Sequencing Chromosome 12

runs db (blast) SOL dbrelational db Choice of suitable seed BACs Running 96 samples For each BAC check db update db update dbcheck db Sequencing to ~10x coverage must decide if OK overlapping to other BAC? % of useful clones (self ligated, coli, etc…) save information Estimate no. of required runs update db Pre-finished data Assembly Pre-finishing update db Finished data Finishing Finding contiguous BACs update db Picking 2-3x384 clones update db

BAC preparation protocol, optimized for 4-6 BAC per week Step 1 (DNA prep)Step 2 (library prep)Step 3 (validation) Growing 1 litre culture Standard 1,2,3 preparation Qiagen silica gel column CsCl gradient Final purifcation Real time PCR for assessing genomic contamination Validated DNA prep DNA shearing /end-repair Making the library (see next slide) Picking from liquid culture ~ 75 clones /384 wells (1/5) (typically 15x384 dishes /BAC) Sequencing to ~ 10x coverage Evaluate quality Store in aliquots at -80 Use one aliquot for titrating the library and to produce the first 96 runs Library ready for sequencing Step 4 (sequencing)

CCANCACA GGTN NTGG ACACNACC NNNNNNNNNNNNN...NNNNNNNNNNNNNN Blunt-end DNA fragment Shotgun BAC library Adaptor ligation GCGGCCGC NNNNNNNNNNNNN...NNNNNNNNNNNNNN GCGGCCGCTGTG GTGTCGCCGGCG NNNNNNNNNNNNN...NNNNNNNNNNNNNN CGCCGGCG Vector ligation

… still selecting seed BACs …so far … 10+3 BACs have been selected as seeds… 2 BACs are ready for submission as finished (validate.pl) 1 BAC is ready for submission as pre-finished (phase2) 3 BACs are in progress, should be ready within 2-3 weeks In order to proceed we need more seed BACs and we have to work out a fast way to obtain the contiguous BACs Current status of the work: