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Mate-Paired library preparation

Comments

Presented scheme illustrates preparation of mate-paired fragments from long DNA. This part of the protocol is identical for Illumina and SOLiD platforms.



• DNA digestion performed by ultrasound treatment (less than 1kb) or on Hydroshear (more than 1kb);



• (optional) Primary size selection decrease a total amount of DNA, less enzymes will be used on the next steps;



• End-repair reaction: three enzymes work simultaneously:
  • T4 DNA polymerase: digestion of 3' protruding ends;
  • Klenow DNA polymerase: extension of 3' recessive ends;
  • T4 PNK: phosphorilation of 5' ends & dephosphorilation of 3' ends, the best substrates are: 5' protruding and blunt ends;



• EcoP15I methylation inactivates EcoP15I sites in genomic DNA (restriction enzyme will not recognize methylated sites). Reaction is not wery efficient, it is better to perform ON;



• Eco CAP adaptor ligation ds adaptor contains EcoP15I recognition site; 100x molar excess to prevent ligation of DNA fragments with each other; it is better to keep ligation volume small, in large volumes circularization is preferable;



• Secondary size selection proper size; removes adaptor dimers;



• Circularization in presence of Internal adapter: should be done at some optimal concentration: too low: self-circularization; too high: long concatemers. Double-stranded DNA is rigid, circularization impossible if fragment length <500bp.
  
  
• EcoP15 I restriction

       5'···CAGCAG(N)25-27···3'
       3'···GTCGTC(N)27-29···5'
  	

  structure of MP-fragments is presented here




• Next steps Illumina are as in fragment library preparation:
  • end-repair;
  • A-tailing;
  • adaptor ligation;
  • selection of biotin-harboring fragments;
  • preamplification;
• Next steps SOLiD are as in fragment library preparation:
  • end-repair;
  • adaptor ligation;
  • nick-translation;
  • selection of biotin-harboring fragments;
  • preamplification;