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Library amplification

Equipment and consumables
Amplification
Size-fractionation
Planning of sequencing

Solutions
Notes
 Comments
Illumina library / SOLiD librarySOLiD library / Illumina library



Equipment and consumables

  • analytical 2% HR TAE gel for PCR serial dilutions;


  • preparative agarose gels for fractionation of amplified library;


  • QIAquick spin-columns (~2 sets/libr.) to use after:
    • PCR;
    • fractionation of amplified library;


  • check equipment:
    • PCR machine;
    • vortex;
    • balance;
    • tabletop centrifuge & picofuge;
    • rotator at NT;


  • check, that you have:
    • powder-free gloves;
    • scalpels & beaker with water to wash scalpel;
    • low size-range DNA marker;
    • H2O;
    • LoBind tubes & tips;




Library amplification

  1. set up RT-PCR reaction (i) to determine the complexity of the library and number of cycles of large-scale PCR and (ii) to estimate preliminary the library quality by melting curve analysis;
    • prepare a mix (in ice-bath):
      total:20µl
      SYBRGreen buffer*, 10x2µl
      dNTPs with dUTP, 12.5mM2µl
      MgCl2, 25mM2µl
      #601**, 10µM0.8µl
      #603**, 10µM0.8µl
      DNA library1µl
      H2O11.4µl
      AmpliTaq Gold, 5u/µl0.1µl
      * SYBR Green PCR core reagents kit from ABI, #4304886;
      ** #601: PCR primer 2.1, #603: =adapter primer 2;
      use #611 & #603 primers for amplification of Mate-Paired library;


      PCR program:
      1. 95°C, 10:00
      2. 95°C, 0:15
      3. 65°C, 0:30
      4. 72°C, 0:30
      5. go to 2, 39 times
      6. melt curve analysis


    • if the number of cycles for the middle of logarifmic stage is "A", then:
      • rough estimated complexity is:
        ~0.1µg / (2A * size[kb] * 103 * 2 * 330g/mol) * NAvogadro[1/mol] = 1011/(2A * size[kb])
      • number of cycles of large-scale PCR is:
        N=A-log2{(Vsample[µl]/Vreaction[µl]) / (1µl / 20µl)}


    • large-scale PCR: mean size of PCR-fragments:
      number of cycles "A" in RT-PCR:
      number of cycles in large-scale PCR N=
      DNA
      #600, 20µM
      #601, 20µM
      2x Master Mix**
      H2O
      *#600: PCR primer 1.1, #601: PCR primer 2.1;
      ** 2x Phusion HF Master Mix (NEB, #F-531L)
      use "PE_PCR primer 1.1" & "PE_PCR primer 2.1" primers for amplification of Mate-Paired library;

      PCR program:
      1. 98°C, 0:30
      2. 98°C, 0:10
      3. 65°C, 0:30
      4. 72°C, 0:30
      5. go to 2, repeat:
      6. 4°C, hold


    • if PCR is performed in 30µl or less — load it on the gel without preliminary purification, otherwice QIAquick purification:
      • PB:

        • // total:
      • elute with 40µl EB (2x21µl)
    PCR-titration program:
  1. 72°C, 20:00 // nick-translation
  2. 95°C, 5:00
  3. 95°C, 0:15
  4. 62°C, 0:15
  5. 70°C, 1:00
  6. go to 2, 14 times
  7. 4°C, hold


  1. prepare PCR mix (in ice-bath):
    1 libr
    Invitrogen PCR SuperMix116µl
    PCR primer 1, 50µM2.4µl
    PCR primer 2, 50µM2.4µl
    total:120µl
  2. put 18µl into the first PCR tube and 10µl into the other 9 tubes (store in ice-bath);
  3. put 2µl of the library into the first tube, make 2x serial dilutions in tubes 1-9 and leave as 0-control the tube 10;
  4. two control tubes for primers:
    • tube 11: 10µl of Invitrogen PCR SuperMix & 0.5µl PCR primer 1, 50µM;
    • tube 12: 10µl of Invitrogen PCR SuperMix & 0.5µl PCR primer 2, 50µM;
  5. analyse on agarose gel;
  6. if desired band start to be visible from the tube "N" (~25ng):
    • rough estimated complexity is:
      {~25ng * 2N-1 / 215} / (size[kb] * 103 * 2 * 330g/mol) * NAvogadro[1/mol] = 3x105x2N/size[kb]
    • number of cycles of large-scale PCR is:
      15-log2{(Vsample[µl]/Vreaction[µl]) / (1µl / 10µl / 2N-1)} = 16 - N - log2{10*Vsample/Vreaction}


  7. large-scale PCR: mean size of PCR-fragments:
    number of tube "N" with appropriate dilution:
    number of cycles in large-scale PCR:
    DNA
    Invitrogen PCR SuperMix
    Pfu polymerase, 2.5u/µl
    PCR primer 1, 50µM
    PCR primer 2, 50µM
  8. QIAquick purification:
    • PB:

      • // total:
    • elute with 40µl EB (2x21µl);




Library size-fractionation

  • prepare 2% high-resolution agarose gel with individual wells (typical output is less then 3µg and may be loaded on one well);
  • for each library prepare labeled (#lib) tubes for size fractions;

  1. run the gel;
  2. cut out desired fragments as one block:
    LibraryOptimalPossible
    Fragment  
    Mate-Paired &500  
    Mate-Paired >500  
    LibraryOptimalPossible
    Fragment  
    Mate-Paired &500impossible
    Mate-Paired >500154-156bp
  3. QG-gel extraction, elute in 50µl (2x26µl):
      
    Gel block1g
    QG buffer4ml
    Isopropanol1.3ml
  4. determine concentration on Qubit;
  5. usually, concentration of library stock solution is in the range of few ng/µl. For storage it worth to add 1/10 of volume of 1% Tween 20. So, library will be in {EBQiagen with 0,1% Tween 20}. Try to minimize thawing/freezing cycles.




Planning of sequencing

  1. estimate complexity of amplified library:
    m[ng] / (2N x size[kb] x 1000 x 2 x 330[g/mol]) x NAvogadro = 109 x m[ng] /( 2N x size[kb])


  2. calculate a planned amount of expected sequencing reads (clustersbeads);

    ParameterValue
    N number of cycles of large-scale PCR
    mamount of purified DNA [ng]
    smean size of purified DNA [kb]
    library complexity [molecules]
    sequencingnumber of channels
    expected clusters
    molecules/clusters ratio
    sequencingfield type
    number of fields/glass
    expected beads
    molecules/beads ratio
    genome size [bp]
    read length [bases]
    M-P distance [bases]
    "0" for fragment sequencing
    mappable reads [%]
    outputraw bases
    mappable bases
    coveragereal
    virtual
    mappable reads [%]
    outputraw bases
    mappable bases
    coveragereal
    virtual


  3. compare complexity "COM" with a planned amount of sequencing reads "READS":
    • COM > 10x READS: everything is fine;
    • COM > READS: it is possible to sequence library, but significant number of reads will be a clone of each other;
    • COM < READS: it worth to prepare additional library (new library, or amplify the rest of material) or change a plans for sequencing;



    Notes

  • Library complexity is a number of independent molecules which containes the library. "Independent" means: obtained from the original DNA material, but not as a result of clonal amplification of the same fragment.


  • Planning:
    • when the initial amount of DNA is extremely low it worth to amplify library BEFORE secondary size-selection;
    • at least 1/3 of the library should be taken for large-scale amplification, because (i) there are still enough material to repeat amplification if something fails, (ii) if take less it will be unreasonable reduction of the library complexity;
    • it is necessary to calculate in advance, that complexity of the amplified library will be at least 10x higher, then planned number of sequencing reads;
    • library complexity may be significantly overestimated if calculate it from the amount of unamplified library, because it contains a lot of unamplifiable fragments, adapters, etc. The most reliable estimation is after gel-purification of amplified library;


  • Amplification:
    • prepare reaction mixture on ice;
    • do not use final 5-10 min 70-72°C extension. It does not help, but may induce problems because of exonuclease activity of proofreading polymerase;
    • start run PCR program, pause on 95°C, install tubes and resume pause;


  • It is important to avoid overamplification, because:
    • most important: overamplified DNA contains hetero-hybrids. It will result in hetero-amplified beads in emulsion PCR;
    • DNA-profile will be distorted during overamplification;




Second-generation sequencing
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