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Efficient Workflow for Sequencing Chromosome 12: Targeting BACs and Data Management

This document outlines a comprehensive protocol for sequencing Chromosome 12 using BACs. It includes steps for selecting suitable seed BACs, database checks and updates, and preparation of libraries for sequencing. The process involves optimizing the workflow for 4-6 BACs per week, including DNA preparation, library validation, and real-time PCR assessments. The key stages are outlined for managing runs and estimating required coverage, with a focus on finding contiguous BACs. Current progress highlights include several BACs ready for submission and the need for additional seed BACs for continuity.

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Efficient Workflow for Sequencing Chromosome 12: Targeting BACs and Data Management

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  1. Sequencing Chromosome 12

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

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

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

  5. Current status of the work: • … 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

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