Troubleshooting Sequencing Data

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1 Troubleshooting Sequencing Data Troubleshooting Sequencing Data No recognizable sequence (see page 7-10) Insufficient Quantitate the DNA. Increase the amount of DNA in the sequencing reactions. See page Inhibitory contaminant in Clean up the. See page Insufficient primer Quantitate the primer. Increase the amount of primer in the sequencing reactions. See page Primer has no annealing site Use a primer that is complementary to the. Poor primer design or incorrect Redesign the primer. See page primer sequence Missing reagent Repeat reactions following the protocol carefully. See page Old or mishandled reagents Use fresh reagents. See page Thermal cycler power failure Repeat reactions. Thermal cycling conditions Calibrate the thermal cycler regularly. Use the correct thermal cycling parameters. Use the correct tube for your thermal cycler. Set ramp rates to 1 C/second. Extension products lost during reaction cleanup Extension products not resuspended Lane tracking failure (ABI 373 or ABI PRISM 377 DNA Sequencer) Electrokinetic injection failure (ABI PRISM 310 Genetic Analyzer) Ensure that correct centrifugation speeds and times are used for precipitation and spin column procedures. See page 3-33 Resuspend sample pellet in loading buffer or TSR carefully. Repeat injections. Data Evaluation and Troubleshooting 7-39

2 Noisy data throughout sequence, with low signal strength (see page 7-11) Noisy data throughout sequence, with good signal strength (see page 7-11 Not enough DNA in the sequencing reactions Use more DNA in the sequencing reactions. Load or inject more of the resuspended sequencing reactions. See Preparing and Loading Samples for Gel Electrophoresis on page 3-50 or Preparing and Loading Samples for Capillary Electrophoresis on page GC-rich or GC-rich region in Increase the denaturation temperature to 98 C. Add DMSO to a final concentration (v/v) of 5%. Double all reaction components and incubate at 98 C for 10 minutes Add 5 10% glycerol or 5 10% formamide to the reactions. Linearize the DNA with a restriction enzyme. Shear the insert into smaller fragments (<200 bp) and subclone. Amplify the DNA using 7-deaza-dGTP in the PCR, then sequence the PCR product. Expired or mishandled reagents Use fresh reagents. See page Thermal cycling conditions Calibrate the thermal cycler regularly. Use the correct thermal cycling parameters. Use the correct tube for your thermal cycler. Set ramp rates to 1 C/second. Lane tracking failure Contaminated Clean up the. See page Multiple s in sequencing reaction Multiple priming sites Multiple primers when sequencing PCR products Primer with N 1 contamination High signal saturating detector Examine your on an agarose gel to see that only one is present. See page Ensure that your primer has only one priming site. Redesign the primer if See page Purify your PCR to remove excess primers. See page Use HPLC-purified primers. Use less DNA in the sequencing reactions or load less on the gel or into the capillary. Incorrect run module Use the correct run module. See page 6-2. Incorrect instrument (matrix) file Use the correct instrument file for your sequencing chemistry. See page 6-7 for information on creating instrument files Data Evaluation and Troubleshooting

3 Noise up to or after a specific point in the sequence (see page 7-12) Poor mobility correction (see page 7-13) Early signal loss (see page 7-14) Mixed plasmid preparation Multiple PCR products Primer-dimer contamination in PCR sequencing Slippage after repeat region in Plasmid DNA Templates on page 3-6 and Determining DNA Quality on page Preparing PCR Products for Sequencing on page 3-12 and Determining DNA Quality on page Optimize your PCR amplification. See page Make sure there is no sequence complementarity between the two PCR primers. Use a sequencing primer that is different from either of the PCR primers. Ensure that your sequencing primer does not overlap the sequence of the PCR primers. Use a Hot Start technique, e.g., AmpliTaq Gold DNA Polymerase. Use an anchored primer. See page Incorrect dye set/primer (mobility) file Use the correct mobility file. See page 6-5. Incorrect Peak 1 Location for data Choose a new Peak 1 Location. See page analysis Gel with very different separation properties from the gel matrices that were used to construct the dye set/primer (mobility) files Region of secondary structure in the GT-rich regions with BigDye terminators (see page 7-34) Use the correct dye set/primer file for your gel type. Refer to the ABI PRISM DNA Sequencing Analysis Software User s Manual. Use a sequencing primer that anneals at a different position. Increase the denaturation temperature to 98 C. Increase the extension temperature by 2 3 C. Decrease the extension temperature in cycle sequencing to 55 C or 50 C. Increase the magnesium ion concentration by 1 mm. Data Evaluation and Troubleshooting 7-41

4 Early signal loss (see page 7-14) Excess dye peaks at the beginning of the sequence in dye terminator chemistries (see page 7-27) GC-rich region in Poor lane tracking, such that tracker line diverges from the data Increase the denaturation temperature to 98 C. Add DMSO to a final concentration (v/v) of 5%. Double all reaction components and incubate at 98 C for 10 minutes Add 5 10% glycerol or 5 10% formamide to the reactions. Linearize the DNA with a restriction enzyme. Shear the insert into smaller fragments (<200 bp) and subclone. Amplify the DNA using 7-deaza-dGTP in the PCR, then sequence the PCR product. Poor quantitation of primer Quantitate the primer. See page Poor quantitation of Quantitate the DNA, especially with PCR products. See page Poor removal of unincorporated dye terminators Choose the Start Point for data analysis to exclude the excess dye peaks. See page Follow the protocols for excess dye terminator removal carefully. See page Refer also to the Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions User Bulletin (P/N ). This document can be obtained from the PE Applied Biosystems WWW site ( ub/precipitation_ub.pdf). When using Centri-Sep spin columns, be careful to load the sample onto the center of the gel surface. Do not touch the gel surface with the pipet tip. See page IMPORTANT When using BigDye terminators, be sure to hydrate the column for at least 2 hours. Spin samples in the centrifuge for the recommended times. Spinning too long precipitates more dyes with the sample. When working with microcentrifuge tubes, aspirate the supernatant rather than decant it. Decanting leaves excess ethanol on the sides of the tube Data Evaluation and Troubleshooting

5 Broad, red peak between base 200 and 350 (see page 7-28) Poor removal of unincorporated dye terminators Follow the protocols for excess dye terminator removal carefully. See page Refer also to the Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions User Bulletin (P/N ). It can be obtained from the PE Applied Biosystems WWW site ( pdf/ga/ub/precipitation_ub.pdf). When using Centri-Sep spin columns, be careful to load the sample onto the center of the gel surface. Do not touch the gel surface with the pipet tip. See page Pull-up peaks/ bleedthrough (see page 7-22) Stop peaks in dye primer chemistry Compressions (see page 7-31) Poor data following a long homopolymer region (see page 7-35) Total signal strength above 4000 Primer-dimer contamination in PCR sequencing (see page 7-24) Default fragments in PCR sequencing of plasmid inserts (see page 7-24) DNA sequence composition (see page 7-30) Sequence-dependent region of anomalous mobility, particularly with dye primer chemistries Slippage IMPORTANT When using BigDye terminators, be sure to hydrate the column for at least 2 hours. Quantitate the DNA (see page 3-17). Use less. Load or inject less of the resuspended sequencing reactions. See Preparing and Loading Samples for Gel Electrophoresis on page 3-50 or Preparing and Loading Samples for Capillary Electrophoresis on page Optimize your PCR amplification. See page Make sure there is no sequence complementarity between the two PCR primers, especially at the 3 end. Use a Hot Start technique for the PCR amplification used to generate the sequencing, e.g., AmpliTaq Gold DNA Polymerase. Use a dye terminator sequencing chemistry. Plasmid DNA Templates on page 3-6 and Determining DNA Quality on page Use a dye terminator sequencing chemistry. See page 2-2. If using dye primer chemistry, try a dye terminator sequencing chemistry. See page 2-2. Increase the denaturing ability of the gel or polymer by using higher run temperatures or denaturing agents such as formamide. Note This can decrease the resolution of the gel or polymer and give shorter read lengths. Try an alternate sequencing chemistry. Use an anchored primer to determine sequence after a homopolymer T region in the sequence (A region in the strand). See page Data Evaluation and Troubleshooting 7-43

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