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The ASAS Genomics Centre

The ASAS Genomics Centre. DNA Sequencing Facility Sanger Sequencing & Troubleshooting By Kristine Boxen ASAS monthly seminars. 15 July 2015. DNA Sequencing Facility Sanger Sequencing & Troubleshooting. 15 July 2015 Kristine Boxen. Automated DNA Sanger sequencing. Classic technology

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The ASAS Genomics Centre

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  1. The ASAS Genomics Centre DNA Sequencing Facility Sanger Sequencing & Troubleshooting By Kristine Boxen ASAS monthly seminars 15 July 2015

  2. DNA Sequencing Facility Sanger Sequencing & Troubleshooting 15 July 2015 Kristine Boxen

  3. Automated DNA Sanger sequencing • Classic technology • Developed in the 1970’s by Frederick Sanger • An important tool in molecular biology • We use a slight variation of the original method called chain-termination • DNA samples are sent to the Centre for processing • Controls are run with every tray and pre checked on a Nanodrop • Chemical reactions called “cycle sequencing” performed on a thermal cycler 9700 using BigDye Terminator v3.1 kits

  4. Automated DNA Sanger sequencing • Incorporation of four fluorescent dyes that bind specifically to the four nucleotides • DNA nucleotides A,C,G,T(adenine, cytosine, guanine and thymine) • Thousands of copies are produced that are different lengths • Samples purified using magnetic bead technology • Samples sequenced on a 3130XL Genetic Analyzer • DNA products separated by size (like agarose gel electrophoresis)

  5. Check samples on a Nanodrop first to measure: • Purity • Concentration Can accurately measure tiny volumes of sample as low as 1 μl. NanoDrop spectrophotometer 4

  6. BigDye Terminator (DT) chemistry d d A T P A G C C T CAG A d d C T P d d G T P A G C C T C A d d T T P AGCC T C A G C C T A G C C A G C A G A • Samples and primer are mixed with BigDye (enzyme, buffer, and nucleotides) We can do this for you, or you can do it yourself • DTs are performed in a single tube making this the method of choice • We use version 3.1 kits from ThermoFisher Scientific 5

  7. Cycle sequencing is linear amplification • Only one primer per reaction • Uses 50 °C annealing temperature • Primermelting temperature (TM) important GeneAmp PCR System 9700 BigDye 6 Diagram from http://www.appliedbiosystems.com/absite

  8. ABI PRISM™ 3130XL DNA Genetic Analyzer • The 3130XL is a workhorse! • Processes 192 samples per 24 hours • Produces up to 192,000 bases of sequence daily • Accuracy is approx 98% 7

  9. ABI PRISM™ 3130XL DNA Genetic Analyzer A look inside the 3130XL! Capillaries are Teflon-coated glass tubes and contain liquid polymer for separation (Replacing older-style slab polyacrylamide gels)

  10. Detection system Deoxy nucleotides A G C T Dideoxy nucleotides - AGTTCGTAGTCAAATGCAT AGTTCGTAGTCAAATGCA AGTTCGTAGTCAAATGC AGTTCGTAGTCAAATG AGTTCGTAGTCAAAT AGTTCGTAGTCAAA ARGON LASER AGTTCGTAGTCAA AGTTCGTAGTCA AGTTCGTAGTC CCD camera AGTTCGTAGT Filter Wheel Primer + AGTTCGTAG T .....TCAAGCATCAGTTTACGTAAGCT.... Template T C A A A T G C A T Four-dye, one-lane technology eliminates variability and increases sample throughput. 9

  11. Capillary image • The image above is a reconstruction or gel image of the data collected. • DNA fragments are separated by size. • The side picture shows a portion of the gel image enlarged where you can observe individual bases.

  12. Data output Data in electropherogram format shows peaks .abi file Free software sequence scanner v1.0 (Life Tech). Data in sequence file format shows text .seq file • 18 2 KNTUL2 16SBR.Seq • 1 TNGATTATCG TGAAAAACGA ACCTAATAGC GGCTGCACCA TTAGGATGTC • 51 CTGATCCAAC ATCGAGGTCG TAAACCCCCT TGTCGATATG GGCTCTAAAA • 101 GGGGATTGCG CTGTTATCCC TAGGGTAACT CGGTCCGTTG ATCGGCGTTG • 151 CCGGATCTTA TTGGTCAGAA TTTCTGTTTA TTAGAGCGGG AGCTCTAGGT • 201 TGTAGGAAAA GTATTCCCGT TCCACGTGGG GGTTTTGTAT TTCCCCGCGG

  13. Recommendations for successful sequencing Good quality sequence data, with sharp peaks, no N’s and high quality value scores * Check sample using a Nanodrop. Check for contaminants. * Check the primer melting temperature. Use a software programme eg Primer Express. * Submit samples at the correct concentrations. Use water not EDTA or TRIS. * Ask for us to add DMSO (dimethyl sulphate) if secondary structure problems. * For Ethanol ppte always use fresh stocks and ensure it is completely removed. Image from Applied Biosystems DNA Sequencing by Capillary Electrophoresis; 2nd Edition, section 8 Troubleshooting

  14. Troubleshooting Contaminants such as excess salt, RNA or protein in your sample: These cause the bands to be distorted and wide and the quality scores are low. The software has problems calling the right bases. A failed reaction: There are many Ns called as the reaction has failed due to template and/or primer problems with high background noise observed. The software cannot call the correct bases. Images from Applied Biosystems DNA Sequencing by Capillary Electrophoresis; 2nd Edition, section 8 Troubleshooting

  15. Troubleshooting – too much salt Effect of too much residual salt Across the gel image the sequence gradually deteriorates with increasing amounts of sodium chloride, NaCl. Lane 1 no added salt Lanes 2-11 salt added in 10mM increments from 10-100mM. Image from ABI Automated DNA Sequencing Chemistry Guide: Data Evaluation and troubleshooting 7-3

  16. Troubleshooting – too much ethanol Effect of too much residual ethanol Excess dye blobs are seen in samples in lane 1 & 2 with incomplete removal of ethanol. Image from ABI Automated DNA Sequencing Chemistry Guide: Data Evaluation and troubleshooting 7-3

  17. Troubleshooting – traces of marker pen Effect of marker pen written on top of trays Always write your name on the side of 96 well trays as problems with leakage. 12 Image from ABI Automated DNA Sequencing Chemistry Guide: Data Evaluation and troubleshooting 7-3

  18. Submitting your template Sample Requirements Please supply your templates as detailed below: My recommendation: Have your samples as close as possible to these concentrations. 12

  19. Current Charges for Sanger Sequencing Book through iLab at http://asas-centres.ilabsolutions.com My recommendation: For full service, 1/4 BigDye is the best option for plasmids. 12

  20. Monthly prizes sponsored by ThermoFisher Scientific Every month we award a prize selected from the best sequencing samples and most improved samples submitted in a given month. Will you be next? 12

  21. ` • Kristine Boxen • (09) 3737599 x 87293 • k.boxen@auckland.ac.nz • Special thanks to ThermoFisher Scientific for sponsorship of monthly prizes and use of images. • TNGATTATCGTGAAAAACGAACCTAATAGCGGCTGCAGACCATTAGGATTTCCTGATCCAAATCGAGGTCGTAGAAACCCCTTTCGTTATGGCTAAAAAGGGGATTGCGAGCTGTTATCCCTAGGGTAAACTCGGTCCGTTGATCGGCGTTGCCGGATCTTATTGGTCAGAATTTCTGTTAATTAAGGAGCGGGAGCTCTAGGTTGTAGGAAAAGTATCCCGTTCAAGGTGGGGTTTTGTATTCCCCGCGGTCGCCCCAACCAAAGACATAGAGTAGGGTTATAGGGGTTTTAACTTGAGGGCTACTTTGGTGTCTAAAGTTCTTAGGGTATCGTTATGGCTAAAAAGGGGATTGCGAGCTGTTATCCCTAGGGTAAACTCGGTCCGTTGATCGGCGTTGCCGGATCTTATTGGTCAGAATTTCTGTTAATTAAGGA

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