Troubleshooting DNA Sequences:

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Troubleshooting DNA Sequences

• Guidelines and Suggestions

http//vermontcancer.org/dna
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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

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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

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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

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Required Template Concentrations
Template Type Required template concentration Total amount in Rxn
Double strand plasmid 50 ng/uL 375 ng
Large plasmid (gt7Kb) 50 ng/uL 525 ng
Single strand plasmid 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
gt1000 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
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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

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Primer Titration Plasmid
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Template Titration Plasmid
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Template Titration PCR Product
PCR Product Size720BP 70 ng added 30ng
added
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Template Concentration It Can MatterSubmitting
template at the incorrect concentration
Submitted as 50 ng/ul Was 350 ng/ul Diluted
to 50 ng/ul
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Sample Type RNAi construct, BAC, Cosmid, gDNA,
Bisulfite treated DNA

• Different Sample Types May Require Different
  Template or primer concentration, Chemistry,
  Profile, and additives

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Sequencing RNAi ConstructsAuto Seq1 Profile,
Chemistry (Default)
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RNAi ConstructGC Rich Profile, 5 DMSO
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RNAi ConstructModified RXN Set-up, RNAi Profile
660 ng Template 10 pMol Primer 8ul BDT v.3.1 10
Betaine (Q Buffer)
98 c 5min 96 c 15 sec 50 c 10 sec 60 c 4 min
50X
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RNAi Construct testing to reduce costsAutoSeq1
(Default)
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RNAi ConstructDefault Chemistry, RNAi Profile
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RNAi ConstructBDTv3.1/dGTP Chemistry, GC
RichProfile
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RNAi ConstructBDTv3.1/dGTP Chemistry, RNAi
Profile
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RNAi Construct LOR scores for three different
approaches
Thermal Profiles
RNAi
AutoSeq1
GC Rich
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BACs, Cosmids, 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
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BACs Default set-up and AutoSeq1
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BACs Modified Set-up, BAC profile
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BAC Sequencing LOR scores for two different
approaches
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gDNA Sequencing Not as easy as you may think!
PCR and Cycle Sequencing combined
1.3 Kb product gDNA 500 ng FWD Primer
500nM REV Primer 100nM 125 uM dNTPs Straight
Big Dye Cycling 950C 30s 950C 15s 500C 15 s
35-45 600C 4 min
Murphy, K., et.al., Clinical Chemistry 511 35-39
(2005)
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Bisulfite SequencingSequencing methylated gDNA
Default Set-up and Profile
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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
4min Chemistry BDT V1.1
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Bisulfite Sequencing Default Set-up and
BiSulSeq ProfileVish Profile
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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
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Sequence Context Constraints

• GC rich, Homoploymeric runs, Repetitive sequence
  (STR)

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Run of GsDefault Set-up and Profile (AutoSeq1)
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Run of GsdGTP Chemistry, AutoSeq1 profile
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GC Rich TemplateGeneric Set up, AutoSeq1 Profile
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GC Rich TemplateBDTv3.1/dGTP (31), GC Rich
profile
Previous stop point
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Repetititve Sequence Template C Defaults
Stops
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Repetititve Sequence Template C BDT v3.1/dGTP
(31) mix, GC Rich Profile
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Repetititve Sequence Template D BDT v3.1/dGTP
(31) mix, GC Rich Profile
Lunatic!!
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Repetititve Sequence Template E BDT v3.1/dGTP
(31) mix, GC Rich Profile
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Repetititve Sequence Template A BDT v3.1/dGTP
(31) mix, GC Rich Profile
Not always a fix!
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Repetititve Sequence Template A BDT v1.1,
Default Profile
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Homopolymeric runs
Throw the Book Primer Location
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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


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What is Phred?

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

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Need to look at the Chromatogram!! (.AB1 File)
Trace viewers PC
Mac Chromas
EditView Finch TV
Finch TV
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Instrument and Processing Related Anomolies

• AND Email notifications

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Bad Injection messageOne 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 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.
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Loss of Resolution In the middle
Reinjection successful!
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Timing of Reinjections
C fluorophore degrading
Reinjections on Monday from a Friday run may need
to be set-up again
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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.
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Spike messageOne 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.
Example
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.
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Template Purification

• Whats best for sample or sequence type

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Impact of Purification Method on Sequence
Quality Gel Purified
Run 1 Run 2
PCR Product size 630BP
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Impact of Purification Method on Sequence
Quality Enzyme Treated
Same sample Exo1/SAP treated PCR Product
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DNA Sequencing Troubleshooting Resources

• VCC DNA Analysis Website
• www.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

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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

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Acknowledgements
VCC DNA Analysis Facility MaryLou Shane Meghan
Brown Scott Tighe
To all users of the VCC DNA Analysis
Facility Special thanks to all the users who
have provided template for research studies and
those who shared their data for this presentation
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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. CCDs 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
CCDs 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.