Double-Ended Shotgun Sequencing of PA14 Daniel G. Lee 10/30/02

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).

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

PA14 Genomic DNA Prep 1.Original PA14 Rif R isolate from LGR ml culture. 3.Alkaline lysis, with: 1.CTAB ppt. 2.2 x Chloroform/Isoamyl Alcohol extraction. 3.3 x Phenol extraction. 4.1 x Phenol/Chloroform/Isoamyl Alcohol extraction. 5.1 x Chloroform/Isoamyl Alcohol extraction. 4.Isoamyl alcohol ppt. 5.Resupended in 5 ml mg/ml (5.87 mg total).

Workflow for Double-Ended Shotgun Sequencing of PA14 PA14 Genomic DNA Prep Plasmid preps of PA14 library. Linear amplification of inserts using dideoxy terminators Sequencing of amplification products Genome-wide alignment with PAO1 order contigs. identify gaps for sequence finishing. identify differences between PA14 and PAO1. Finishing and Annotation Contig assembly (PHRED and PHRAP) Shear PA14 DNA and Size Fractionate Ligate PA14 fragments into vector and transform E. coli

Plasmid Library Construction 1. Shear DNA using nitrogen (cleavage more random than sonication). 2. Fill-in to produce blunt ends. 3. Size fractionate on low- melt agarose gel. 1-3 kb fragments (700 bp). 3-7 kb fragments 4. Ligate. 5. Transform. 6. Pick colonies.

Plasmid Preps 1. O/N cultures in 96-well plates. 2. Freeze cell pellets. 3. Alkaline-lysis mini-preps in 96-well plates. 604/650 plates done. 4. Dry DNA pellets O/N. 5. Resuspend DNA in H 2 O. 6. Transfer to 384-well plate. QC by agarose gel.

Sequencing Reactions 1. Set up reaction mix: Labelled ddNTPs. dNTPs Buffer Taq Forward or reverse sequencing primer. 2. Aliquot rxn mix to 384- well PCR plate; freeze. 3. Add 3  l DNA to each well (or 3  l vector for “PCR control”). 4. “PCR”

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

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

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.

ccg-aatt-cggctttacg aatgccggcattacg tt--ggc-ttacgaccctttg-ggt t--ggc-ttacg--gactaggggtacca CCG-AATTCCGGCTTTACGACGACTTGGGGTACCA Contig Assembly

PA14 Sequencing: Current Status (as of 10/17/02) 1.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 2.Total number of “contigs”: < 2000? 1 contig ~ 44 kb 1 contig ~ 35 kb ~ 10 contigs > 25 kb ~ 12 contigs 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 > 50 kb.

Workflow for Double-Ended Shotgun Sequencing of PA14 PA14 Genomic DNA Prep Plasmid preps of PA14 library. Linear amplification of inserts using dideoxy terminators Sequencing of amplification products Genome-wide alignment with PAO1 order contigs. identify gaps for sequence finishing. identify differences between PA14 and PAO1. Finishing and Annotation Contig assembly (PHRED and PHRAP) Shear PA14 DNA and Size Fractionate Ligate PA14 fragments into vector and transform E. coli

Comparisons of PAO1 and PA14 A B A: gaps in regions corresponding to PAO1 sequence. B: gaps in PA14-specific regions.

1.Software Packages available from TIGR for Alignments. MUMmer 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. 2.DisplayMUMs for graphical analysis of MUMmer output. Tools for Genome-Wide Alignments of PAO1 and PA14

1. PROmer - amino acid translation in all 6 frames for protein/peptide alignments. Useful for comparative genome annotation. 2. Jonathan’s automated suite of annotation tools (Hrp project) Tools for Annotation of PA14

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).

Finishing A B Methods: PCR. Cosmids (Directed sequencing) (Altered rxn. Conditions) Considerations: Type of gap. Anticipated size of gap. Quality/nature of sequence at junction.

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)

Requirements for Publication 1.“Finished” PA14 sequence. a) Sufficient quality. b) No gaps? 2.Annotation. 3.Comparison to PAO1. 4. What else? a) Virulence data? b) Proteomics? c) Others?

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