AlanFung:Protocol/Construction of Solexa sequencing Library

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Revision as of 03:21, 13 November 2009 by >Alan6017518 (→‎TBE Gel Size Selection)
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Construction of Solexa sequencing library from padlock captured PCR products using NEBNext DNA Sample Prep Master Mix Set 1 Kit (E6040S/L)

  • Info Read the FAQ!!!
  • Protocol
  • The NEBNext DNA SPMMS 1 (I) is compatible with protocols requiring a 3´ A overhang on the library molecules to facilitate ligation to adapters containing a 5´ T overhang, such as Illumina’s Genomic DNA Sample Prep protocol for the Genome Analyzer II.
  • For 60bp run, we can use the library size of >180bp
  • For 80bp runs, the lower limit for the sequencing libraries should be 200bp.
  • If the library size is too small, some of the 80bp reads will reach to the other end of the adaptor sequences. we don't want to waste the sequencing $$ on the regions we don't want
  • The total size of the adaptors is 105bp, and there is another ~25bp of the capturing arm
  • The upper limit should be ~50bp above the lower limit

Overview[edit]

  • Fragmentation and end-polishing
  • Size Selection
  • Ligation
  • PCR of sequencing Library
  • QPCR quantification


Fragmentation and end-polishing (Make blunt ends with 5'P)[edit]

  • The PCR amplicons (200ng-1ug) are fragmented with Covaris sonicator to ~100bp
  • End-Repair Reactions
Fragmented DNA 85ul
10X End Repair Bufer 10ul
End Repair Enzyme Mix 5ul
  • Incubate tubes at RT for 30minutes.
  • Perform a Qiaquick purification and elute with 39ul EB buffer.
  • NOTE: For blunt-end ligation, the A-Tailing reaction should be skipped. Doing TA ligation is preferable since it eliminates the chance of getting chimeric reads.
  • A-Tailing Reactions
Blunet-end DNA 37ul
10X dA-Tailing Reaction Buffer 5ul
Klenow Fragment (3'-5' exo-) 3ul
H2O 5ul

Incubated at 37C for 30min, purified with Qiaquick columns, eluted with 40 ul EB.

  • Measure concentration with nanodrop

Size selection using Invitrogen 2% SizeSelect gel[edit]

  • Fill any unused well with 30ul EB Buffer
  • Mix 15ul EB buffer with 0.5ul ladder in a 0.2ml tube
  • Load 20u end-polished DNA sample into two lanes(use 3 or more lanes if the starting DNA is more than 1ug)
  • Run the SizeSelect program for 12~14 min. Pause when the 125bp band just move into the middle collection well
  • Use pipette to extract all solution in each of the sample well, and quickly refill the wells with 20ul EB.
  • Resume the electrophoresis for additional 15sec, extract DNA and refill the wells with 20ul EB. *Resume the electrophoresis for additional 15sec, extract DNA (all the extracted DNA from the same sample are mixed in a tube)
  • Refill the wells with 20ul EB, and run the electrophoresis for additional 3 minutes. Take a picture of the gel to document the extracted fragment sizes.
  • Concentrate the DNA with a SpeedVac (no heat) to ~36ul (takes about 1hour).

Ligation. Blunt-end and TA ligations use the same protocol but slightly different adaptor sequences.[edit]

  • Set up ligation reaction. Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time.
ul
End-repaired & size selected DNA 36
40uM adaptor2 2
5X Quick Ligase Buffer 10
Quick Ligase 2
  • Incubate at room temperature for 15 minutes, purified with MinElute columns, eluted with 22ul EB.

PCR of sequencing library[edit]

Solexa_PCR_up: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

AmpR6.3Sol: CAAGCAGAAGACGGCATACGAGCTCTTCGGAACGATGAGCCTCCAAC

AmpF6.3rSol: CAAGCAGAAGACGGCATACGAGCTCTTCCAGATGTTATCGAGGTCCGA

Ligation products 10 10
10uM solexa PCR up 2 2
10uM AmpR6.3Sol 2 -
10uM AmpF6.3Sol - 2
2X iProof master mix (Bio-Rad) 50 50
50X SYBR Green I 0.4 0.4
H2O 36 36
  • Setup 2 master mix
F R
Ligation products
10uM solexa PCR up 13.2 13.2
10uM AmpR6.3Sol 13.2 -
10uM AmpF6.3Sol - 13.2
2X iProof master mix (Bio-Rad) 330 330
50X SYBR Green I 2.64 2.64
H2O 237.6 237.6

PCR program: 98 °C 30sec -> 8 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold. File:20091112 125359.jpg

  • TBE Gel verification (10well)
  • 15ul h2o+4.5ul 6x loading dye+0.5ul 25bp ladder
  • 15ul h2o+3ul 6X loading dye+2ul sample

File:ZhangLab 2 2009-11-12 14hr 46min.jpg File:ZhangLab 2 2009-11-12 14hr 48min.jpg

  • Mix the amplicons with two sets of primers, purified with Qiaquick columns with 32ul EB buffer

TBE Gel Size Selection[edit]

8-DF:46.3ng/ul
9-DF:27.9ng/ul 
8-FS:30.6ng/ul 
9-FS:24.7ng/ul 
  • Combining might lead to overload, run one gel for each library

Without Set CV-F[edit]

   * Run samples in 3 lanes and ladder in 2 lanes in in a 5-well TBE gel
   * Load 2 lanes with 25bp ladder Mix in a .2ml tube(30ul H2O+9ul 6X loading dye+1ul 25bp ladder)/2
   * Mix (30ul library + 15ul 6X loading dye + 15ul h20)/3 load to 3 wells