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Fragment library preparation

Equipment and consumables
DNA preparation
Primary size-selection
End repair
A-tailing
Adaptors ligation
Ligation of the P1 & P2 adaptors
Secondary size-selection
UDGase treatment

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



Equipment and consumables

  • analytical agarose gels for:
    • DNA analysis: 1% TAE;
    • shearing test: 2% HR TAE;


  • preparative agarose gels for:
    • primary fractionation;
    • secondary fractionation;


  • QIAquick spin-columns (~5 sets/libr.) to use after:
    • primary gel-fractionation (may be several columns);
    • end reparing;
    • A-tailing;
    • adaptor ligation;
    • nick-translation;
    • secondary gel-fractionation;


  • check equipment:
    • PCR machine;
    • vortex;
    • Hielscher UTR200 sonicator;
    • 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;




DNA preparation

  • prepare analytical agarose gels for (i) DNA analysis & (ii) sharing test;

  1. check DNA on 1% TAE gel (~0.1µg aliquote):
    • integrity;
    • quantity (use plasmid DNA as a control);
    • RNA contamination;
  2. if necessary, perform RNase digestion;
  3. share DNA on Hielscher UTR200;
  4. check shearing by electrophoresis in 2% high-resolution TAE gel;
  5. if necessary, concentrate DNA:
  6. dilute DNA in 30µl of EB·;



Primary size-selection (optional)

    primary size-selection helps:
  • reduce amount of DNA, so less enzymes & adapters will be spended for library preparation;
  • remove low-molecular weight contamination, which may exhaust enzymes & adapters;


    • skip this stage if library is prepared from the small amount of DNA


  • prepare 2% high-resolution agarose gel with combined wells (up to 3µg for well and 1µg for gap);
  • for each library prepare labeled (#lib, #lib_a, #lib_b, ...) tubes for size fractions;

  1. run the gel;
  2. cut out 50-150bp as one block (#lib) and larger fragments with ~100bp intervals (#lib_a, #lib_b, ...);
  3. freeze reserve bands in liquid nitrogen & store at -20°C;
  4. 50-150bp band: QG-gel extraction, elute in 50µl (2x26µl):
    6.3ml
    Gel block1g
    QG buffer4ml
    Isopropanol1.3ml
  5. determine DNA concentration on Qubit



End repair (home-made set)

  • prepare 20°C water bath in foam plastic box;

  1. combine in 1.7ml tube
     
    sheared DNA50µl
    10x T4 DNA ligase buff. with 10mM ATP (NEB)1x
    dNTP's mix, 25mM0.25mM
    T4 DNA pol., 3u/µl0.1u/µl
    Klenow DNA pol., 5u/µl0.03u/µl
    T4 PNK, 10u/µl0.3u/µl
    H2OmQ
  2. 30min 20°C in water bath;
  3. QIAquick-QG purification:
    • QG:
    • Iso:

      • // total:
    • elute with 34µl EB (2x18µl);


A-tailing

  • prepare 37°C water bath in foam plastic box;

  1. combine in 1.7ml tube:
     
    DNA34µl
    10x Klenow buffer1x
    dATP, 1mM0.2mM
    Klenow 3'->5' exo minus, 5u/µl 
    H2OmQ
  2. 30min 37°C in water bath;
  3. MinElute-QG purification:
    • QG:
    • Iso:

      • // total:
    • elute with 10µl EB;


Adapters ligation

  • prepare 20°C water bath in foam plastic box;

  1. combine in the tube with DNA (10x molar excess of adapters):
     
    DNA10µl
    Adapter mix, 13pmol/µl10x excess
    2x DNA ligase buffer1x
    DNA ligase ???u/µl 
  2. 15 min at 20°C in water bath;
  3. QIAquick purification:
    • PB:

      • // total:
    • elute with 30µl EB (2x16µl);



Ligation of P1 & P2 adapters

  • prepare 25°C water bath in foam plastic box;

  1. combine in the tube with DNA (30x molar excess of adapters):
     
    DNA34µl
    P1 (ds) adapter, 50pmol/µl30x excess
    P2 (ds) adapter, 50pmol/µl30x excess
    2X NEB Quick Ligase buffer1x
    40x NEB Quick Ligase1x
  2. 20 min at 25°C in water bath;
  3. QIAquick purification:
    • PB:

      • // total:
    • elute with 34µl EB (2x18µl);



Secondary size-selection

    purpose:
  • when the initial amount of DNA is extremely low it worth to amplify libraryto amplify library BEFORE size-selection;
  • remove adapters, which may induce high background during amplification;
  • select optimal size range for sequencing: 60-120bp w/o adapters; 190-180 with adapters;


    • when the initial amount of DNA is extremely low it worth to take fragments of all "sequencible" sizes: 110-700770-250bp (with adapters).


  • prepare 2% high-resolution agarose gel with combined wells (up to 3µg for well and 1µg for gap);
  • for each library prepare labeled (#lib & #lib_R) tubes for size fractions;


  1. run the gel;
  2. cut out 190-180bp as one block (#lib) and larger fragments as reserve block (#lib_R);
  3. freeze reserve bands in liquid nitrogen & store at -20°C;
  4. 190-180bp band: QG-gel extraction, elute in 50µl (2x26µl):
      
    Gel block1g
    QG buffer4ml
    Isopropanol1.3ml
  5. determine concentration on Qubit
  6. store at -20°C;




UDGase treatment

NB! only for Uridine-containing libraries

  1. add 5.7µ of 10 UNG buffer;
  2. add 1u UNG per 1µg of DNA (but not less, thuro;l);
  3. 30min at 37°C;
  4. store at -20°C;



This is unamplified library. Before Cluster preparationemulsion PCR it is necessary to perform Library amplificationLibrary amplification.




    Solutions

    UDG Reaction Buffer, 10x

    10x UDG buffer; store at -20°C
      1ml5ml
    Tris-HCl, pH8.0200mM1M200µl1ml
    Dithiothreitol10mM100mM100µl500µl
    EDTA10mM0.5M20µl100µl
    H2O mQ680µl3.4ml







    Notes

  • Short AT-rich DNA fragments are extremely sensitive to denaturation. It is necessary to avoid denaturating factors: (i) heating; (ii) low-salt buffers; (iii) alkaline pH.


  • DNA fractionation on agarose gel:
    • best way of running the gel: 100V for the first 5 min, 30V for the next 25 min, then 100-120V;
    • one empty well should be between marker and sample;
    • marker should occupy the whole well. It permits to estimate the band inclination (normally, upper side runs slower, than lower side);
    • do not load samples with extremely different amount of DNA on the same gel;
    • for sensitive samples use "1 gel   =>   1 sample";


  • Size-selection:
    • when there are enough DNA, it worth to limit sequencing fragments by 60-120bp, because it helps to increase the density of the clusters (clusters are smaller, more homogenious by size and intensity)to make amplification on beads more homogenious;
    • if the amount of starting material is limited, it worth to take all sizes, which may be amplified and sequenced (Illumina: up to 600bp w/o adaptorsSOLiD: up to 250bp w/o adaptors);


  • Low Range Ultra Agarose is for the analysis of short fragments:
    • it is not as rigid as the normal agarose. It is important, that agarose gel is prepared in advance and cooled at 4°C for at least 1h before the experiment. Otherwise, leakage is possible;
    • it worth to check the wells after removing of combs by loading ~2µl of 1x Xylene cyanol bufer;
    • OrangeG dye runs as a fragment of ~10bp on 2% HR gel;


  • Good interruption points (store at -20°C):
    • after DNA shearing;
    • after primary size selection;
    • after adaptor ligation;
    • after secondary size-selection;

      • it would be better do not interrupt process from "end repair" to "adaptor ligation";






Second-generation sequencing
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