Brandon:LabNotes/Project1/2015-9-18: Difference between revisions

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>Bsos
>Bsos
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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[CTCTCTAT]TCGTCGGCAGCGTC i5 barcode + flowcell adaptor
  AATGATACGGCGACCACCGAGATCTACAC(CTCTCTAT)TCGTCGGCAGCGTC i5 barcode + flowcell adaptor
  AATGATACGGCGACCACCGAGATCTACAC[TATCCTCT]TCGTCGGCAGCGTC  
  AATGATACGGCGACCACCGAGATCTACAC(TATCCTCT)TCGTCGGCAGCGTC  
  AATGATACGGCGACCACCGAGATCTACAC[GTAAGGAG]TCGTCGGCAGCGTC                 
  AATGATACGGCGACCACCGAGATCTACAC(GTAAGGAG)TCGTCGGCAGCGTC                 
                                                                           GCGATCGAGGACGGC [AGATGTGTATAAGAGACAG]  read1
                                                                           GCGATCGAGGACGGC [AGATGTGTATAAGAGACAG]  read1
                                         TCGTCGGCAGCGTC{TCCACGC}[TATAGCCT]{GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG]  i5_transposon + bc
                                         TCGTCGGCAGCGTC{TCCACGC}(TATAGCCT){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG]  i5_transposon + bc
                                         TCGTCGGCAGCGTC{TCCACGC}[ATAGAGGC]{GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG]   
                                         TCGTCGGCAGCGTC{TCCACGC}(ATAGAGGC){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG]   
                                                                        
                                                                        
   
   
  i7 universal connector
  i7 universal connector
                                         CACCGTCTCCGCCTC [AGATGTGTATAAGAGACAG] read 2
                                         CACCGTCTCCGCCTC [AGATGTGTATAAGAGACAG] read 2
  GTCTCGTGGGCTCGG{CTGTCCCTGTCC}[CGAGTAAT]{CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG]
  GTCTCGTGGGCTCGG{CTGTCCCTGTCC}(CGAGTAAT){CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG]
  GTCTCGTGGGCTCGG{CTGTCCCTGTCC}[TCTCCGGA]{CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG]
  GTCTCGTGGGCTCGG{CTGTCCCTGTCC}(TCTCCGGA){CACCGTCTCCGCCTC}[AGATGTGTATAAGAGACAG]
   
   
                                      
                                      
                                                             GGCTCGGGTGCTCTG[ATTCCGCT]TAGAGCATACGGCAGAAGACGAAC i7 bc+ flowcell adaptor
                                                             GGCTCGGGTGCTCTG(ATTCCGCT)TAGAGCATACGGCAGAAGACGAAC i7 bc+ flowcell adaptor
                                                             GGCTCGGGTGCTCTG[GCTCCGAC]TAGAGCATACGGCAGAAGACGAAC
                                                             GGCTCGGGTGCTCTG(GCTCCGAC)TAGAGCATACGGCAGAAGACGAAC
  GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC} <- read2
  GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC} <- read2
  GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC}[TAATGAGC]{CCTGTCCCTGTC}GGCTCGGGTGCTCTG i7_transposon = bc
  GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC}(TAATGAGC){CCTGTCCCTGTC}GGCTCGGGTGCTCTG i7_transposon = bc
  GACAGAGAATATGTGTAGA{CTCCGCCTCTGCCAC}[AGGCCTCT]{CCTGTCCCTGTC}GGCTCGGGTGCTCTG
  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