Fragment library preparation

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Illumina / SOLiDSOLiD / Illumina

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NB! pre-PCR operation

Scheme of fragment library preparation

NB! protocol is for >50ng of starting DNA. There is a special adaptation for <50ng: Fragment library preparation from small amounts of DNA
NB! use exclusively LoBind tubes and filtered tips for library preparation
NB! protocol for SOLiD should be checked

This protocol is for preparation of unamplified library. Before Cluster preparationemulsion PCR it is necessary to perform Library amplification.


[править] Equipment and consumables

check, that you have:

» scalpels;
» low size-range DNA marker;
» analytical agarose gels for:
DNA analysis: 1% TAE
shearing test: 2% High Resolution TAE
» preparative agarose gels for:
primary fractionation
secondary fractionation
» QIAquick spin-columns to use after:
primary gel-fractionation (may be several columns)
end repair
adaptor ligation
nick-translation
secondary gel-fractionation
» MinElute column to use after:
A-tailing
» foam plastic box for water bath
» Hielscher UTR200 or Covaris S2 sonicator
» rotator at room temperature




[править] DNA preparation

prepare analytical agarose gels for:

  • DNA analysis
  • shearing test


  1. check DNA on 1% TAE gel (~0.1µg aliquot):
    • integrity[1];
    • quantity[2];
    • RNA contamination;
  2. if necessary, perform RNase digestion[3];
  3. make shure, that you know desirable size range for fragments;
  4. sheare DNA on Hielscher UTR200[4] or Covaris S2 (later is preferable);
  5. check shearing by electrophoresis in 2% high-resolution TAE gel;
  6. if necessary, concentrate DNA:
    • for <10µg: QIAquick purification (3x QG, 1x Isopropanol);
    • for > 8µg of DNA: NaCl/EtOH precipitation;
  7. dilute DNA in 30µl of EB




[править] Primary size-selection[5]

Primary size-selection is obligatory for search of rear rearrangements, trans-splicing, etc, because it is prevents formation of heteroduplexes[6]. In other cases it is optional, skip if library is prepared from <0.5µg of DNA


prepare:

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


  1. run the gel (120V per 28cm);
  2. cut out main band as one block (#lib) and larger fragments with ~100bp intervals (#lib_a, #lib_b):
    ExperimentMaterialmain bandreserve bands
    sequencing de novo,
    small genomes (bacteria, etc.)
       
    sequencing de novo,
    large genomes (eucariotes, etc.)
       
    genome resequencing; PE 2x50bp, PE 2x75bp[7]10µg<DNA160-230"230-330", "330-430"
    50ng<DNA<10µg; PE 2x50bp[8]130-200"200-300", "300-400"
    50ng<DNA<10µg; PE 2x75bp[9]160-250"250-350", "350-450"
    DNA<50ng; PE 2x50bp, PE 2x75bplarge size range should be taken; it is impossible to prevent heteroduplex formation. So, it is necessary to sacrifice either "heteroduplex prevention" or "maximal library complexity"
    exome sequencing5µg<DNA, PE 2x75bp160-230"230-330", "330-430"

    * NB! this table for "randomly sheared DNA". If DNA has predetermined sizes (e.g., oligonucleotide library) particular these sizes should de sequenced

  3. freeze reserve bands in liquid nitrogen and store at -20°C;
  4. main band: QG-gel extraction, elute in 50µl EB (2x26µl):
    volume6.3ml
    Gel block1xV1g
    QG buffer4xV4ml
    Isopropanol1.3xV1.3ml

  5. determine DNA concentration on Qubit:
    » when start from >5µg: BR kit
    » when start from <5µg: HS kit




[править] End repair (home-made set)

  • prepare 20°C water bath in foam plastic box[10]


  1. combine in 1.7ml tube
     
    sheared DNA
    H2OmQ
    ER buffer, 9.08x1x
    ER enzyme mix, 12x~1x

  2. 30min 20°C in water bath;
  3. QIAquick-QG purification:
    DNA < 200bpDNA > 200bp

    volume630µl
    DNA1xV100µl
    QG buffer4xV400µl
    Isopropanol1.3xV130µl

    volume500µl
    DNA1xV100µl
    QG buffer3xV300µl
    Isopropanol1xV100µl

    » elute with 34µl EB (2x18µl)




[править] A-tailing (only Illumina)

  • prepare 37°C water bath in foam plastic box


  1. combine in 1.7ml tube:
     
    DNA
    A-tailing buffer, 8.33x1x
    H2OmQ
    Klenow 3'->5' exo minus, 5u/µl 

  2. 30min 37°C in water bath;
  3. MinElute-QG purification (autocalculation table for "End repair"):
    » elute with 10µl EB


[править] Adapters ligation

  • prepare 20°C water bath in foam plastic box
  • combine DNA and adaptors before adding ligation buffer, let stand for 1 min


  1. combine in the tube with DNA (1030x molar excess of adapters):
    » combine DNA and adaptors before adding ligation buffer, mix and let stand for 1 min

     
    DNA10µl
    Adapter mix*, 13pmol/µl10x excess
    2x DNA ligase buffer1x
    DNA ligase ???u/µl 
    * Adapter mix:
    » one-side sequencing: genomic adapter
    » both-sides sequencing: PE genomic adapter
    it is better always to prepare library for both-sides sequencing (PE genomic adapter). It may be also sequenced from one side.

     
    DNA34µl
    P1 (ds) adapter, 50pmol/µl30x excess
    P2 (ds) adapter, 50pmol/µl30x excess
    2X NEB Quick Ligase buffer1x
    40x NEB Quick Ligase1x

  2. mix carefully, 30 min at 20°C in water bath;
  3. QIAquick purification:
    volume600µl
    DNA1xV100µl
    PB buffer5xV500µl

    » elute with 30µl EB (2x16µl)




[править] Secondary size-selection[11]

  • select optimal size range for sequencing: 60-120bp w/o adapters; 120-180 with adapters
  • when the initial amount of DNA is extremely low (<50ng) it is worth to
» amplify library BEFORE size-selection
» take fragments of all "sequensable" sizes. Illumina: 110-700SOLiD: 100-250bp (with adapters).


prepare:

  • 2% high-resolution agarose gel with combined wells (up to 3µg for well and 1µg for gap)
  • for each library — labeled (#lib and #lib_R1, #lib_R2) tubes for size fractions


  1. run the gel (120V per 28cm);
  2. cut out main band as one block (#lib) and lreserve blocks (#lib_R1, #lib_R2):
    sequencing taskmain bandreserve bands
    single-read (50bp), enough DNA (>500ng) 120-220"220-270", "270-320"
    PE (2x50bp), genome seq.200-250"250-350", "350-450"
    PE (2x75bp), genome seq.270-320"320-400", "400-470"
    PE (2x50bp), RNA seq.200-220"220-320", "320-420"
    table should be corrected
  3. cut out main band as one block (#lib) and larger fragments as reserve blocks (#lib_R1, #lib_R2);
  4. freeze reserve bands in liquid nitrogen and store at -20°C;
  5. main band: QG-gel extraction, elute in 30µl (2x16µl):
    volume500µl
    DNA1xV100µl
    QG buffer3xV300µl
    Isopropanol1xV100µl

  6. determine concentration on Qubit (HS kit)
  7. store at -20°C




[править] UDGase treatment

NB! only for Uridine-containing libraries


  1. add 3.4µl of 10x UDG buffer;
  2. add UNG; 1u/µl:
    • for >0.5µg of DNA: 1µl(1u) per 1µg of DNA
    • for <0.5µg of DNA: 0.5µl(0.5u)
  3. 30min at 37°C;
  4. store at -20°C




[править] Solutions

[править] ER buffer, 9.08x

End repair buffer, 9.08x; store at -20°C

conc.stock500µl
T4 DNA ligase buff. with 10mM ATP (NEB)9.08x10x454µl
dNTP's mix, 2.31mM25mM46µl



[править] ER enzyme mix, 12x

End repair enzyme mix, 12x; store at -20°C

conc.100µl
T4 DNA pol., 3u/µl1.4u/µl45.5µl
Klenow DNA pol., 5u/µl0.45u/µl9µl
T4 PNK, 10u/µl4.5u/µl45.5µl



[править] A-tailing buffer, 8.33x

A-tailing buffer, 8.33x; store at -20°C

conc.stock500µl
Klenow buffer8.33x10x417µl
dATP1.66mM10mM83µl


[править] UDG Reaction Buffer, 10x

store at -20°C

  1ml5ml
Tris-HCl, pH8.0200mM1M200µl1ml
Dithiothreitol10mM100mM100µl500µl
EDTA10mM0.5M20µl100µl
H2O mQ680µl3.4ml




[править] Notes

Protocol may be used for preparation libraries for

» one-side (fragment, single read) sequencing
» both-sides (Paired End — PE) sequencing



Short AT-rich DNA fragments are extremely sensitive to denaturation. It is necessary to avoid denaturating factors:

» heating
» low-salt (<0.1mM salt) buffers
» alkaline pH



[править] 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 (per 28cm chamber)
» one empty well should be between marker and sample, two empty wells between samples
» marker should occupy the whole well. It permits to estimate the band inclination (normally, upper part runs slower, than lower part)
» do not load samples with extremely different amounts of DNA on the same gel
» for sensitive samples use "one gel   =>   one sample"



[править] Size-selection

  • see also [[Library_preparation|Table with a typical sequencing tasks].
  • genomic sequencing
» when there is enough DNA, it is worth to limit sequencing fragments by 80-130bp (one 50bp read) or 130-180bp (PE, 2x50bp), because it helps to increase the density of the clusters (clusters are smaller, more homogeneous by size and intensity)to make amplification on beads more homogeneous
» if the amount of starting material is limited, it is worth to take all sizes, which may be amplified and sequenced (Illumina: up to 600SOLiD: up to 250bp w/o adaptors)
  • RNA profiling: PE, 2x50bp sequencing: 130-150bp w/o adaptors



[править] 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 is worth to check the wells after removing of combs by loading ~5µl of 0.5x Xylene cyanol buffer
» OrangeG dye runs as a fragment of ~10bp on 2% HR gel



[править] Good interruption points

store at -20°C

» during DNA preparation: after RNase digestion; shearing; purification
» after primary size selection (purified, or in QG-buffer)
» after adaptor ligation
» after secondary size-selection


it would be better not to interrupt process from "end repair" till "adaptor ligation".


  1. should be no low molecular weight smear
  2. use 0.1µg of plasmid DNA as a control
  3. it is possible to shear DNA after RNase treatment w/o purification
  4. for unclear reasons, sometimes shearing works less efficient — shearing control is obligatory
  5. primary size-selection helps to:
    • prevents formation of heterohybrids
    • reduce amount of DNA, so that less enzymes and adapters are spent for library preparation
    • remove low-molecular weight contamination, which may exhaust enzymes and adapters
  6. if small-size border is more than "1/2 of large-size border"
  7. the aim is to obtain 180-210bp size range after secondary size selection. This range is selected because:
    » narrow range is preferable for analysis of small InDel's
    » it is unpracticall try to cut out of the gel range below 30bp
    » to save DNA material, selected range should be near the maximum of size distribution after ultrasound shearing
  8. the aim is to obtain 130-180bp size range after secondary size selection. This range is selected because:
    » the range is wider, than for "10µg<DNA", to increase the library complexity
    » "small size border" is coinside with that for the secondary size selection to prevent heteroduplex formation
  9. the aim is to obtain 180-230bp size range after secondary size selection. This range is selected because:
    » the range is wider, than for "10µg<DNA", to increase the library complexity
    » "small size border" is coinside with that for the secondary size selection to prevent heteroduplex formation
  10. important not to increase temperature, otherwise exonuclease activity of T4 polymerase will be too high
  11. Purpose of secondary size-selection is to remove adapters, which may induce high background during amplification


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