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==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. [[Image:2010-09-10-ligation-PCR.png|450px]] 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 [[Image:2010-09-10-Lancelet-300bp-lib-size-selection.png|300px]] [[Image:2010-09-10-Lancelet-800bp-lib-size-selection.png|350px]] [[Image:2010-09-10-GM20431-chr-lib-size-selection.png|300px]] [[Image:2010-09-15_mutiplexed_libraries.png|400px]] ===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 *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
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