instrument, then prepare and run reactions Applied Biosystems 3500 or 3130 Series Genetic Analyzer Data Analysis FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 11

12 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol Materials and Equipment Kit Contents The table below describes the contents of the two bacterial kits. Kit Component Description FAST MicroSEQ S rdna PCR Kit (PN ) MicroSEQ S rdna Sequencing Kit (PN ) FAST MicroSEQ S PCR Master Mix Positive Control Negative Control MicroSEQ S Forward Sequence Mix MicroSEQ S Reverse Sequence Mix One tube containing 2 PCR Master Mix sufficient for 60 amplifications One tube of positive control DNA (E. coli) at 1 ng/μl, sufficient for 10 positive-control assays One tube of negative control (water) Two tubes sufficient for a total of 55 reactions Two tubes sufficient for a total of 55 reactions Kit Storage Guidelines Kit On Receipt Storage Conditions After First Use PCR 15 to 25 C 2 to 8 C in a PCR cleanroom Sequencing 15 to 25 C 15 to 25 C Avoid excess freeze-thaw cycles. Aliquot reagents in smaller amounts, if necessary. Before each use of the kit, allow the frozen reagents to thaw at room temperature or on ice. IMPORTANT! Do not heat the reagents. Whenever possible, keep thawed reagents on ice during use. Mix the reagents by gently vortexing the tubes. Centrifuge the tubes briefly to collect all liquid at the bottom of the tube. Equipment and Materials Not Included Equipment and materials are required in addition to the reagents supplied in the FAST MicroSEQ S rdna Bacterial Identification Kit. To obtain a current list of equipment and additional materials, go to click System (under MicroSEQ in the right menu), then click MicroSEQ System List of Other Required Materials. 12 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

13 Preparing DNA Preparing DNA Isolating Genomic DNA from Samples For information on good laboratory practices for PCR, see Appendix C, Good PCR Practices on page Isolate bacterial genomic DNA from bacterial colonies, using Applied Biosystems PrepMan Ultra Sample Preparation Reagent. Follow the instructions in the PrepMan Ultra Sample Preparation Reagent Protocol. IMPORTANT! The ideal colony size is 1 to 2 mm. For smaller colonies, decrease the amount of PrepMan Ultra sample preparation reagent to 50 μl from the 100 μl suggested in the protocol. 2. When the supernatant is not in use, Applied Biosystems recommends storing it at 15 to 25 C. Before use, thaw, then vortex and centrifuge the stored supernatant. Alternatively, the supernatant can be covered and stored at 4 C for up to 1 month. Diluting Genomic DNA for PCR Prepare a 1:100 dilution of the PrepMan Ultra supernatant: Note: The minimum recommended dilution for the PrepMan Ultra supernatant is 1: Pipette 495 μl of nuclease-free water into a 1.5-mL microcentrifuge tube. 2. Add 5 μl of the PrepMan Ultra supernatant. Note: If the PrepMan Ultra supernatant is colored (typically a shade of black or green), PCR inhibition may occur. See Troubleshooting on page When the supernatant is not in use, Applied Biosystems recommends storing it at 15 to 25 C. Before use, thaw, then vortex and centrifuge the stored supernatant. Alternatively, the supernatant can be covered and stored at 4 C for up to 1 month. FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 13

14 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol Performing PCR to Amplify the 16S rdna Region Before preparing the PCR reactions, review Appendix C, Good PCR Practices on page 35 and Kit Storage Guidelines on page 12 for sample and reagent handling instructions. Preparing PCR Reactions 1. Vortex the diluted supernatant to mix the tube contents. 2. Using the volumes shown in the table, prepare samples and controls in MicroAmp reaction tubes or 96-well plates. IMPORTANT! Select the appropriate tubes or 96-well plates for your thermal cycler. See your instrument user guide, or go to click System (under MicroSEQ in the right menu), then click MicroSEQ System List of Other Required Materials. Reaction Type Volume for One Reaction Negative controls 15 μl of FAST PCR Master Mix 15 μl of negative control (provided with kit) Samples 15 μl of FAST PCR Master Mix 15 μl of 1:100 dilution of PrepMan Ultra supernatant Positive controls 15 μl FAST PCR Master Mix 15 μl of positive-control DNA (provided with kit) Note: To help avoid cross-contamination, Applied Biosystems recommends pipetting components in the following order: negative controls, samples, positive controls. Where possible, leave empty cells between different reaction types. 3. Use strip caps and the capping tool, or adhesive film and the sealing tool, to cap the tubes or plate (see Sealing the plate with strip caps or Sealing the plate with adhesive film on page 15). Vortex, spin briefly, then place the tubes or the plate in the thermal cycler. Sealing the plate with strip caps IMPORTANT! Apply significant downward pressure on the sealing tool in all steps to form a complete seal. Note: Use of strip caps instead of 96-well adhesive plate covers may help reduce cross-contamination. To use the rolling capping tool: 14 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

15 Performing PCR to Amplify the 16S rdna Region Roll the capping tool across all strips of caps on the short edge, then the long edge of the tray. Roll the capping tool around all outer rows of strips of caps. Rolling capping tool To use the rocking capping tool: Slip your fingers through the handle with the holes in the tool facing down. Place the holes in the tool over the first eight caps in a row. Rocking capping tool Rock the tool back and forth a few times to seal the caps. Repeat for remaining caps in the row, then for all remaining rows. Sealing the plate with adhesive film IMPORTANT! Apply significant downward pressure on the applicator to completely seal the wells. Pressure is required to activate the adhesive on the optical cover. 1. Place an optical adhesive cover on the plate, then rub the flat edge of the applicator back and forth along the long edge of the plate. Applicator 2. Rub the flat edge of the applicator back and forth along the short edge (width) of the plate. Long edge of plate Short edge of plate 3. Rub the edge of the applicator horizontally and vertically between all wells. 4. Rub the edge of the applicator around all outside edges of the plate using small back and forth motions to completely seal around the outside wells. 5. Vortex the plate on the low setting for 5 seconds. If you see liquid on the well sidewalls, spin down the plate at 2000 g for 20 seconds using a centrifuge with a plate adapter. IMPORTANT! Make sure reagents are in the bottom of the wells. FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 15

16 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol Performing the Amplification Run 1. Set the appropriate ramp mode for your thermal cycler: Veriti 96-Well Thermal Cycler Default Note: To use the default mode, select Browse/New Methods in the thermal cycler main menu, select New, then edit the thermal cycling conditions. See the Veriti Thermal Cycler User Guide for details Fast (F96) GeneAmp PCR System 9700 Maximum (Max) Note: An amplification run using a 9700 thermal cycler can take up to 20 minutes longer than a run using the Veriti or 9800 Fast thermal cycler. 2. Set the thermal cycling conditions: Initial Step Each of 30 Cycles Melt Anneal Final Extension Final Step HOLD CYCLE HOLD HOLD 95 C 10 sec 95 C 0 sec 64 C 15 sec 72 C 1 min 4 C 3. Set the reaction volume to 30 μl, then start the run. 4. Before removing the caps or adhesive film, briefly centrifuge the tubes or plate. Note: Centrifuging helps avoid cross-contamination from liquid remaining on the caps or plate covers. Note: You can cover and store the PCR products at 15 to 25 C until you are ready to use them. Note: PCR products are stable for 6 months or longer at 15 to 25 C. Analyzing PCR Products (Optional) Analyze PCR products to confirm the presence of amplified DNA, or to estimate the PCR product yield. To analyze PCR products: 1. Load 5 μl of PCR product per lane on one of the following agarose gel separations: 2% agarose gel separation if you use another ready-to-use gel system (such as E-gel from Invitrogen) or if you prepare your own gel 1.2% agarose gel separation if using a Lonza Bioscience FlashGel cassette Note: Read and understand the SDSs provided by the manufacturer before you store, handle, or work with any chemicals or hazardous materials. 16 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

17 Performing PCR to Amplify the 16S rdna Region 2. Use the Mass Standard Ladder to estimate the PCR product yield. In a positive control or sample, a single fragment ranging from 460 to 560 bp in size should be detected on a gel. Actual fragment size depends on the bacterial species. No product should be visible in a negative control reaction. IMPORTANT! If your samples show no PCR product, PCR inhibition is the most likely cause. See Troubleshooting on page 25. Purifying PCR Products for Cycle Sequencing Remove unused dntps and primers from each PCR product using one of the following products: ExoSAP-IT (USB) Montage PCR Filter Unit (Millipore) Note: Read and understand the SDSs provided by the manufacturer before you store, handle, or work with any chemicals or hazardous materials. Note: Be sure you follow the guidelines for the starting sample volume for cleanup as directed in the product literature. FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 17

18 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol Performing Cycle Sequencing Cycle sequencing occurs when successive rounds of denaturation, primer annealing, and primer extension in a thermal cycler result in the incorporation of dye terminators into extension products. The products are then loaded into a genetic analyzer to determine the 16S rdna sequence. For additional information about cycle sequencing chemistries, refer to the Automated DNA Sequencing Chemistry Guide (PN ). Preparing Cycle Sequencing Reactions 1. Before removing the tube or plate caps, briefly centrifuge the purified PCR products. Note: Centrifuging helps avoid cross-contamination from liquid remaining on the caps or plate covers. 2. In reaction tubes or a 96-well plate, prepare separate forward- and reversesequencing reactions for each PCR product and control: Forward-sequencing reaction Combine 7 μl of purified PCR product or control + 13 μl of the forward sequence mix Reverse-sequencing reaction Combine 7 μl of purified PCR product or control + 13 μl of the reverse sequence mix IMPORTANT! Select the appropriate tubes or 96-well plates for your thermal cycler. See your instrument user guide, or go to click System (under MicroSEQ in the right menu), then click MicroSEQ System List of Other Required Materials. Note: To help avoid cross-contamination, Applied Biosystems recommends pipetting components in the following order: negative controls, samples, positive controls. 3. Cover and store the unused portions of the purified PCR products at 15 to 25 C until you are ready to use them. Note: PCR products are stable for 6 months or longer at 15 to 25 C. 4. Cap the tubes or the plate, then place the tubes or the plate in the thermal cycler. Note: Use of strip caps instead of 96-well adhesive plate covers may help reduce cross-contamination. Performing the Cycle Sequencing Run 1. Set the appropriate ramp mode for your thermal cycler: Veriti 96-Well Thermal Cycler Default 9800 Fast (F96) thermal cycler 18 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

19 Performing Cycle Sequencing GeneAmp PCR System 9700 Maximum (Max) 2. Set the thermal cycling conditions: Initial Step Each of 25 Cycles Melt Anneal Extend Final Step HOLD CYCLE HOLD 96 C 1 min 96 C 10 sec 50 C 5 sec 60 C 1 min 15 sec 3. Set the reaction volume to 20 μl, then start the run. 4 C 4. Before removing the tube or plate caps, briefly centrifuge the extension products. Note: Centrifuging helps avoid cross-contamination from liquid remaining on the caps or plate covers. 5. If necessary, cover and store the extension products at 4 C overnight or at 15 to 25 C for up to 1 week before purifying them. Purifying Extension Products After cycle sequencing, use one of the following products to remove excess dye terminators, non-incorporated nucleotides, and primers from the extension products. Select an appropriate purification product depending on whether you performed cycle-sequencing in tubes or a plate. Follow the guidelines and procedures supplied with the kits. For... Purify using one of the following... Tubes Performa DTR Gel Filtration Cartridges (Edge Biosystems) DyeEx 2.0 Spin Kit (Qiagen) CentriSep Column (Applied Biosystems) 96-well plates Performa DTR Ultra 96-Well Plate Kit (Edge Biosystems) DyeEx 96 Kit (Qiagen) FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 19

20 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol Performing Electrophoresis of Extension Products Configuring the Instrument for Electrophoresis IMPORTANT! Use only the 50-cm capillary array length regardless of the instrument that you are using. Refer to your instrument user guide for more information. 1. Configure your data collection software as described in the MicroSEQ ID Analysis Software Version 2.0[or greater] Getting Started Guide, Appendix A, Using Autoanalysis with Applied Biosystems 3500 or 3130 Series Genetic Analyzers. 2. Configure the instrument with the parameter settings specified in the following table. Instrument Applied Biosystems 3130/3130xl Applied Biosystems 3500/3500xL Procedure Specify: Filter Set E Run Module StdSeq50_POP6_1 Base-caller KB.bcp DyeSet/Primer (Mobility File) KB_3130_POP6_BDT v1.mob Create a plate using the MSID plate template in the 3500 Series Data Collection software. This plate template contains an instrument protocol/run module (POP-6 polymer) and a basecalling protocol optimized for MicroSEQ ID applications. You can use POP7 polymer with the StdSeq50_POP7 run module and the KB_3130_POP7_BDT v1.mob file. However, this instrument configuration reduces data quality within the first 40 bases on the 5' end of the sequence. Refer to the MicroSEQ ID Analysis Software Online Help for more information. Note: If you are not using a 3500 or 3130 series genetic analyzer, select the appropriate parameter settings from the following table. Instrument Filter Set Run Module Basecaller DyeSet/Primer (Mobility File) ABI PRISM 310 E Seq POP6 (1 ml) E.md4 KB.bc p KB_310_POP6_BDT v1_50std.mob ABI PRISM 3100 ABI PRISM 3100-Avant Applied Biosystems 3730 Applied Biosystems 3730xl E StdSeq50_POP6_1 KB.bc p E StdSeq50_POP7 KB.bc p KB_3100_POP6_BDT v1.mob KB_3730_POP7_BDT v1.mob 20 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

21 Performing Electrophoresis of Extension Products Performing the Electrophoresis Run IMPORTANT! If the electrophoresis run time will be longer than 24 hours (for example, if you are injecting more than 40 wells on a 4-capillary instrument or more than 160 wells on a 16-capillary instrument) evaporation might occur. Therefore, Applied Biosystems recommends the addition of formamide to the reactions. See Appendix B, Preventing Evaporation During Electrophoresis, on page Before removing the tube caps or plate cover, briefly centrifuge the extension products. 2. Prepare reactions: a. Pipette at least 15 μl of each purified extension product or control into separate wells in a 96-well plate. b. Pipette 15 μl of Hi-Di formamide into each blank well that will be injected together with samples. 3. Cover the plate, centrifuge, then load the plate into your instrument. Start the run. Note: Centrifuging removes bubbles from the bottom of the wells. 4. Cover and store the unused portion of the purified extension products at 4 C overnight or at 15 to 25 C for up to 1 week. When the run is complete, review the data using the MicroSEQ ID Analysis Software. Note: If you are not using autoanalysis with a 3500 or 3130 series genetic analyzer, refer to the MicroSEQ ID Analysis Software Getting Started Guide for data analysis instructions. FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 21

22 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol Analyzing Data MicroSEQ ID Analysis Software MicroSEQ ID Analysis Software analyzes sequences obtained with any of the MicroSEQ Microbial Identification Kits. The software assembles the 16S region rdna sequence for the unknown, then compares the sequence with 16S region rdna sequences. For the FAST MicroSEQ S rdna Bacterial Identification Kit, data is compared to the MicroSEQ ID 16S rdna 500 Library. Based on the comparison, the software provides a potential ID for the unknown bacterial species. With the software, you can perform: Basecalling with assignment of quality values Clear-range determination, which lets you exclude data near sequence ends (typically poor-quality data) from analysis Assembly and alignment of sequences to generate a high-quality consensus sequence Comparison of the consensus sequence to the MicroSEQ ID proprietary libraries to generate a list of the closest matches, including percentage match scores Exports of projects and consensus sequences to facilitate data-sharing between collaborators The software also has features that assist with 21 CFR Part 11 compliance requirements. For more information, refer to the MicroSEQ ID Analysis Software Online Help, MicroSEQ ID Analysis Software Quick Reference Card and the MicroSEQ ID Analysis Software Getting Started Guide for software version 2.0 or later. MicroSEQ Proprietary Libraries All MicroSEQ ID Library sequences are carefully validated. Polymorphic positions are taken into account and included in library species. Custom Libraries MicroSEQ ID Analysis Software allows you to create custom libraries using data generated by the MicroSEQ ID software, or using sequences from public databases. Custom libraries are easy to import and export, making information sharing convenient. During the analysis process, you can search proprietary and custom libraries simultaneously to determine 3 to 20 closest matches to the sequence of your unknown bacterial species. MicroSEQ Reports MicroSEQ ID Analysis Software generates four detailed reports: Analysis QC Report Allows you to quickly scan the unknowns in a project to gather information about the samples, including the top percent identity match and specimen score to measure data quality. Figure 1 on page 23 shows an example Analysis QC Report. 22 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

23 Analyzing Data Library Search Report Provides more detailed information about the libraries that were searched, including a list of all the top matches and the total number of bases searched. Figure 2 on page 24 shows an example Library Search Report. Audit Trail Report Tracks changes made to projects after analysis. Electronic Signature History Report Provides a summary of the electronic signatures used in a project. All reports can be generated on project and specimen levels. In addition, the software allows you to create custom reports. For information, refer to the MicroSEQ ID Analysis Software Online Help for software version 2.0 or later. Figure 1 Example Analysis QC Report FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 23

24 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol Figure 2 Example Library Search Report 24 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

25 Troubleshooting Troubleshooting Problem Possible Cause Solution Amplification Problems No PCR product No biomass or Fungal sample or PCR inhibition or Cells were not disrupted by the PrepMan Ultra method or Incorrect dilution 1. If no PCR product is seen, use more bacterial cells. 2. If the problem persists, use the MicroSEQ D2 LSU rdna Fungal Identification Kits. 3. If the problem persists, make one or more new dilutions of the PrepMan Ultra supernatant, then run several PCR reactions of each dilution to increase your chance of obtaining a PCR product of the correct size. If the PrepMan Ultra supernatant is: Clear Make smaller dilutions (1:40 or 1:10) of the original PrepMan Ultra supernatant Colored (typically a shade of black or green) Make smaller dilutions (1:40 or 1:10) of the original PrepMan Ultra supernatant Make a 1:1000 dilution of the original PrepMan Ultra supernatant 4. If you do not obtain a PCR product from any of the diluted samples, try one of the following solutions: Use a DNA extraction kit to isolate pure DNA or Use the bead-beating method to isolate fungal genomic DNA or bacterial genomic DNA. FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 25

26 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol Problem Possible Cause Solution Sequencing Problems Signal is too high Absence of signal/blank electropherogram The sequence is short The first part of the sequence is very bright and off-scale and the remainder has very low intensity Both results and raw data show occasional high spikes for all four dye colors Too much amplicon in the sequencing reaction Sample evaporation High starting amount of DNA or Too much DNA template in the sequencing reaction Bubbles in the capillary Dilute the purified extension product with Hi-Di Formamide, then perform a new run. If you ran purified extension product that was: Not diluted Dilute the purified extension product at a ratio of 1:1. Diluted at a 1:1 ratio Dilute the purified extension product at a 1:10 ratio. Diluted at a 1:10 ratio Dilute the purified extension product at a 1:40 ratio. See Appendix B, Preventing Evaporation During Electrophoresis, on page 33. See Appendix B, Preventing Evaporation During Electrophoresis, on page Decrease the amount of bacterial cell material using one of the following methods: Use a smaller colony or pellet Further dilute the PrepMan Ultra supernatant 2. If the problem persists, estimate PCR product yield on agarose gel and use 5 to 20 ng of amplicon for sequencing as described on Analyzing PCR Products (Optional) on page 16. Check the instrument maintenance and troubleshooting guides. 26 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

27 Troubleshooting Problem Possible Cause Solution Sequencing Problems (continued) Large regions of overlapping sequence or Cannot call bases for large regions of sequence Small regions of overlapping sequence Pipetting more than 1 template per well or DNA sample is contaminated (that is, the DNA is derived from more than one species of bacteria) or The organism has multiple copies of the rdna gene, and some copies have insertions or deletions In bacterial species with multiple copies of the rrna gene, the gene can be polymorphic, resulting in overlap of up to 1% of the sequence 1. Prepare new reactions, then repeat electrophoresis. 2. If the problem persists, sub-culture the organism to pure culture, then repeat identification. 3. If the problem persists, clone the PCR product (using a kit such as the Invitrogen Topo PCR Cloning Kit) before performing sequencing. No action needed. FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 27

28 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol Frequently Asked Questions Sensitivity and Quantitation What is the sensitivity of the FAST MicroSEQ S rdna Bacterial Identification Kit? As long as you start from a visible colony or cell pellet, MicroSEQ kits will work. Can I use the FAST MicroSEQ S rdna Bacterial Identification Kit to quantify bacteria? No. The PCR is an endpoint assay. Sample Preparation and Storage Which kit should I use to identify yeast samples? Use the FAST MicroSEQ D2 LSU rdna Fungal Identification Kit or the MicroSEQ D2 LSU rdna Fungal Identification Kit to sequence and identify yeast samples. What is the best way to prepare yeast samples? Prepare yeast samples using the PrepMan Ultra Sample Preparation Reagent or bead-beating method, just as you prepare bacterial samples. Extra dilutions of the fungal DNA supernatant are sometimes necessary. Are there alternative methods for preparing genomic DNA? If the PrepMan Ultra kit method does not successfully disrupt cells, you can use the bead-beating method to isolate genomic DNA. Alternatively, you can use a DNA extraction kit (available from various vendors) to isolate pure DNA. Can I use less PrepMan Ultra Sample Preparation Reagent if I start with a smaller colony? Yes. The ideal colony size is 1 to 2 mm. For smaller colonies, you can decrease the amount of PrepMan Ultra Sample Preparation Reagent to 50 μl from the suggested 100 μl in the PrepMan Ultra Sample Preparation Reagent Protocol. Can I enrich my genomic DNA by using less PrepMan Ultra Sample Preparation Reagent? Yes. However, be careful not to overload the PCR mix. Enriched samples tend to have more cellular and other debris, which can interfere with PCR. At what temperature should I store my PrepMan Ultra-isolated DNA? Store isolated DNA at 15 to 25 C (you may safely keep it at room temperature or 4 C overnight). 28 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

29 Frequently Asked Questions Contamination How can I tell if my sequence is representative of a single species? The DNA sequence from a single species should be distinct (easy to call base pairs), without significant regions of overlapping sequence. If my initial DNA sample is contaminated (that is, it comes from multiple species), how can I sequence my PCR product? Clone the PCR product using a kit such as the Topo PCR Cloning Kit from Invitrogen. Overlapping Sequences My sequence has large regions of overlap (>5% mixed bases). What does this mean? See Troubleshooting, Large regions of overlapping sequence on page 27. My sequence has small regions ( 1%) of overlap. What does this mean? See Troubleshooting, Small regions of overlapping sequence on page 27. PCR Product Size Can I always expect the same size PCR product for all species? PCR products can vary from the expected product size, depending on the species. Expected product sizes for the: MicroSEQ Fungal Kits 1 band at 300 to 500 bp MicroSEQ 500 Kits 1 band at 460 to 560 bp MicroSEQ Full Gene Kit 1 band at 460 to 560 bp and 2 bands at 700 to 800 bp Can I increase the number of cycles to increase the PCR yield? Yes, but doing so can cause additional background signal from the negative control. Species Libraries How are species in the Applied Biosystems libraries validated? Refer to the MicroSEQ kit Validation Statement link at Where does Applied Biosystems obtain the strains used to determine the reference sequences in the Applied Biosystems libraries? The strains are derived from major culture collections such as the American Type Culture Collection (ATCC) and the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) (German Collection of Microorganisms and Cell Cultures). FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 29

30 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol What is the difference between the libraries for the MicroSEQ Full Gene Kit and the MicroSEQ 500 Kits? The sequences in the library for the MicroSEQ 500 Kits are ~500 bp, which is the expected size of the PCR products for this kit. The sequences in the library for the MicroSEQ Full Gene Kit are ~1440 bp, the maximum sequence length that the kit allows you to determine. How many entries are included in the MicroSEQ ID Analysis Software libraries? See the Applied Biosystems MicroSEQ web site at for current information on number of entries. Additional Documentation Where can I find additional information about MicroSEQ ID Analysis Software? Information about the MicroSEQ ID Analysis Software is available in the: Documentation for MicroSEQ ID Analysis Software version 2.0 or later: MicroSEQ ID Analysis Software Quick Reference Card MicroSEQ ID Analysis Software Getting Started Guide MicroSEQ ID Analysis Software Online Help Applied Biosystems MicroSEQ web site at Note: For additional documentation, see How to Obtain Support on page page FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

31 Additional Supported Applied Biosystems Instruments A To take advantage of the reduced amplification and sequencing times allowed by the FAST PCR chemistry, Applied Biosystems recommends using the Veriti 96-Well Fast Thermal Cycler and the Applied Biosystems 3500 or 3130 series Genetic Analyzer with the MicroSEQ kits. However, the MicroSEQ kits can also be used with: GeneAmp PCR System 9700 Applied Biosystems 3730 and 3730xl DNA Analyzers ABI PRISM 3100 and 3100-Avant Genetic Analyzers ABI PRISM 310 Genetic Analyzer FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 31

32 Appendix A Additional Supported Applied Biosystems Instruments 32 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

33 Preventing Evaporation During Electrophoresis B Applied Biosystems recommends the use of Hi-Di formamide to prevent sample evaporation during long electrophoresis runs. If your run time is: 24 hours or less, addition of formamide is not necessary Between 24 and 48 hours, see Preparing a Diluted Sample below Longer than 48 hours, see Drying-Down and Resuspending the Sample on page 34 Preparing a Diluted Sample 1. Prepare reactions using a 1:1 ratio of purified extension product and formamide: a. In a 96-well plate, pipette 10 μl Hi-Di formamide into each well to which you will add purified extension products or controls. b. Pipette 10 μl Hi-Di formamide into each blank well that will be injected together with the samples. c. Add 10 μl of purified extension product or control to each well filled in step 1a, then mix by pipetting up and down. Note: If after a 1:1 dilution you do not detect a sequencing ladder due to a low signal, rerun the sample without diluting. 2. Centrifuge the plate, load the plate into your instrument, then start the run. Note: Centrifuging removes bubbles from the bottom of the wells. Note: See Configuring the Instrument for Electrophoresis on page 20 for details. 3. Cover and store the unused portion of the purified extension products overnight at 4 C or for up to 1 week at 15 to 25 C. Next, when the run is complete, review the data using the MicroSEQ ID Analysis Software. Note: If you are not using a 3500 or 3130 series Genetic Analyzer, refer to the MicroSEQ ID Analysis Software Getting Started Guide for data analysis instructions. FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 33

34 Chapter B Preventing Evaporation During Electrophoresis Drying-Down and Resuspending the Sample 1. Centrifuge the tubes or plate containing the purified extension products in a speed vac. Note: Centrifuge time and speed depend on the number of samples and the type of speed vac used. Typical times range from 30 to 60 minutes. IMPORTANT! Do not over-dry the DNA pellet, and do not use heat to dry the pellet. 2. Resuspend the DNA in 15 μl of Hi-Di Formamide. Note: Formamide disrupts hydrogen bonds in double-stranded DNA, inhibiting secondary structure and DNA conglomeration, and resulting in cleaner and more consistent electrophoresis runs. 3. Centrifuge the plate, load the plate into your instrument, then start the run. Note: Centrifuging removes bubbles from the bottom of the wells. Note: See Configuring the Instrument for Electrophoresis on page 20 for details. Next, when the run is complete, review the data using the MicroSEQ ID Analysis Software. Note: If you are not using a 3500 or 3130 series Genetic Analyzer, refer to the MicroSEQ ID Analysis Software Getting Started Guide for data analysis instructions. 34 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

35 Good PCR Practices C When preparing samples for PCR amplification: Wear clean gloves and a clean lab coat (not previously worn while handling amplified PCR products or during sample preparation). Change gloves whenever you suspect that they are contaminated. Maintain separate areas and dedicated equipment and supplies for: Sample preparation and PCR setup PCR amplification and analysis of PCR products Never bring amplified PCR products into the PCR setup area. Centrifuge PCR samples briefly whenever residual sample may be present on the inside lid (such as after dropping a tube or plate, or when there is condensation on the tube or plate from heating or thawing). Open and close all sample tubes and reaction plates carefully. Try not to splash or spray PCR samples. Keep reactions and components capped or sealed as much as possible. Use a positive-displacement pipette or aerosol-resistant pipette tips. Clean lab benches and equipment periodically with freshly diluted 10% bleach solution. Clean the PCR set-up area with DNAZap reagent. FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 35

36 Appendix C Good PCR Practices 36 FAST MicroSEQ S rdna Bacterial Identification Kit Protocol

37 Safety D This appendix covers: Chemical safety General chemical safety SDSs Chemical waste safety Biological hazard safety Safety alerts Chemical alerts FAST MicroSEQ S rdna Bacterial Identification Kit Protocol 37