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Troubleshooting DNA Sequences: Guidelines and Suggestions.

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Presentation on theme: "Troubleshooting DNA Sequences: Guidelines and Suggestions."— Presentation transcript:

1 Troubleshooting DNA Sequences: Guidelines and Suggestions

2 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

3 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

4 Recommended Template Concentrations: DNA TemplateQuantity Double strand DNA500ng Single strand DNA50ng PCR product size: 0-200bp12ng PCR product size: 200-500bp24ng PCR product size: 500-1000bp50ng PCR product size: >1000bp60ng RNAi construct700ng Cosmid, BAC1ug Genomic2-3ug

5 Normal Conditions: Default Profile AutoSeq1 Profile 96°1min 96 ° 10 sec 50 ° 5 sec25x 60 ° 4 min C.S. Rxn conditions Ds-500ng PCR (6ng/100bp Product) 3.2 pmol Primer 1/8 dilution BDv3.1

6 Primer Titration: Plasmid

7 Template Titration: Plasmid

8 Template Titration: PCR Product PCR Product Size=~720BP 70 ng added 30ng added

9 Sample Type: RNAi construct, BAC, Cosmid, gDNA, Bisulfite treated DNA Different Sample Types May Require Different Template or primer concentration, Chemistry, Profile, and additives

10 Sequencing RNAi Constructs: Auto Seq1 Profile (Default)

11 RNAi Construct: GC Rich Profile, 5% DMSO

12 RNAi Construct: Modified RXN Set-up, RNAi Profile 660 ng Template 10 pMol Primer 8ul DDT v.3.1 10% Betaine (Q Buffer) 98 c 5min 96 c 15 sec 50 c 10 sec 60 c 4 min 50X

13 RNAi Construct: AutoSeq1 (Default):

14 RNAi Construct: Default Chemistry, RNAi Profile:

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

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

17 RNAi Construct: LOR scores for three different approaches AutoSeq1 GC Rich RNAi Thermal Profiles:

18 BAC’s: Default set-up and AutoSeq1

19 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

20 BAC’s: Modified Set-up, BAC profile

21 BAC Sequencing: LOR scores for two different approaches

22 Bisulfite Sequencing: Sequencing methylated gDNA Default Set-up and Profile

23 Bisulfite Sequencing: Suggested Set-up and profile BiSulSeq Profile 95°1min 96 ° 10 sec 52 ° 10sec30x 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 4min Chemistry BDT V1.1

24 Bisulfite Sequencing: Default Set-up and BiSulSeq Profile “Vish Profile”

25 Cosmids: BacDSRG Profile 96°5min 96 ° 30 sec 50 ° 20sec50x 60 ° 4 min C.S. Rxn conditions DNA- 1ug 10 pmol Primer straight BDv3.1

26 Sequence Context Constraints: GC rich, Homopolymeric runs, Repetitive sequence (STR)

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

28 Run of G’s: dGTP Chemistry, AutoSeq1 profile

29 GC Rich Template: Generic Set up, AutoSeq1 Profile

30 GC Rich Template: BDTv3.1/dGTP (3:1), GC Rich profile Previous stop point

31 Repetititve Sequence: Template C Defaults Stops

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

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

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

35 Repetititve Sequence: Template A BDT v3.1/dGTP (3:1) mix, GC Rich Profile Not always a fix!

36 Repetititve Sequence: Template A BDT v1.1, GC Rich Profile

37 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 PerformanceDifficult 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.-

38 Repetititve Sequence: Template A Amersham Sequence Finishing Kit

39 Instrument Related Anomolies: Solutions!

40 Drop-Out Peaks:

41 “Waterfall”: Results in Drop-out Peaks: Inflection point

42 Reinjection Helps: Drop out Peaks Gone

43 Premature Loss of Resolution:

44 Premature Loss of Resolution: Simply reinject sample

45 Loss of Resolution: In the middle Reinjection successful!

46 Timing of Reinjections: C fluorophore degrading Reinjections on Monday from a Friday run may need to be Set-up again

47 Chemistry: What’s best for sample or sequence type

48 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

49 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

50 Impact of Purification Method on Sequence Quality: Gel Purified Run 1 Run 2 PCR Product size= ~630BP

51 Impact of Purification Method on Sequence Quality: Enzyme Treated Same sample: Exo1/SAP treated PCR Product

52 DNA Sequencing Troubleshooting Resources:

53 How to Make a Query:

54 Comments to Query:

55 Another Great Resource: Nucleics

56 Nucleics: Possible Solutions

57 Other Resources: A Short List /troubleshooting.asp d=2256 “Focus on Plasmids”

58 Conclusions: Successful Sequencing is Dependent on: Template Quality Template Quantity Upfront Identification of Sample Type Upfront identification of Sequence context constraints

59 Acknowledgements: MaryLou Shane Romaica Omaruddin Meghan Brown VCC DNA Analysis Facility 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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