TCB No May Technical Bulletin. GS FLX and GS Junior Systems. Short Fragment Removal for the Amplicon Library Preparation Procedure

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1 TCB No May 2013 Technical Bulletin GS FLX and GS Junior Systems Short Fragment Removal for the Amplicon Library Preparation Procedure Introduction Some library preparation methods may result in the creation of short, undesirable DNA fragments that are amplified during empcr Amplification along with the desired amplicon target. This scenario may yield poor sequencing results as the shorter undesired fragments will preferentially amplify, thereby decreasing the total number of Passed Filter Reads of the desired amplicon target. Therefore, it is crucial in these cases to remove the short contaminating fragments in order to generate the highest number of Passed Filter Reads for the desired amplicon. For life science research only. Not for use in diagnostic procedures. Last save on 13-May-2013 Page 1 of 8

2 Procedure The Sample Library Quality Control procedure described below can first be used to determine if your amplicon library is contaminated with short fragments before proceeding. The Sample Library Quality Control procedure must be performed again if small fragment removal is done to check if short fragments have been successfully removed from your library preparation. Sample Library Quality Control 1. Once an AMPure XP Bead size exclusion is performed, it is important to assess if short fragments have been successfully removed from your library preparation. In order to assess this, the following amplification protocol should be followed as a quality control step. Prepare the PCR master mix below for each final, cleaned library sample tested: Reagent Volume Molecular Biology Grade Water 39 µl 10x PCR Buffer with MgCl 2 5 µl Nucleotide Mix (10 mm each) 2 µl Forward Primer (100 µm)* 1 µl Reverse Primer (100 µm)* 1 µl Amplicon Sample (2 x 10 8 molecules/µl) 1 µl FastStart Taq DNA Polymerase 1 µl Total 50 µl *Be sure to use the correct primers for your library type. Lib-A and Lib-L libraries require different primer sequences: QC Primers for Lib-A Amplicons: Forward Primer: 5 -CGT ATC GCC TCC CTC GCG CCA- 3 Reverse Primer: 5 -CTA TGC GCC TTG CCA GCC CGC- 3 QC Primers for Lib-L Amplicons: Forward Primer: 5 -CCA TCT CAT CCC TGC GTG TC-3 Reverse Primer: 5 -CCT ATC CCC TGT GTG CCT TG-3 2. Run the following PCR program: Page 2 of 8

3 Temperature Time Cycles 94 C 11 min 1x 94 C 1 min 60 C 1 min 20x 72 C 1 min 72 C 10 min 1x 4 C hold 3. Once the PCR program is complete, add 1 µl of Exonuclease I and incubate at 37 C for 30 minutes. 4. Run 1 µl of the amplified size-selected DNA and 1 µl of PCR product not processed with AMPure XP beads on either a single Bioanalyzer DNA 7500 LabChip or a FlashGel System for DNA with a FlashGel 2.2% DNA cassette. 5. If the presence of short DNA species is confirmed, remove the unwanted DNA by repeating the DNA cleanup procedure. Be sure to perform another quality control PCR amplification to confirm that the second cleanup was successful. Library Cleanup with AMPure XP Beads Although there are several ways to cleanup library samples, the Agencourt AMPure XP Beads have been found to effectively remove unwanted short fragments from libraries. The AMPure XP Bead buffer must be replaced with the Sizing Solution found in the GS Rapid Library Buffers Kit (part number ) due to the variability of the size exclusion across different AMPure XP Bead lots. Determining AMPure XP Bead:DNA Ratio for Targeted Sized Fragment Removal While the Amplicon Library Preparation Method Manual uses a 1.6:1 Bead:DNA ratio for Library Purification, a more stringent small fragment removal procedure may also be used. The following procedure can be carried out using a DNA ladder (DNA Molecular Weight Marker XIV) to determine the optimal Bead:DNA ratio for your amplicon size. 1. Vortex the AMPure XP Bead bottle for 20 seconds, or until the beads are completely resuspended. 2. Aliquot 1 ml of AMPure XP Beads into a new microcentrifuge tube. You will use this aliquot for the rest of the procedure. Label six 1.7 ml tubes for the six Bead:DNA ratios to be included in the assay, from 1.8:1 to 0.80:1, in increments of 0.2, per Table 1. Page 3 of 8

4 Target Ratio Volume of AMPure XP Beads (µl) Volume of DNA (µl) 1.8 : : : : : : Table 1: Volumes of AMPure XP Beads and DNA to use for determining optimal ratio for targeted size fragment removal. Volumes can be scaled based on total material available for use. 3. Aliquot 72 µl of AMPure XP Beads in each labeled microcentrifuge tube. 4. Place the tubes on the Magnetic Particle Concentrator (MPC). 5. When the beads have completely pelleted on the side of the tube, leave the tube on the MPC and discard all supernatant, without disturbing the beads. 6. Add 72 µl of Sizing Solution to the beads, vortex for 5 seconds. 7. To each microcentrifuge tube add the appropriate volume of your amplified material as determined by the calculation given in column 3 of Table Vortex all tubes and incubate at room temperature (+22 to +25 C) for 5 minutes. 9. Using the MPC, pellet the beads against the wall of the tubes. This may take several minutes due to the high viscosity of the suspension. 10. Remove the supernatant and wash the beads twice with 500 µl of 70% ethanol, incubating for 30 seconds each time. 11. Remove all the supernatant from each tube and allow the AMPure XP Beads to air dry completely. To reduce drying time, place the tubes in a heating block at +37 C. Visible cracks in the pellet are an indication the beads are dry. 12. Remove the tubes from the MPC, add 10 µl of 1x TE to each tube, and vortex to resuspend the beads. 13. Using the MPC, pellet the beads against the wall of the tubes once more, and transfer the supernatants containing the size-selected DNA to a set of new, appropriately labeled microcentrifuge tube. 14. Run 1 µl of each size-selected DNA on either a single Bioanalyzer DNA 7500 LabChip or a FlashGel System for DNA with a FlashGel 2.2% DNA cassette. 15. Select the smallest ratio that will remove most of the shorter fragments present in your sample while preserving your DNA of interest. Page 4 of 8

5 Short Fragment Removal Once you have chosen a Bead:DNA ratio that will remove the undesired fragments, you can proceed with targeted, short fragment removal for all applicable amplicon libraries. 1. Set a heat block at 37 C. 2. Prepare 70% ethanol in the amount needed (400 μl per sample). For 10 ml, add 7 ml of 100% ethanol to 3 ml Molecular Biology Grade Water, and vortex. 3. Briefly centrifuge the PCR tubes containing your library. 4. Vigorously vortex the bottle of AMPure XP Beads and aliquot the volume needed to cleanup all samples in a new 1.7 ml microcentrifuge tube. 5. Carefully pipet 72 μl of AMPure XP Beads into a 1.7 ml tube. 6. Place the tube on the Magnetic Particle Concentrator (MPC). 7. When the beads have completely pelleted on the side of the tube, leave the tube on the MPC and discard all supernatant, without disturbing the beads. 8. Add 72 µl of Sizing Solution to the beads, vortex for 5 seconds. 9. Determine the appropriate volume of sample to add to the AMPure XP Beads to generate the desired Bead:DNA ratio. Use the formula provided in Table 1, column 3 to determine this volume. If you do not have enough PCR product to add, bring up the volume with Molecular Biology Grade Water. 10. Add the appropriate amount of DNA to the AMPure XP Beads. Mix thoroughly by vortexing for 5 seconds. 11. Incubate for 10 minutes at room temperature. 12. Place the tubes in the MPC and incubate for 5 minutes at room temperature. 13. With the tubes still in the MPC, carefully remove and discard the supernatant without disturbing the beads. 14. Remove the tubes from the MPC and add 200 μl of freshly prepared 70% ethanol to each tube. 15. Vortex the tubes for 5 seconds. The pellet may not resuspend completely; this is acceptable. 16. Place the tubes on the MPC and incubate 1 minute. 17. With the tubes still on the MPC, carefully remove and discard the supernatant without disturbing the beads. 18. Repeat Steps 14 to 17. Remove as much of the supernatant as possible. 19. Place the open tubes on a heat block set at 37 C until the pellet is completely dry (about 5 minutes). Do not leave the tubes on the heat block longer than necessary to avoid over drying. 20. Remove the tubes from the heat block. 21. Add 10 μl of 1x TE to each tube. Vortex 5 sec or until the pellet is completely resuspended. 22. Place the tubes in the MPC and incubate for 2 minutes at room temperature. 23. With the tubes still in the MPC, carefully transfer the supernatants to a set of new 1.7 ml tubes. 24. Save 1 µl of cleaned amplicon library for a PCR amplification quality control. 25. Store the cleaned DNA library samples individually at -15 to -25 C until ready to proceed to the empcr Amplification. Page 5 of 8

6 Library Quality Assessment Once it has been determined that the DNA cleanup has been successful in removing all undesired, short fragments, the amplicon library can be used in empcr Amplification, see Figure 1 and 2 for examples of successful cleaning patterns. Short fragment removal is critical for the success of amplicon sequencing. This targeted removal will ensure that your amplicon of interest will be used to generate as many DNA library beads as possible. A B Figure 1: Bioanalyzer traces of DNA library A: before cleanup and B: after AMPure XP Bead cleanup. Page 6 of 8

7 Figure 2: FlashGel System for DNA with a FlashGel 2.2% DNA cassette. Ladder sizes are 4000 bp, 2000 bp, 1250 bp, 800 bp, 500 bp, 300 bp, 200 bp and 100 bp. Samples loaded (from left to right) untreated, 1.8x, 1.6x, 1.4x, 1.2x, 1x and 0.8x. Part Numbers for Required Reagents Reagent Vendor Catalog Number DNA Molecular Weight Marker XIV Roche Applied Science Molecular Biology Grade Water many possible n/a 10x PCR Buffer with MgCl 2 Roche Applied Science see below Nucleotide Mix (10 mm each) Roche Applied Science see below Primers many possible n/a FastStart Taq DNA Polymerase Roche Applied Science see below Exonuclease I New England Biolabs M0293S or M0293L Page 7 of 8

8 Published by : Roche Diagnostics GmbH Sandhofer Straße Mannheim Germany 2013 Roche Diagnostics All rights reserved.notice to Purchaser For patent license limitations for individual products please refer to: For life science research only. Not for use in diagnostic procedures. Trademarks 454, 454 SEQUENCING, GS FLX, GS JUNIOR, EMPCR, and FASTSTART are trademarks of Roche. All other product names and trademarks are the property of their respective owners. Page 8 of 8

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