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Fragment library preparation

Comments

Illumina

SOLiD



Protocols are quite similar.
• DNA digestion performed by ultrasound treatment or by hydrodinamic forces;



• 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;



• A-tailing in Illumina protocole prevents self ligation and ligation of one fragment to another. It seems, that A-tailing reaction has a prefernce to some end-nucleotides;



• Adaptor ligation
  • Illumina: one Y-shape adapter is used. One oligonucleotide of Y-adapter ligates to 5'-ends of all DNA molecules, another oligonucleotide — to 3'-ends. Advantages of such approach:
    • 100% of DNA fragments acquire two diffrent oligonucleotides on 5' and 3' ends;
    • definite orientation of adaptors;
    • Y-adapter is phosphorilated, so nick-translation is not necessary.
    Y-adaptor has 3' T-protruding nucleotide on the double-stranded stem. It prevents self-ligation of adaptors. A single-stranded 3'-end of Y-adaptor is blocked for extension and ligation to prevent background during ligation and PCR.
  • SOLiD: two different double-stranded adaptors. For combinatorial reasons only 1/2 of molecules would have different adaptors on the ends. Contrary to Illumina approach, adaptor may be in both orientations. Blunt-ends of adaptors are dephosphorilated to prevent adaptor-adaptor ligation;



• Nick translation have to be done in SOLiD protocol because: adaptors was dephosphorilated, so they covalently attache only to 5'-ends of DNA fragments. Nick-translation can't happene on PCR-stage, because polymerase is heat-activated (fragment denaturated before nick-translation);



• Size-selection remove adaptor dimers. Fragments with similar length gives more uniform clonal amplification.



• Preamplification increases the number of library molecules. On the clonal amplification step not each molecule is converted to the cluster (Illumina) or bead (SOLiD). The problem, related with preamplification is, that the same molecule may be sequenced several times (this reduces efficiency of sequencing).
  • SOLiD. PCR-1 primer is shorter than adaptor. DNA fragments with P1-P1 adaptors do not amplify because of suppression of PCR by panhandle structure. DNA fragments with P2-P2 adaptors amplified, but they have no chance to stick to paramagnetic beads during ePCR. SOLiD protocol is relatively sensitive to overamplification, because library is loaded in emulsion in double-stranded form;
  • Illumina. Preamplification is obligatory, because one of the bridge-amplification sites is introduced during preamplification (Y-adaptor is shorter, than one of the PCR-primers). Illumina protocol is relatively unsensitive to overamplification, because library is loaded on flowcell in a single-stranded form.