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