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 –
Download ReportTranscript 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 –
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 • 60 ° 4 min 25x 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 Product PCR 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 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 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 Thermal Profiles: RNAi AutoSeq1 GC Rich 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 • 60 ° 4 min C.S. Rxn conditions PCR 10ng 3.2 pmol Primer 1/8 dilution BDv3.1 30x AB Recommendation: 96c 1min, 25X of 96c 10s, 50c 4min Chemistry BDT V1.1 Bisulfite Sequencing: Default Set-up and BiSulSeq Profile “Vish Profile” Cosmids: BacDSRG 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, 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 Difficult Template Type Kit Performance >20bp polynucleotide Repeat Difficult Template Type Kit Performance 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. - Requires small sample size (1ng): generates ~1ug Use 2-4 ul of 5ul RXN volume Repetititve Sequence: Template A Amersham 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 Raw Q20=705 AB BDT v3.1-end 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?i d=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: VCC DNA Analysis Facility MaryLou Shane Romaica Omaruddin Meghan Brown 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