troubleshooting dna sequences: guidelines and suggestions
TRANSCRIPT
Troubleshooting DNA Sequences:
Guidelines and Suggestions
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
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 Anomolies:
• Sample Purification prior to Sequencing
• Troubleshooting Resources
Recommended Template Concentrations:
DNA Template Quantity
Double strand DNA 500ng
Single strand DNA 50ng
PCR product size: 0-200bp 12ng
PCR product size: 200-500bp 24ng
PCR product size: 500-1000bp
50ng
PCR product size: >1000bp 60ng
RNAi construct 700ng
Cosmid, BAC 1ug
Genomic 2-3ug
Normal Conditions: Default Profile
AutoSeq1 Profile
• 96° 1min
• 96 ° 10 sec• 50 ° 5 sec 25x• 60 ° 4 min
C.S. Rxn conditions
Ds-500ng
PCR (6ng/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
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 (Default)
RNAi Construct:GC Rich Profile, 5% DMSO
RNAi Construct:Modified RXN Set-up, RNAi Profile
660 ng Template10 pMol Primer8ul DDT v.3.110% Betaine(Q Buffer)
98 c 5min
96 c 15 sec50 c 10 sec60 c 4 min
50X
RNAi Construct:AutoSeq1 (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: Default set-up and AutoSeq1
Cosmids, Bacs, 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: Modified Set-up, BAC profile
BAC Sequencing: LOR scores for two different approaches
Bisulfite Sequencing:Sequencing methylated gDNA
Default Set-up and Profile
Bisulfite Sequencing: Suggested Set-up and profile
BiSulSeq Profile
• 95° 1min
• 96 ° 10 sec• 52 ° 10sec 30x• 60 ° 4 min
C.S. Rxn conditions
PCR 10ng
3.2 pmol Primer
1/8 dilution BDv3.1
AB Recommendation: 96c 1min, 25X of 96c 10s, 50c 4minChemistry BDT V1.1
Bisulfite Sequencing: Default Set-up and BiSulSeq Profile
“Vish Profile”
Cosmids:
BacDSRG Profile
• 96° 5min
• 96 ° 30 sec
• 50 ° 20sec 50x
• 60 ° 4 min
C.S. Rxn conditions
DNA- 1ug
10 pmol Primer
straight BDv3.1
Sequence Context Constraints:
GC rich, Homopolymeric 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
Stop
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, GC Rich Profile
New to the Market:Amersham Phi 29 Sequencing Finishing Kit
Requires small sample size
(1ng): generates ~1ug
Use 2-4 ul of 5ul RXN volume
Difficult Template Type
Kit Performance Difficult Template Type
Kit Performance
>20bp polynucleotide
Repeat
Dinucleotide
Repeat
Poly G + AC/CA ++
Poly C + AG/GA ++
Poly T - AT/TA -
Poly A - CG/GC ++
Secondary
Structure
++ CT/TC ++
GC Rich Temp. ++ GT/TG ++
AT Rich Temp. -
Repetititve Sequence: Template AAmersham Sequence Finishing Kit
Instrument Related Anomolies:
Solutions!
Drop-Out Peaks:
“Waterfall”: Results in Drop-out Peaks:
Inflection point
Reinjection Helps: Drop out Peaks Gone
Premature Loss of Resolution:
Premature Loss of Resolution:Simply reinject sample
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
Chemistry:
What’s best for sample or sequence type
PCR Product: AB BDT V1.1 vs. V3.1
BDT v1.1- BigDye® Terminator v1.1 Cycle Sequencing Kits are designed for specialty applications that require optimal basecalling adjacent to the primer and for sequencing short PCR product templates………..AB website description
BDT v3.1- BigDye Terminator v3.1 Cycle Sequencing Kit's robust, highly flexible chemistry is ideal for de novo, resequencing, and finishing with PCR product, Plasmid, Fosmid, and BAC templates…….AB website descritption
PCR Product: Chemistry Test
AB BDT v1.1
AB BDT v3.1
AB BDT v1.1-end AB BDT v3.1-end
Raw Q20=705 Raw Q20=712
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:
How to Make a Query:
Comments to Query:
Another Great Resource:Nucleics
Nucleics: Possible Solutions
Other Resources: A Short List• http://biowww.net/
• http://cancer.ucsd.edu/Research/Shared/dna/troubleshooting.asp
• http://www.library.kent.edu/resource.php?id=2256
“Focus on Plasmids”
Conclusions: Successful Sequencing is Dependent on:
• Template Quality
• Template Quantity
• Upfront Identification of Sample Type
• Upfront identification of Sequence context constraints
Acknowledgements:
MaryLou ShaneRomaica OmaruddinMeghan Brown
VCC DNA Analysis FacilityTo 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