Explanation of Sequencing Results Comments:

To understand the Service's comments for your sequencing results, it is best to look at the raw data using one of the following recommended sequence analysis programs and to read the DNA Sequencing Troubleshooting Guide.pdf.

To view the raw data (.ab1 files):
You will need one of these programs, free of charge, from the website links below:

• Editview: available upon request from the core (for MAC classic/OS9 or earlier)
• Finch TV: http://www.geospiza.com/Products/finchtv.shtml (for MAC OS X, Linux, or Windows XP/2000)
• Sequence Scanner: http://marketing.appliedbiosystems.com/mk/get/SSS_login?isource=fr_E_Pg_Prod_AB_Gbl_SeqScan_20050920 (for PC Windows 2000/XP)

Sequence Scanner is the best of these programs, but it is only for Windows 2000/XP. To determine your best sequencing approach and to troubleshoot your sequencing results, see the "How-To Guides" option at the top of the home page or go to the downloads link on left.

The purpose of comments are to indicate: 

1. Problems that need to be considered when interpreting the data
2. Suggestions of how to improve your DNA preparation to get higher quality data

General Troubleshooting Steps:

Troubleshooting	Recommendation
DNA Template Quality	Quantify DNA by Agarose Gel
Primer \ Primer site	Confirm the primer sequence is correct and matches the primer site Look at primer mass spec profile for N-1 from primer manufacturer (if leading / lagging peaks present) Test primer by amplifying known target from another template Check that the sequencing primer dilution is correct
Difficult Region for Sequencing	GC-rich template : Resubmit as premium and request on the order to use Big Dye dGTP mix chemistry Homopolymeric stretch: sequence from other end, redesign primer to span homopolymeric stretch. Difficult region with significant repeat sequence, contact Core for more details. We can optimize sequencing chemistries for difficult templates.

Explanation of Comments:

Comment	Possible Causes	Recommendations
Artifact	Sharp spike in electrophorogram Dust \ particulate in polymer Dust \ particulate originating in sample	The Service will automatically repeat these reactions to rule out artifact due to polymer
Failed	No signal no DNA no primer no primer site	Revisit the Guidelines for Submitting Samples Table for the correct concentrations of DNA and primer. Check DNA on an agarose gel with mass standard (e.g. Mass Ladder) Check primer dilution Check that primer works on a different template where it worked well
Increased background	Significant background and / or low signal Dirty DNA (residual salt, ethanol, or inhibitors from DNA cleanup) DNA is a mixture	See the Service's DNA preparation guidelines link on left Check DNA concentration on an agarose gel.
Insertion / deletion "Indel" (PCR product)	Sequence becomes mixed at position of insertion or deletion	To obtain sequence of PCR products with an indel Sequence in from the opposite end to confirm indel Compare data to reference sequence
Leading / Lagging N-1 peak	Mixture of full length (N) and shorter by one base (N-1) in length resulting in leading / lagging peaks Poor quality primer Degraded primer 3' end of sequencing primer is in homopolymer region	Examine the mass spectrometry profile for the sequencing primer for the presence of N and N-1 products. IDT provides a mass spec profile for every primer they synthesize Redesign primers if 3' end has homopolymer sequence or primer anneals in homopolymer stretch
Mixed clone	Sequence becomes mixed after cloning site More than one clone present in sample	For mixed clone, always re-streak out transformant at least once. Re-streak colony at least once and re-isolate plasmid DNA Re-transform plasmid prep, streak out new transformants, and make new plasmid prep
Mixed product	Mixed sequence Non-specific PCR due to primer design DNA is a mixture	Use "Hot-Start" reagents that are only activated by heating of PCR reaction to >90°C Hot-Start DNA polymerase TriLink Biotechnologies (www.trilinkbiotech.com) make the "CleanAmp" dNTPs and primers. We have tested the CleanAmp dNTPs and they work very well
Mixed start	Mixed start is a short sequence that overlaps the main sequence at the start and appears as a mixture, often longer than a primer dimer product. In addition to the main sequence, the short products origninate from PCR product: Multiple primer sites or sub-optimal PCR resulting in short products Sequencing: Sequencing primer anneals near 3' end of template and reads out resulting in short products	Revisit primer design: Use a structure (hairpin) and self-dimer tool to check primer. The IDT website and the Applied Biosystems program Primer Express have useful tools. Use "Hot-Start" reagents that are only activated by heating PCR reaction to >90°C Hot-Start DNA polymerase TriLink Biotechnologies (www.trilinkbiotech.com) make the "CleanAmp" dNTPs and primers. We have tested the CleanAmp dNTPs and they work very well.
Ok	Indicates pGEM sequencing standard meets \ exceeds CRL and QV20+ quality control \ assurance guidelines set by the Core for clinical and HT sequencing services.
Polymerase stutter	Sequence becomes mixed after homopolymeric stretch or repeat sequence region polymerase stutters due to low processivity > 8 nucleotides in PCR products >16 nucleotides in plasmids	Sequence in from opposite end of sequence Use a primer that spans the homopolymer stretch and is anchored at the 3' end in non-repeat / non-homopolymeric region to continue sequencing the DNA segment
Primer dimer	Primer dimer is a short sequence that overlaps the main sequence at the start and appears often as a mixture and / or significantly higher raw signal Caused by sequencing primer annealing to itself A primer dimer product was made during the PCR reaction Primer dimer (cutoff): To better visualize the sequence after the primer dimer region on the electrophorogram, the Core cuts off this initial region and this is noted in the results comments.	Revisit primer design: Use a structure (hairpin) and self-dimer tool to check primer. The IDT website and the Applied Biosystems program Primer Express have useful tools. Use "Hot-Start" reagents that are only activated by heating PCR reaction to >90°C Hot-Start DNA polymerase TriLink Biotechnologies (www.trilinkbiotech.com) make the "CleanAmp" dNTPs and primers. We have tested the CleanAmp dNTPs and they work very well. Increase fidelity of PCR Increase anealing temperature for higher specificity Touchdown-PCR
Sequence-dependent die out / signal drop	Signal declines or drops significantly, resulting in shorter sequence read length and higher background. Caused by High GC-content Repeat-sequence Hairpin	For GC-rich templates GC-rich in a region leading up to die out, resubmit sample as Premium and request on the order that the sequencing be done with Big Dye dGTP mixed chemistry For difficult templates with repeat sequence regions We can optimize sequencing chemistries for difficult templates, contact the Core for details.
Signal off-scale or very strong	Maximum limit of detection exceeded resulting in signal pullup, early die out, and increased background Too much DNA resulting in increased background and earlier die out of signal	Revisit the Guidelines for Submitting Samples Table for the correct concentrations of DNA and primer. For mid-level reactions, reduce amount of DNA 2+ fold If sequencing PCR product or Restriction Fragments, revisit guidelines of ng DNA per KB
Very weak signal	Raw data is < 500 RFU resulting in very poor quality sequence with high background Dirty DNA Suboptimal DNA Primer concentration incorrect	Revisit the Guidelines for Submitting Samples Table for the correct concentrations of DNA and primer. Check DNA on an agarose gel with mass standard (e.g. Mass Ladder) Check primer dilution Check that primer works on a different template where it worked well
Weak signal	Raw data is approximately < 1500 RFU resulting in short and low quality sequence with background Suboptimal DNA Primer concentration incorrect	Revisit the Guidelines for Submitting Samples Table for the correct concentrations of DNA and primer Check DNA on an agarose gel with mass standard (e.g. Mass Ladder) Check primer dilution Check that primer works on a different template where it worked well