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