Kun:LabNotes/GenomeSeq/2010-9-10

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Library construction (cont.)[edit]

  • 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[edit]

   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[edit]

  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[edit]

  • 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
  • Summary of sequencing reads from HL075
       Library             # reads        Read 1 (bps)     Read 2 (bps)
  B.Jap.1.HL075.300bp    15,777,276      1,262,182,080    1,151,741,148
  B.Jap.1.HL075.600bp    21,712,503      1,737,000,240    1,585,012,719
  B.Jap.2.HL075.300bp     7,869,372        629,549,760      574,464,156
  B.Jap.2.HL075.600bp    11,668,673        933,493,840      851,813,129
  B.Jap.3.HL075.300bp     9,224,920        737,993,600      673,419,160
  B.Jap.3.HL075.600bp     4,558,403        364,672,240      332,763,419
  Ep.1.HL075.300bp       16,246,074      1,299,685,920    1,185,963,402
  Ep.1.HL075.600bp       20,184,984      1,614,798,720    1,473,503,832
  Ep.2.HL075.300bp       13,410,708      1,072,856,640      978,981,684
  Ep.3.HL075.300bp       14,658,006      1,172,640,480    1,070,034,438
  Ep.3.HL075.600bp        9,171,575        733,726,000      669,524,975
  • Summary of sequencing reads from HL081
   Library	           # reads            Read 1           Read 2
   B.Jap.1_HL081_300bp	  35,647,069	  2,851,765,520	   2,780,471,382
   B.Jap.2_HL081_300bp	  18,495,776	  1,479,662,080	   1,442,670,528
   B.Jap.3_HL081_300bp   19,012,632	  1,521,010,560	   1,482,985,296
   Ep.G.1_HL081_300bp	  31,792,517	  2,543,401,360	   2,479,816,326
   Ep.G.2_HL081_300bp	  25,361,111	  2,028,888,880	   1,978,166,658
   Ep.G.3_HL081_300bp	  34,333,102	  2,746,648,160	   2,677,981,956
  • Summary of sequencing reads from HL085
   Library	           # reads          Read 1           Read 2
  B.Jap.1_HL085_300bp    6,213,998     497,119,840     39,769,587,200
  B.Jap.2_HL085_300bp    3,149,820     251,985,600     20,158,848,000
  B.Jap.3_HL085_300bp    3,182,502     254,600,160     20,368,012,800
  Ep.G.1_HL085_300bp     6,060,892     484,871,360     38,789,708,800
  Ep.G.2_HL085_300bp     4,825,659     386,052,720     30,884,217,600
  Ep.G.3_HL085_300bp     5,709,191     456,735,280     36,538,822,400