Brandon:LabNotes/Project1/2015-9-18: Difference between revisions
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r5_barcoded_transposon r5_barcoded_transposon | r5_barcoded_transposon r5_barcoded_transposon | ||
5'- TCGTCGGCAGCGTCTCCACGC[r5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' | 5'- TCGTCGGCAGCGTCTCCACGC[r5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' | ||
3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[r5]CGCACCTCTGCGACGGCTGCT -5' | |||
r7_barcoded_transposon r7_barcoded_transposon | r7_barcoded_transposon r7_barcoded_transposon |
Revision as of 17:48, 21 September 2015
combinatorial method sequences detail
{sequences added by them} [moasic end Tn5 binding] (barcode) r5_barcoded_transposon 5'- TCGTCGGCAGCGTC{TCCACGC}(XXXXXXXX){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG] -3' 3'- TCTACACATATTCTCTGTC/5Phos/ -5' r7_barcoded_transposon 5'- GTCTCGTGGGCTCGG{CTGTCCCTGTCC}(XXXXXXXX){CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG] -3' 3'- TCTACACATATTCTCTGTC/5Phos/ -5' After single insertion (Tn5 have only i5 or i7 bound, fragments, 9 base pair gap) r5_barcoded_transposon NNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 5'- TCGTCGGCAGCGTCTCCACGC[r5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNN -3' NNNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[r5]CGCACCTCTGCGACGGCTGCT -5' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNN -5' r7_barcoded_transposon NNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[r7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNN -3' NNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[r7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNN -5' After tagementation fragment options: -8 r5 barcodes, 12 r7 barcodes in 8X12 array for 96 unique barcoded fragments. -Insertion into bottom strand will just reverse the orientation, i5,i7 will be flipped. r5_barcoded_transposon r7_barcoded_transposon 5'- TCGTCGGCAGCGTCTCCACGC[r5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[r7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' r7_barcoded_transposon r5_barcoded_transposon 5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[r7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[r5]CGCACCTCTGCGACGGCTGCT -5' r5_barcoded_transposon r5_barcoded_transposon 5'- TCGTCGGCAGCGTCTCCACGC[r5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[r5]CGCACCTCTGCGACGGCTGCT -5' r7_barcoded_transposon r7_barcoded_transposon 5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[r7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[r7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' After fill in at 72 for 3-5 minutes (gap filling). r5_barcoded_transposon r7_barcoded_transposon 5'- TCGTCGGCAGCGTCTCCACGC[r5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[r7]GGACAGGGACAGCCGAGCCCACGAGAC -3' 3'- AGCAGCCGTCGCAGAGGTGCG[r5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[r7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' r7_barcoded_transposon r5_barcoded_transposon 5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[r7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGCCGTCCTCGATCGC[r5]GCGTGGAGACGCTGCCGACGA -3' 3'- CAGAGCACCCGAGCCGACAGGGACAGG[r7]GTGGCAGAGGCGGAGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[r5]CGCACCTCTGCGACGGCTGCT -5' r5_barcoded_transposon r5_barcoded_transposon 5'- TCGTCGGCAGCGTCTCCACGC[r5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGCCGTCCTCGATCGC[r5]GCGTGGAGACGCTGCCGACGA -3' 3'- AGCAGCCGTCGCAGAGGTGCG[r5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[r5]CGCACCTCTGCGACGGCTGCT -5' r7_barcoded_transposon r7_barcoded_transposon 5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[r7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[r7]GGACAGGGACAGCCGAGCCCACGAGAC -3' 3'- CAGAGCACCCGAGCCGACAGGGACAGG[r7]GTGGCAGAGGCGGAGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[r7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' Now PCRable, adding on dual barcodes. -8 i5 barcodes, 12 i7 barcodes in 8X12 array for 96 unique barcoded fragments. -with previously added barcoded fragments makes 9216 unique compartments i5_adapter 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3' 5'- TCGTCGGCAGCGTCTCCACGC[r5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[r7]GGACAGGGACAGCCGAGCCCACGAGAC -3' 3'- AGCAGCCGTCGCAGAGGTGCG[r5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[r7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5' 3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' i7_adapter Amplification with primer 1 and primer 2 (optional) primer_1 5'- AATGATACGGCGACCACCGA -3' 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCTCCACGC[r5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[r7]GGACAGGGACAGCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]AGCAGCCGTCGCAGAGGTGCG[r5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[r7]CCTGTCCCTGTCGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' 3'- AGCATACGGCAGAAGACGAAC -5 primer_2 Read primers for sequencing: i5_nXTv2_index_read -----> read_1 -----> index_read_1 -----> AATGATACGGCGACCACCGAGATCTACAC GCGATCGAGGACGGCAGATGTGTATAAGAGACAG CTGTCTCTTATACACATCTGAGGCGGAGACGGTG 5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCTCCACGC[r5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[r7]GGACAGGGACAGCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3' 3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]AGCAGCCGTCGCAGAGGTGCG[r5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[r7]CCTGTCCCTGTCGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5' GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC <----- read_2 NOTES: i5 universal connector AATGATACGGCGACCACCGAGATCTACAC i5_nXTv2 index primer AATGATACGGCGACCACCGAGATCTACAC(CTCTCTAT)TCGTCGGCAGCGTC i5 barcode + flowcell adaptor AATGATACGGCGACCACCGAGATCTACAC(TATCCTCT)TCGTCGGCAGCGTC AATGATACGGCGACCACCGAGATCTACAC(GTAAGGAG)TCGTCGGCAGCGTC GCGATCGAGGACGGC [AGATGTGTATAAGAGACAG] read1 TCGTCGGCAGCGTC{TCCACGC}(TATAGCCT){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG] i5_transposon + bc TCGTCGGCAGCGTC{TCCACGC}(ATAGAGGC){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG] i7 universal connector CACCGTCTCCGCCTC [AGATGTGTATAAGAGACAG] read 2 GTCTCGTGGGCTCGG{CTGTCCCTGTCC}(CGAGTAAT){CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG] GTCTCGTGGGCTCGG{CTGTCCCTGTCC}(TCTCCGGA){CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG] GGCTCGGGTGCTCTG(ATTCCGCT)TAGAGCATACGGCAGAAGACGAAC i7 bc+ flowcell adaptor GGCTCGGGTGCTCTG(GCTCCGAC)TAGAGCATACGGCAGAAGACGAAC GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC} <- read2 GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC}(TAATGAGC){CCTGTCCCTGTC}GGCTCGGGTGCTCTG i7_transposon = bc GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC}(AGGCCTCT){CCTGTCCCTGTC}GGCTCGGGTGCTCTG CTGTCTCTTATACACATCT{GAGGCGGAGACGGTG} -> index 1