TROUBLESHOOTING DNA SEQUENCING DATA

TROUBLESHOOTING DNA SEQUENCING DATA

GENOMICS
DNA Sequencing
Genotyping / GeneScan
Real-Time PCR
Oligo Synthesis
GeneChip Probe Array
Custom Spotted Array

PROTEOMICS
Protein Sequencing
Mass Spectrometry

BIOINFORMATICS
GCG / SeqWeb
Molecular Modeling
Internet Resources

Observation	Possible Causes	Recommended Actions
No recognizable sequence	Insufficient template	Quantitate the DNA template. Increase the amount of DNA in the sequencing reactions.
	Inhibitory contaminant in template.	Clean up the template.
	Insufficient primer	Quantitate the primer. Increase the amount of primer in the sequencing reactions.
	Primer has no annealing site	Use a primer that is complementary to the temple.
	Poor primer design or incorrect primer sequence	Redesign the primer.
	Missing reagent	Repeat reactions following the protocol carefully.
	Old or mishandled reagents	Use fresh reagents.
	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 1ƒC/second.
	Extension products lost during reaction cleanup.	Ensure that correct centrifugation speeds and times are used for precipitation and spin column procedures.
	Extension products not re-suspended	Re-suspend sample pellet in loading buffer or TSR carefully.
	Lane tracking failure (ABI 373 or ABI PRISM 377 DNA Sequencer)	Check lane tracking. Re-track and re-extract lanes if necessary.
	Electrokinetic injection failure (ABI PRISM 310 Genetic Analyzer)	Repeat injections.
Noisy data throughout sequence, with low signal strength	Not enough DNA in the sequencing reactions	Use more in the sequencing reactions.
	Not enough DNA in the sequencing reactions	Load or inject more of the re-suspended sequencing reactions.
	GC-rich template or GC-rich region in template	Increase the denaturation temperature to 98ƒC.
		Add DMSO to a final concentration (v/v) of 5%.
		Incubate the reaction at 96ƒC for 10 minutes before cycling.
		Double all reaction components and incubate at 98ƒC for 10 minutes before cycling.
		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.
	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 1ƒC/second.
	Lane tracking failure	Check lane tracking. Re-track and re-extract lanes if necessary.
Noisy data throughout sequences, with good signal strength	Contaminated template	Clean up the template.
	Multiple templates in sequencing reactions	Examine your template on an agarose gel to see that only one template is present.
	Multiple priming sites	Ensure that your primer has only one priming site. Redesign the primer if necessary.
	Multiple primers when sequencing PCR products	Purify your PCR template to remove excess primers.
	Primer with N-1 contamination	Use HPLC-purified primers.
	High signal saturating detector	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.
	Incorrect instrument (matrix) file	Use the correct instrument file for your sequencing chemistry.
Noise up to or after a specific point in the sequence	Mixed plasmid preparation	Ensure that you have only one template.
	Multiple PCR products	Ensure that you have only one template.
	Primer-dimer contamination in PCR sequencing	Optimize your PCR amplification.
		Make sure there is no sequence complimentarity 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.
	Slippage after repeat region in template	Try an alternate sequencing chemistry.
	Slippage after repeat region in template	Use an anchored primer.
Poor mobility correction	Incorrect dye set/primer (mobility) file	Use the correct mobility file.
	Incorrect Peak 1 Location for data analysis	Choose a new Peak 1 Location.
	Gel with very different separation properties from the gel matricies that were used to construct the dye set/primer (mobility) files	Use the correct dye set/primer file for your gel type.
Early signal loss	Region of secondary structure in the template	Sequence the opposite strand.
		Use a sequencing primer that anneals at a different position.
		Try an alternate sequencing chemistry.
		Incubate the reaction at 96ƒC for 10 minutes before cycling.
		Increase the denaturation temperature to 98ƒC.
		Increase the extension temperature by 2-3ƒC.
	GT-rich regions with Big Dye terminators	Decrease the extension temperature in cycle sequencing to 55ƒC or 50ƒC.
		Increase the magnesium ion concentration by 1mM.
		Sequence the opposite strand.
		Try an alternate sequencing chemistry.
Early signal loss	GC-rich region in template	Increase the denaturation temperature to 98ƒC.
		Add DMSO to a final concentration of (v/v) of 5%.
		Incubate reaction at 96ƒC for 10 minutes before cycling.
		Double all reaction components and incubate at 98ƒC for 10 minutes before cycling.
		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 lane tracking, such that tracker line diverges from the data	Check lane tracking. Re-track and re-extract lanes if necessary.
	Poor quantitation of primer	Quantitate the primer.
	Poor quantitation of template	Quantitate the DNA template, especially with PCR products.
Excess dye peaks at the beginning of the sequence in dye terminator chemistries	Poor removal of unincorporated dye terminators	Choose the Start Point for data analysis to exclude the excess dye peaks.
		Follow the protocol for excess dye terminator removal carefully. Refer also to the Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reaction User Bulletin (P/N 4304655). This document can be obtained from the PE Applied Biosystems WWW site (http://www2.perkin-elmer.com/ab/techsupp/pdf/ga/ub/Precipitation_UB.pdf).
		When using Centry-Sep spin colunms, be careful to load the sample onto the center of the gel surface. Do not touch the gel surface with the pipet tip. 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 samples.
		When working with microcentrifue tubes, aspirate the supernatant rather than decant it. Decanting leaves excess ethanol on the sides of the tube.
Broad, red peak between base 200 and 350	Poor removal of unincorporated dye terminators	Follow the protocols for excess dye terminator removal carefully Refer also to the Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reaction User Bulletin (P/N 4304655). This document can be obtained from the PE Applied Biosystems WWW site (http://www2.perkin-elmer.com/ab/techsupp/pdf/ga/ub/Precipitation_UB.pdf).
	Poor removal of unincorporated dye terminators	When using Centry-Sep spin colunms, be careful to load the sample onto the center of the gel surface. Do not touch the gel surface with the pipet tip. IMPORTANT: When using BigDye terminators, be sure to hydrate the column for at least 2 hours.
Pull-up peaks/bleedthrough	Total signal strength above 4000	Quantitate the DNA template. Use less template.
Pull-up peaks/bleedthrough	Total signal strength above 4000	Load or inject less of the re-suspended sequencing reactions.
Stop peaks in dye primer chemistry	Primer-dimer contamination in PCR sequencing	Optimize you PCR amplification.
		Make sure there is no sequence complimentarity between the two PCR primers, especially at the 3’ end.
		Use a Hot Start technique for the PCR amplification used to generate the sequencing template, e.g., AmpliTaq Gold DNA Polymerase.
		Use a dye terminator sequencing chemistry.
	Default fragments in PCR sequencing of plasmid inserts	Ensure that you have only one template.
	DNA sequence composition	Use a dye terminator sequencing chemistry.
Compressions	Sequence-dependent region of anomalous mobility, particularly with dye primer chemistries	If using primer chemistry, try a dye terminator sequencing chemistry.
		Sequence the opposite strand.
		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.
Poor data following a long homopolymer region	Slippage	Try an alternate sequencing chemistry.
Poor data following a long homopolymer region	Slippage	Use an anchored primer to determine sequence after a homopolymer T region in the sequence (A region in the template strand).

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