Double-Ended Shotgun Sequencing of PA14

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Double-Ended Shotgun Sequencing of PA14

• Daniel G. Lee
• 10/30/02

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Determination of PA14 Genomic Sequence and
Whole-Genome Alignment with PAO1

• The complete genome of a related P. aeruginosa
  strain, PAO1, has been determined. The genome
  size is 6.2 Mb.
• PAO1 is less virulent than PA14 in almost all of
  our model hosts.
• PA14 contains additional DNA (sometimes large
  islands of DNA) not found in PAO1. Some of these
  additional genes may be responsible for the
  enhanced virulence of PA14.
• A complete PA14 genomic sequence will allow us
  to
• identify all the (DNA) differences between PA14
  and PAO1 (and later evaluate their contribution
  to virulence).
• Simplify the bioinformatics component of the PA14
  Unigene library.
• Design a microarray (whole-genome or
  PA14-specific).

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PA14 Sequencing - Outline

1. Sequencing workflow.
2. Finishing.
3. Annotation and whole-genome alignment.
4. Integration with PA14 insertion library.
5. Requirements for publication.

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PA14 Genomic DNA Prep

1. Original PA14 RifR isolate from LGR.
2. 500 ml culture.
3. Alkaline lysis, with
4. CTAB ppt.
5. 2 x Chloroform/Isoamyl Alcohol extraction.
6. 3 x Phenol extraction.
7. 1 x Phenol/Chloroform/Isoamyl Alcohol extraction.
8. 1 x Chloroform/Isoamyl Alcohol extraction.
9. Isoamyl alcohol ppt.
10. Resupended in 5 ml TE _at_1.174 mg/ml (5.87 mg
    total).

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Workflow for Double-Ended Shotgun Sequencing of
PA14
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Plasmid Library Construction

• Shear DNA using nitrogen (cleavage more random
  than sonication).
• Fill-in to produce blunt ends.
• Size fractionate on low-melt agarose gel.
• 1-3 kb fragments (700 bp).
• 3-7 kb fragments
• Ligate.
• Transform.
• Pick colonies.

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Plasmid Preps

• O/N cultures in 96-well plates.
• Freeze cell pellets.
• Alkaline-lysis mini-preps in 96-well plates.
• 604/650 plates done.
• Dry DNA pellets O/N.
• Resuspend DNA in H2O.
• Transfer to 384-well plate.
• QC by agarose gel.

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Sequencing Reactions

• Set up reaction mix
• Labelled ddNTPs.
• dNTPs
• Buffer
• Taq
• Forward or reverse sequencing primer.
• Aliquot rxn mix to 384-well PCR plate freeze.
• Add 3 ml DNA to each well (or 3 ml vector for
  PCR control).
• PCR

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DNA Sequencing

1. EtOH ppt. PCR reactions.
2. Dry.
3. Rssp. in H2O.
4. Add previously characterized PCR reactions as
   sequencing controls.
5. ABI Prism sequencers (liquid polymer capillary
   sequencer, 96 reactions at a time).

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• ABI sequencer outputs electropherograms.
• PHRED determines identity of base as well as
  quality score.

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Contig Assembly

1. Electropherograms.
2. PHRED - determines base identity and quality
   score for each position.
3. PHRAP - aligns sequences to assemble contigs,
   determines consensus sequence and quality score
   for each position.

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Contig Assembly
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PA14 SequencingCurrent Status (as of 10/17/02)

• Total amount of sequence 6 Mb (6.5 X coverage)
• 72,000 sequences (36,000 clones).
• 390 (out of 650) 96-well plates sequenced.
• 604 plates mini-prepped
• Total number of contigs lt 2000?
• 1 contig 44 kb
• 1 contig 35 kb
• 10 contigs gt 25 kb
• 12 contigs 20-25 kb
• most contigs are 5-10 kb.
• Library consists of 1 kb inserts (current plans
  to introduce a library of 3-6 kb inserts).

As of 10/21/02, one contig 73 kb, many gt 50 kb.
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Workflow for Double-Ended Shotgun Sequencing of
PA14
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Comparisons of PAO1 and PA14
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Tools for Genome-Wide Alignments of PAO1 and PA14

• Software Packages available from TIGR for
  Alignments.
• MUMmer 2.1 - aligns MUMs (maximal unique matches)
  for two input sequences (two 3-4 Mb genomes
  aligned in under 30 seconds, using less than 100
  Mb of memory, on a typical desktop computer
  running Unix/Linux).
• NUCmer - alignments of highly similar sequences
  that may have large rearrangements (i.e. -- a
  group of assembly contigs vs. a complete genome).
• PROmer - amino acid translation in all 6 frames
  for protein/peptide alignments. Useful for
  comparative genome annotation.
• DisplayMUMs for graphical analysis of MUMmer
  output.

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Tools for Annotation of PA14

1. PROmer - amino acid translation in all 6 frames
   for protein/peptide alignments. Useful for
   comparative genome annotation.
2. Jonathans automated suite of annotation tools
   (Hrp project)

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Approaches for Finishing

1. PCR amplification and directed sequencing of
   gapped regions.
2. Isolation of cosmid clones spanning gaps,
   subcloning, sequencing of subclones (using
   universal primers).
3. (Direct genomic sequencing).
4. (Altering sequencing reaction conditions for
   regions that are difficult to sequence through).

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Finishing

• Methods
• PCR.
• Cosmids
• (Directed sequencing)
• (Altered rxn. Conditions)

• Considerations
• Type of gap.
• Anticipated size of gap.
• Quality/nature of sequence at junction.

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Integration with PA14 Unigene Library
Subject for BLAST Searches Verify PA14 Sequences (close gaps, improve sequence quality) Assign Insert Coordinates Assign Identity of Disrupted ORF
PAO1
PA14 Contigs
Finished PA14 Sequence (annotated)
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Requirements for Publication

• Finished PA14 sequence.
• Sufficient quality.
• No gaps?
• Annotation.
• Comparison to PAO1.

• 4. What else?
• Virulence data?
• Proteomics?
• Others?

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ACKNOWLEDGEMENTS

• MGH
• N. Liberati
• S. Miyata
• J. Urbach
• F. Ausubel
• X. He
• M. Saucier
• L. Rahme

Harvard Partners Genome Center K.
Montgomery G. Grills L. Li W. Brown J. Decker R.
Elliot L. Gendal K. Osborn A. Parerra C. Xi P.
Juels R. Kucherlapati