Kun:LabNotes/GenomeSeq/2010-9-10: Difference between revisions

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*Each of the four libraries was sequenced in two lanes in the HL072 flowcell.  
*Each of the four libraries was sequenced in two lanes in the HL072 flowcell.  
*No read was obtain from the Lancelet-800bp library. Perhaps this is beyond the size limit of the Illumina sequencer.
*No read was obtain from the Lancelet-800bp library. Perhaps this is beyond the size limit of the Illumina sequencer.
*Summary of sequencing reads from HL072
        Data file                      # reads        Base pairs
  B_Jap1_300bp_HL072_sequence.txt 4,696,365      281,781,900
  B_Jap1_600bp_HL072_sequence.txt 7,558,134      453,488,040
  B_Jap2_300bp_HL072_sequence.txt 2,181,495      130,889,700
  B_Jap2_600bp_HL072_sequence.txt 3,784,750      227,085,000
  B_Jap3_300bp_HL072_sequence.txt 2,562,222      153,733,320
  B_Jap3_600bp_HL072_sequence.txt 1,542,170        92,530,200
  EP1_300bp_HL072_sequence.txt         4,181,616      250,896,960
  EP1_600bp_HL072_sequence.txt         6,825,994      409,559,640
  EP2_300bp_HL072_sequence.txt         3,327,675      199,660,500
  EP3_300bp_HL072_sequence.txt         4,331,030      259,861,800
  EP3_600bp_HL072_sequence.txt         3,124,016      187,440,960

Revision as of 18:53, 17 September 2010

Library construction (cont.)

  • The libraries made on 9/9 were good. I need to repeat the ligation using the ligation products made on 8/26 for BJ3.3 and BJ3.8. Then I will do ligation PCR with the barcoded primers on all the ligation products.

Ligation

   Purified ligation products from 8/36     18 μl
   Quick Ligation Reaction Buffer (5X)       6 μl
   20 μM DNA Adaptors                        x μl
   Quick T4 DNA Ligase                       2 μl
   H2O                                     4-x ul
   Incubate RT for 15 minutes. 
   Purified with 1V AMPure beads.    

PCR

  Adapter ligated DNA         20 μl
  PCR_F(10uM)                  2 μl
  PCR_R.N2IndX(10uM)           2 μl  
  50X SYBR Green I           0.8 μl
  H2O                         25 μl
  2X Phusion HF MM            50 μl
                              -----
                             100 μl
   Barcodes     300bp       800bp
   B.Jap-1      Ind1        Ind1+7 
   B.Jap-2      Ind2        Ind2+8
   B.Jap-3      Ind3        Ind3+9
   Ep.G-1       Ind4        Ind4+10
   Ep.G-2       Ind5   
   Ep.G-3       Ind6        Ind6+12
   1A           Ind1+7
   1F           Ind2+10(?)
   1G           Ind3+11
  98C 30 ->(98C 10s -> 65C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x4 -> 72C 2min.
  Took all except BJ2.3/BJ3.3/BJ2.8/BJ3.8 out after 9 cycles.
  File:2010-09-10-ligation-PCR.png
  Purified 50ul of each amplicon with 50ul AmPure beads, eluted with 40ul H2O
   Conc(ng/ul)  300bp       800bp
   B.Jap-1      12           6 
   B.Jap-2      12           9
   B.Jap-3      3            5
   Ep.G-1       10           8
   Ep.G-2       4   
   Ep.G-3       7            7
   1A           8
   1F           5
   1G           6
   
   Combine 
   B.Jap1.3, B.Jap2.3, B.Jap3.3, EP1.3, EP2.3, EP3.3 => Lancelet 300bp library
   B.Jap1.8, B.Jap2.8, B.Jap3.8, EP1.8, EP3.8 => Lancelet 800bp library
   1A, 1F, 1G => GM20431 Chr. library
   Perform size selection, cut out two different bands from the Lancelet 800bp library. 
   Estimate size: 
       Lancelet-300bp: 250-350bp
       Lancelet-800bp: 900-1000bp
       Lancelet-600bp: 600-700bp
   File:2010-09-10-Lancelet-300bp-lib-size-selection.png File:2010-09-10-Lancelet-800bp-lib-size-selection.png
   File:2010-09-10-GM20431-chr-lib-size-selection.png
   File:2010-09-15 mutiplexed libraries.png

Sequencing

  • Each of the four libraries was sequenced in two lanes in the HL072 flowcell.
  • No read was obtain from the Lancelet-800bp library. Perhaps this is beyond the size limit of the Illumina sequencer.
  • Summary of sequencing reads from HL072
        Data file                       # reads        Base pairs
  B_Jap1_300bp_HL072_sequence.txt	4,696,365       281,781,900
  B_Jap1_600bp_HL072_sequence.txt	7,558,134       453,488,040
  B_Jap2_300bp_HL072_sequence.txt	2,181,495       130,889,700
  B_Jap2_600bp_HL072_sequence.txt	3,784,750       227,085,000
  B_Jap3_300bp_HL072_sequence.txt	2,562,222       153,733,320
  B_Jap3_600bp_HL072_sequence.txt	1,542,170        92,530,200
  EP1_300bp_HL072_sequence.txt	        4,181,616       250,896,960
  EP1_600bp_HL072_sequence.txt	        6,825,994       409,559,640
  EP2_300bp_HL072_sequence.txt	        3,327,675       199,660,500
  EP3_300bp_HL072_sequence.txt	        4,331,030       259,861,800
  EP3_600bp_HL072_sequence.txt	        3,124,016       187,440,960