Troubleshooting DNA Sequences:

Guidelines and Suggestions

http://vermontcancer.org/dna

Biodesktop Workshops:

• Scheduled: 300 HSRF– Jan. 29th at 10-11AM (Friday)

• Rama Kocherlakota– Basics of submitting orders– Retrieving data– Creating a team to share data– Opportunity to provide input

SESSION OUTLINE:• Guidelines: Generic Set up and Profiles

• Impact of Template and primer ratio???

• Suggestions for different sample types and Sequence Context: Chemistry, Profile, Additives– Sample types: PCR, Plasmid, BAC, Cosmid, RNAi construct, Bisulfite

treated gDNA, gDNA– Sequence Context: GC Rich, Homopolymeric Runs, Repetitive Sequence

• Instrument and Processing Anomolies:

• Retreiving data off the biodesktop: How, What…..

• Sample Purification prior to Sequencing

• Troubleshooting Resources

Sequencing Instruments: AB 3100-Avant, 3130XL both

Capillary Based

• Advantages– Higher throughput– Can reinject samples– Higher separation

efficiency– Better resolution– No Plates!

• Disadvantages– Sensitive to charged

ions– Sensitive to

microparticulates or bubbles

Required Template Concentrations:

Template Type Required templateconcentration

Totalamount in Rxn

Double strandplasmid

50 ng/uL 375 ng

Large plasmid (>7Kb) 50 ng/uL 525 ng

Single strandplasmid

25 ng/uL 75 ng

PCR 0-200 bp

5 ng/uL 10 ng

200-500 bp 5 ng/uL 20 ng

500-1000 bp 5 ng/uL 40 ng

>1000 bp 5 ng/uL 45 ng

PCR (Exo-Sap) N/A 1-2 ul

BAC and gDNA 1ug/uL 1-2 ug

*This amount of template is needed for each primed sequencing reaction

Normal Conditions: Default Profile

AutoSeq1 Profile

• 96° 1min

• 96 ° 10 sec• 50 ° 5 sec 25x• 60 ° 4 min

C.S. Rxn conditions

DS plasmid-375 ng

PCR (5ng/100bp Product)

3.2 pmol Primer

1/8 dilution BDv3.1

Primer Titration: Plasmid

Template Titration: Plasmid

Template Titration: PCR ProductPCR Product Size=~720BP

70 ng added

30ng added

Template Concentration: It Can MatterSubmitting template at the

incorrect concentration

Submitted as:50 ng/ulWas:350 ng/ul

Diluted to:50 ng/ul

Sample Type: RNAi construct, BAC, Cosmid, gDNA, Bisulfite treated DNA

Different Sample Types May Require Different Template or primer

concentration, Chemistry, Profile, and additives

Sequencing RNAi Constructs:Auto Seq1 Profile, Chemistry (Default)

RNAi Construct:GC Rich Profile, 5% DMSO

RNAi Construct:Modified RXN Set-up, RNAi Profile

660 ng Template10 pMol Primer8ul BDT v.3.110% Betaine(Q Buffer)

98 c 5min

96 c 15 sec50 c 10 sec60 c 4 min

50X

RNAi Construct: testing to reduce costsAutoSeq1 (Default):

RNAi Construct:Default Chemistry, RNAi Profile:

RNAi Construct:BDTv3.1/dGTP Chemistry, GC RichProfile:

RNAi Construct:BDTv3.1/dGTP Chemistry, RNAi Profile:

RNAi Construct: LOR scores for three different approaches

AutoSeq1

GC Rich

RNAi

ThermalProfiles:

BAC’s, Cosmid’s, Genomic:

BAC DSRG Profile

• 96° 5min

• 96 ° 30 sec• 50 ° 20sec• 60 ° 4 min

C.S. Rxn conditions

DNA- 1ug

10 pmol Primer

straight BDv3.1

50X

BAC’s: Default set-up and AutoSeq1

BAC’s: Modified Set-up, BAC profile

BAC Sequencing: LOR scores for two different approaches

gDNA Sequencing: Not as easy as you may think!

1.3 Kb productgDNA = 500 ngFWD Primer: 500nMREV Primer: 100nM125 uM dNTP’sStraight Big Dye

Cycling:950C 30s

950C 15s500C 15 s 35-45600C 4 min

PCR and Cycle Sequencing combined:

Murphy, K., et.al., Clinical Chemistry 51:1 35-39 (2005)

Bisulfite Sequencing:Sequencing methylated gDNA

Default Set-up and Profile

Bisulfite Sequencing: Suggested Set-up and profile

BiSulSeq Profile: “ Vish”

• 95° 1min

• 96 ° 10 sec• 52 ° 10sec 30x• 60 ° 4 min

C.S. Rxn conditions

PCR 5ng/100 bp

3.2 pmol Primer

1/8 dilution BDv3.1

“Vish II”: 96c 1min, 25X of 96c 20s,50c 5s, 54c 4minChemistry BDT V1.1

Bisulfite Sequencing: Default Set-up and BiSulSeq Profile

“Vish Profile”

Cosmids:

BAC DSRG Profile

• 96° 5min

• 96 ° 30 sec• 50 ° 20sec• 60 ° 4 min

C.S. Rxn conditions

DNA- 1ug

10 pmol Primer

straight BDv3.1

50x

Sequence Context Constraints:

GC rich, Homoploymeric runs, Repetitive sequence (STR)

Run of G’s:Default Set-up and Profile (AutoSeq1)

Run of G’s:dGTP Chemistry, AutoSeq1 profile

GC Rich Template:Generic Set up, AutoSeq1 Profile

GC Rich Template:BDTv3.1/dGTP (3:1), GC Rich profile

Previous stop point

Repetititve Sequence: Template C Defaults

Stops

Repetititve Sequence: Template C BDT v3.1/dGTP (3:1) mix, GC Rich Profile

Repetititve Sequence: Template D BDT v3.1/dGTP (3:1) mix, GC Rich Profile

Lunatic!!

Repetititve Sequence: Template E BDT v3.1/dGTP (3:1) mix, GC Rich Profile

Repetititve Sequence: Template A BDT v3.1/dGTP (3:1) mix, GC Rich Profile

Not always a fix!

Repetititve Sequence: Template A BDT v1.1, Default Profile

Homopolymeric runs:

“Throw the Book”

Primer Location

Retrieving Data:• An email is sent notifying the user that data is ready.

The staff of the DNA facility has the ability to edit this message to include specific remarks about how your samples ran, so please look at this message!

• The format for the sequence data files: Well Location_template-primer, (A01_pGem-M13For).

In these messages, we often refer to your individual samples by the injection number (A01), not the full name.

Instructions

*

*

What is Phred?

• Basecaller (used by genome centers)• Provides quality score for each base • Generates two files

Need to look at the Chromatogram!! (.AB1 File)

Trace viewers:PC: Mac:

Chromas EditView Finch TV Finch TV

Instrument and Processing Related Anomolies:

AND Email notifications

Bad Injection message:One of your samples had a bad injection. It is being re-injected at no

charge and we will send the data to you when it is ready.

Example:

Causes SolutionsGenerally, this is due to incomplete excess dye clean up of the cycle sequence reaction.

If the DNA facility cleaned the reaction, we consider this an error on our part and we will repeat the cycle sequence reaction and the sequence run for free.If the clean up was done by the user, we charge for the sample, but we will be happy to help with troubleshooting if the problem continues.

Dye blobs can sometimes occur when the cycle sequence reaction was a very low signal.

Optimize the template and primer for a successful cycle sequence reaction.

Loss of Resolution: In the middle

Reinjection successful!

Timing of Reinjections:

C fluorophore degrading

Reinjections on Monday from a Friday run may need to be set-up again

Dye-Blob message:• Your sequence sample, injection number (A01) has a dye leak which interferes with the basecalling in

that region. These cannot be corrected with a reinjection of the sample. It is our policy to repeat the cycle sequence reaction and sequence run once for free to eliminate this problem. If the dye leak was not a problem for you we will not repeat the sample. If it is a problem, resubmit the sample on the BioDesktop and put “re-run for free due to a dye leak” in the comment section of the order form. Please make sure that we have enough template DNA to repeat the reaction.

Example:

Causes Solutions

Generally, this is due to incomplete excess dye clean up of the cycle sequence reaction.

If the DNA facility cleaned the reaction, we consider this an error on our part and we will repeat the cycle sequence reaction and the sequence run for free.If the clean up was done by the user, we charge for the sample, but we will be happy to help with troubleshooting if the problem continues.

Dye blobs can sometimes occur when the cycle sequence reaction was a very low signal.

Optimize the template and primer for a successful cycle sequence reaction.

Spike message:One of your samples had spikes. It is being re-injected at no charge

and we will send the data to you when it is ready.

Causes Solutions

This is generally believed to be a sequencing instrument artifact. It is caused when a small bubble or crystals in the polymer migrate through the capillary with the sample and cause a sharp peak of multicolors. These can mask the true data peaks.

Re-injection of the sample usually gets rid on the spike.

Example:

Template Purification:

What’s best for sample or sequence type

Impact of Purification Method on Sequence Quality: Gel Purified

Run 1

Run 2

PCR Product size= ~630BP

Impact of Purification Method on Sequence Quality: Enzyme Treated

Same sample: Exo1/SAP treated PCR Product

DNA Sequencing Troubleshooting Resources:

• VCC DNA Analysis Websitewww.vermontcancer.org/dna

• DSRG Sequencing Troubleshooting Web Resource www.abrf.org/index.cfm/stwr.home

• DNA Sequencing Resource: Nucleics http://www.nucleics.com/nucleics-dna-sequencing-site.html

• http://biowww.net/

• DNA Sequencing: Optimizing the Process and Analysis

• DNA Facility Staff

Conclusions: Successful Sequencing is Dependent on:

• Template Quality

• Template Quantity

• Upfront Identification of Sample Type

• Upfront identification of Sequence context constraints

• Read Email messages sent by facility staff

Acknowledgements:

VCC DNA Analysis Facility:

MaryLou Shane

Meghan Brown

Scott Tighe

To all users of the VCCDNA Analysis Facility

Special thanks to all the users who have providedtemplate for research studies and those who shared their data for this presentation

VersaDoc 4000MP:Keep in Mind these very important facts about X-Ray film vs CCD imagers:

1] X-Ray film exposures are not quantitative although many people believe they are, it is not the case. The dynamic range is so narrow, and is non-linear that to assume quantitative results can be very misleading. When a band is black, it is black! Please make note of the Gamma curve for X-Ray film.

2] The VersaDoc has a CCD imager in it. CCD’s are unique as they are true photon counters. The higher the bits, the higher the dynamic linear range. The VersaDoc uses a 16 bit CCD! That is what they were originally designed for, and that is why they are quantitative. Many reviewers also know this as well.3] Some people believe that CCD’s are not as sensitive as X-Ray film. This also is not the case. If you add the substrate to the blot and image immediately, the result is essentially the same. The difference people notice often comes from a time delay to walk to the imagers after you add substrate. That is why we suggest adding your substrate in the lab that the VersaDoc Imager is located in.