Brandon:LabNotes/Project1/2015-9-18: Difference between revisions
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==combinatorial method sequences detail == | ==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 | i5 universal connector | ||
AATGATACGGCGACCACCGAGATCTACAC i5_nXTv2 index primer | AATGATACGGCGACCACCGAGATCTACAC i5_nXTv2 index primer | ||
AATGATACGGCGACCACCGAGATCTACAC | AATGATACGGCGACCACCGAGATCTACAC(CTCTCTAT)TCGTCGGCAGCGTC i5 barcode + flowcell adaptor | ||
AATGATACGGCGACCACCGAGATCTACAC | AATGATACGGCGACCACCGAGATCTACAC(TATCCTCT)TCGTCGGCAGCGTC | ||
AATGATACGGCGACCACCGAGATCTACAC | AATGATACGGCGACCACCGAGATCTACAC(GTAAGGAG)TCGTCGGCAGCGTC | ||
GCGATCGAGGACGGC [AGATGTGTATAAGAGACAG] read1 | GCGATCGAGGACGGC [AGATGTGTATAAGAGACAG] read1 | ||
TCGTCGGCAGCGTC{TCCACGC} | TCGTCGGCAGCGTC{TCCACGC}(TATAGCCT){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG] i5_transposon + bc | ||
TCGTCGGCAGCGTC{TCCACGC} | TCGTCGGCAGCGTC{TCCACGC}(ATAGAGGC){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG] | ||
i7 universal connector | i7 universal connector | ||
CACCGTCTCCGCCTC [AGATGTGTATAAGAGACAG] read 2 | CACCGTCTCCGCCTC [AGATGTGTATAAGAGACAG] read 2 | ||
GTCTCGTGGGCTCGG{CTGTCCCTGTCC} | GTCTCGTGGGCTCGG{CTGTCCCTGTCC}(CGAGTAAT){CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG] | ||
GTCTCGTGGGCTCGG{CTGTCCCTGTCC} | GTCTCGTGGGCTCGG{CTGTCCCTGTCC}(TCTCCGGA){CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG] | ||
GGCTCGGGTGCTCTG | GGCTCGGGTGCTCTG(ATTCCGCT)TAGAGCATACGGCAGAAGACGAAC i7 bc+ flowcell adaptor | ||
GGCTCGGGTGCTCTG | GGCTCGGGTGCTCTG(GCTCCGAC)TAGAGCATACGGCAGAAGACGAAC | ||
GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC} <- read2 | GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC} <- read2 | ||
GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC} | GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC}(TAATGAGC){CCTGTCCCTGTC}GGCTCGGGTGCTCTG i7_transposon = bc | ||
GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC} | GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC}(AGGCCTCT){CCTGTCCCTGTC}GGCTCGGGTGCTCTG | ||
CTGTCTCTTATACACATCT{GAGGCGGAGACGGTG} -> index 1 | CTGTCTCTTATACACATCT{GAGGCGGAGACGGTG} -> index 1 |
Revision as of 08:39, 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