Strategies for de novo DNA sequencing Anna Blomstergren Royal Institute of Technology Department of Biotechnology Stockholm 2003 © Anna Blomstergren Department of Biotechnology Royal Institute of Technology Alba Nova University Center SE-106 91 Stockholm Sweden Printed at Universitetsservice US AB Box 700 14 SE-100 44 Stockholm Sweden ISBN 91-7283-608-3 Anna Blomstergren 2003. Strategies for de novo DNA sequencing. Department of Biotechnology, Albanova University Center, Royal Institute of Technology, Stockholm, Sweden. ISBN 91-7283-608-3 Abstract The development of improved sequencing technologies has enabled the field of genomics to evolve. Handling and sequencing of large numbers of samples require an increased level of automation in order to obtain high throughput and consistent quality. Improved performance has lead to the sequencing of numerous microbial genomes and a few genomes from higher eukaryotes and the benefits of comparing sequences both within and between species are now becoming apparent. This thesis describes both the development of automated purification methods for DNA, mainly sequencing products, and a comparative sequencing project. The initially developed purification technique is dedicated to single stranded DNA containing vector specific sequences, exemplified by sequencing products. Specific capture probes coupled to paramagnetic beads together with stabilizing modular probes hybridize to the single stranded target. After washing, the purified DNA can be released using water. When sequencing products are purified they can be directly loaded onto a capillary sequencer after elution. Since this approach is specific it can be applied to multiplex sequencing products. Different probe sets are used for each sequencing product and the purifications are performed iteratively. The second purification approach, which can be applied to a number of different targets, involves biotinylated PCR products or sequencing products that are captured using streptavidin beads. This has been described previously, but here the interaction between streptavidin and biotin can be disrupted without denaturing the streptavidin, enabling the re-use of the beads. The relatively mild elution conditions also enable the release of sensitive biotinylated molecules. Another project described in this thesis is the comparative sequencing of the 40 kb cag pathogenicity island (PAI) in four Helicobacter pylori strains. The results included the discovery of a novel gene, present in approximately half of the Swedish strains tested. In addition, one of the strains contained a major rearrangement dividing the cag PAI into two parts. Further, information about the variability of different genes could be obtained. © Anna Blomstergren, 2003 Keywords: DNA sequencing, DNA purification, automation, solid-phase, streptavidin, biotin, modular probes, Helicobacter pylori, cag PAI. What’s past is prologue... William Shakespeare LIST OF PUBLICATIONS This thesis is based on the following manuscripts, which in the text will be referred to by their roman numerals: I. Anna Blomstergren, Deirdre O’Meara, Morten Lukacs, Mathias Uhlén and Joakim Lundeberg (2000), Cooperative oligonucleotides in purification of cycle sequencing products, Biotechniques 29(2), 352- 363. II. Anna Blomstergren, Anders Holmberg, Morten Lukacs and Joakim Lundeberg (2003), Automated purification of multiplex cycle sequencing products suitable for capillary electrophoresis, submitted. III. Anders Holmberg, Anna Blomstergren, Morten Lukacs, Joakim Lundeberg and Mathias Uhlén (2003), Reversible biotin-streptavidin interaction with release using non-ionic aqueous solutions at elevated temperatures, submitted. IV. Anna Blomstergren, Annelie Lundin, Christina Nilsson, Lars Engstrand, Joakim Lundeberg (2003), Comparative analysis of the complete cag pathogenicity island sequence in four Helicobacter pylori isolates, Gene, in press. TABLE OF CONTENTS INTRODUCTION ......................................................................1 1 Historical background ..........................................................2 2 Structure and properties of DNA ........................................3 3 Sequencing methods..............................................................5 3.1 Sanger sequencing...................................................................................... 5 3.2 Maxam and Gilbert..................................................................................... 8 3.3 Pyrosequencing .......................................................................................... 8 3.4 Single molecule sequencing ..................................................................... 11 3.5 Sequencing by hybridization (SBH)......................................................... 12 4 Major strategies for genome and transcript sequencing.13 4.1 Sequencing of genomic DNA................................................................... 13 4.1.1 Complete genome sequencing ......................................................... 13 4.1.1.1 Clone-by-clone shotgun sequencing ....................................... 14 4.1.1.2 Whole-genome shotgun sequencing ....................................... 16 4.1.1.3 Creating shotgun libraries ....................................................... 18 4.1.1.4 Gap closure ............................................................................. 20 4.1.2 Sequencing specific regions of the genome..................................... 21 4.1.2.1 Primer walking........................................................................ 21 4.1.2.2 Directed PCR amplification .................................................... 22 4.2 Sequencing of transcripts (cDNA) ........................................................... 23 5 Sequencing technologies .....................................................24 5.1 Amplification of templates....................................................................... 24 5.1.1 Cultivation of plasmids and M13..................................................... 24 5.1.2 PCR amplification ........................................................................... 26 5.1.3 Rolling circle amplification ............................................................. 27 5.2 Generation of Sanger fragments............................................................... 28 5.3 Purification of sequencing products ......................................................... 29 5.3.1 Precipitation techniques................................................................... 29 5.3.2 Filtration methods............................................................................ 30 5.3.3 Magnetic bead techniques................................................................ 31 5.3.3.1 Hybridization based techniques .............................................. 31 5.3.3.2 Streptavidin-biotin .................................................................. 31 5.3.3.3 Unspecific capture of DNA..................................................... 32 5.3.3.4 Capture of dideoxynucleotides................................................ 33 5.4 Separation and detection........................................................................... 33 5.4.1 Electrophoresis ................................................................................ 33 5.4.2 Mass spectrometry ........................................................................... 34 5.5 Automation............................................................................................... 35 6 Data analysis ........................................................................35 6.1 Quality assessment ................................................................................... 36 6.2 Assembly.................................................................................................. 36 6.3 Comparing sequences............................................................................... 37 6.4 Annotation................................................................................................ 38 PRESENT INVESTIGATIONS..............................................41 7 Solid-phase purification of DNA........................................42 7.1 Hybridization based technique for the purification of cycle sequencing products................................................................................................. 42 7.2 Purification using the biotin-streptavidin system ..................................... 44 7.3 Comparison of the two assays .................................................................. 47 8 Comparative sequencing of H. pylori ................................47 8.1 Helicobacter pylori................................................................................... 47 8.2 The cag pathogenicity island.................................................................... 49 8.3 Strategy for comparing the cag PAI in four clinical isolates of H. pylori 51 8.4 Nucleotide and amino acid sequence variation ........................................ 53 8.5 Major rearrangements............................................................................... 53 9 Concluding remarks............................................................55 10 Acknowledgments................................................................56 11 References ............................................................................58 Original Papers I-IV ................................................................73 Strategies for de novo