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Troubleshooting DNA Sequences:

. Biodesktop Workshops:. Scheduled: 300 HSRFJan. 29th at 10-11AM (Friday)Rama KocherlakotaBasics of submitting ordersRetrieving dataCreating a team to share dataOpportunity to provide input. . SESSION OUTLINE:. Guidelines: Generic Set up and ProfilesImpact of Template and primer ratio???Suggestions for different sample types and Sequence Context: Chemistry, Profile, AdditivesSample types: PCR, Plasmid, BAC, Cosmid, RNAi construct, Bisulfite treated gDNA, gDNASequence Conte9458

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Troubleshooting DNA Sequences:

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    1. Troubleshooting DNA Sequences: Guidelines and Suggestions Data sets submitted US 18 France 2 Belgium 4 Germany 1 Italy 1 Brazil 4 Japan 3 23/26= 88%Data sets submitted US 18 France 2 Belgium 4 Germany 1 Italy 1 Brazil 4 Japan 3 23/26= 88%

    2.

    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 and Processing Anomolies: Retreiving data off the biodesktop: How, What….. Sample Purification prior to Sequencing Troubleshooting Resources

    4. 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

    5. Required Template Concentrations:

    6. 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

    7. Primer Titration: Plasmid

    8. Template Titration: Plasmid

    9. Template Titration: PCR Product

    10. Template Concentration: It Can Matter Submitting template at the incorrect concentration

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

    12. Sequencing RNAi Constructs: Auto Seq1 Profile, Chemistry (Default)

    13. RNAi Construct: GC Rich Profile, 5% DMSO

    14. RNAi Construct: Modified RXN Set-up, RNAi Profile

    15. RNAi Construct: testing to reduce costs AutoSeq1 (Default):

    16. RNAi Construct: Default Chemistry, RNAi Profile:

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

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

    19. RNAi Construct: LOR scores for three different approaches

    20. BAC’s, Cosmid’s, 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

    21. BAC’s: Default set-up and AutoSeq1

    22. BAC’s: Modified Set-up, BAC profile

    23. BAC Sequencing: LOR scores for two different approaches

    24. gDNA Sequencing: Not as easy as you may think!

    25. Bisulfite Sequencing: Sequencing methylated gDNA Default Set-up and Profile

    26. 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

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

    28. 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

    29. Sequence Context Constraints: GC rich, Homoploymeric runs, Repetitive sequence (STR)

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

    31. Run of G’s: dGTP Chemistry, AutoSeq1 profile

    32. GC Rich Template: Generic Set up, AutoSeq1 Profile

    33. GC Rich Template: BDTv3.1/dGTP (3:1), GC Rich profile

    34. Repetititve Sequence: Template C Defaults

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

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

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

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

    39. Repetititve Sequence: Template A BDT v1.1, Default Profile

    40. Homopolymeric runs:

    41. 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.

    42. What is Phred? Basecaller (used by genome centers) Provides quality score for each base Generates two files

    43. Need to look at the Chromatogram!! (.AB1 File)

    44. Instrument and Processing Related Anomolies: AND Email notifications

    45. Bad Injection message: One 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.

    46. Loss of Resolution: In the middle

    47. Timing of Reinjections:

    48. 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.

    49. Spike message: One 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.

    50. Template Purification: What’s best for sample or sequence type

    51. Impact of Purification Method on Sequence Quality: Gel Purified

    52. Impact of Purification Method on Sequence Quality: Enzyme Treated

    53. 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

    54. 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

    55. Acknowledgements:

    56. VersaDoc 4000MP:

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