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

From ZhangLabWiki
Jump to navigation Jump to search
>Bsos
>Bsos
 
(4 intermediate revisions by the same user not shown)
Line 1: Line 1:
==combinatorial method sequences detail ==
==combinatorial method sequences detail==




*method originally from paper on haplotyping using contiguity preserved transposition [http://www.nature.com/ng/journal/v46/n12/full/ng.3119.html#supplementary-information Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing]
*method used in [http://www.sciencemag.org/content/348/6237/910.full#ref-17 Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing] using nuclei instead of haplotyping
*sequence detail for methods used in above papers
ungrouped sequences are nXTv2 i5/i7 sequences
{sequences added by them}
[moasic end Tn5 binding]
(barcode)
p5_barcoded_transposon
5'- TCGTCGGCAGCGTC{TCCACGC}(XXXXXXXX){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG] -3'
                                                    3'- TCTACACATATTCTCTGTC/5Phos/ -5'
p7_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)
p5_barcoded_transposon
NNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'                                      5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNNN -3'
NNNNNNNNNNNNNN        GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5'                                      3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNN -5'
p7_barcoded_transposon
NNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'                                            5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNNN -3'
NNNNNNNNNNNNNN        GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5'                                            3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNN -5'
After tagementation fragment options:
-8 p5 barcodes, 12 p7 barcodes in 8X12 array for 96 unique barcoded fragments.
-Insertion into bottom strand will just reverse the orientation, i5,i7 will be flipped.
p5_barcoded_transposon                                                                        p7_barcoded_transposon
5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'
                                        3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN        GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5'
p7_barcoded_transposon                                                                              p5_barcoded_transposon
5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'
                                              3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN        GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5'
p5_barcoded_transposon                                                                        p5_barcoded_transposon
5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'
                                        3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN        GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5'
p7_barcoded_transposon                                                                              p7_barcoded_transposon
5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'
                                              3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN        GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5'
After fill in at 72 for 3-5 minutes (gap filling).
p5_barcoded_transposon                                                          p7_barcoded_transposon
5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC -3'
3'- AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5'
p7_barcoded_transposon                                                                p5_barcoded_transposon
5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGCCGTCCTCGATCGC[p5]GCGTGGAGACGCTGCCGACGA -3'
3'- CAGAGCACCCGAGCCGACAGGGACAGG[p7]GTGGCAGAGGCGGAGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5'
p5_barcoded_transposon                                                          p5_barcoded_transposon
5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGCCGTCCTCGATCGC[p5]GCGTGGAGACGCTGCCGACGA -3'
3'- AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5'
p7_barcoded_transposon                                                                p7_barcoded_transposon
5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC -3'
3'- CAGAGCACCCGAGCCGACAGGGACAGG[p7]GTGGCAGAGGCGGAGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]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[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC -3'
                                  3'- AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]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[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                                                                                                                    3'- AGCATACGGCAGAAGACGAAC -5
                                                                                                                                                                                          primer_2
Read primers for sequencing:
for index reads, long reads, ~37 or more bp, were used for reading both indexes, for each index primer
        i5_nXTv2_index_read ----->                                                  read_1 ----->                              index_read_1 ----->
    AATGATACGGCGACCACCGAGATCTACAC                            GCGATCGAGGACGGCAGATGTGTATAAGAGACAG              CTGTCTCTTATACACATCTGAGGCGGAGACGGTG
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                                                GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC
                                                                                                                <----- read_2 
Sequences used:
[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/Nextera_XT_V2_sequences_and_outline Nextera XT v2] used for i5/i7 barcoding
below sequences from [http://www.nature.com/ng/journal/v46/n12/full/ng.3119.html#supplementary-information Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing]
Oligo Sequence (5'-3')
P5_i5_1_Universal_Connector_A_C15_ME         TCGTCGGCAGCGTCTCCACGCTATAGCCTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P5_i5_2_Universal_Connector_A_C15_ME         TCGTCGGCAGCGTCTCCACGCATAGAGGCGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P5_i5_3_Universal_Connector_A_C15_ME         TCGTCGGCAGCGTCTCCACGCCCTATCCTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P5_i5_4_Universal_Connector_A_C15_ME         TCGTCGGCAGCGTCTCCACGCGGCTCTGAGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P5_i5_5_Universal_Connector_A_C15_ME         TCGTCGGCAGCGTCTCCACGCAGGCGAAGGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P5_i5_6_Universal_Connector_A_C15_ME         TCGTCGGCAGCGTCTCCACGCTAATCTTAGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P5_i5_7_Universal_Connector_A_C15_ME         TCGTCGGCAGCGTCTCCACGCCAGGACGTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P5_i5_8_Universal_Connector_A_C15_ME         TCGTCGGCAGCGTCTCCACGCGTACTGACGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P7_i7_1_Universal_Connector_B_D15_ME         GTCTCGTGGGCTCGGCTGTCCCTGTCCCGAGTAATCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_2_Universal_Connector_B_D15_ME         GTCTCGTGGGCTCGGCTGTCCCTGTCCTCTCCGGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_3_Universal_Connector_B_D15_ME         GTCTCGTGGGCTCGGCTGTCCCTGTCCAATGAGCGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_4_Universal_Connector_B_D15_ME         GTCTCGTGGGCTCGGCTGTCCCTGTCCGGAATCTCCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_5_Universal_Connector_B_D15_ME         GTCTCGTGGGCTCGGCTGTCCCTGTCCTTCTGAATCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_6_Universal_Connector_B_D15_ME         GTCTCGTGGGCTCGGCTGTCCCTGTCCACGAATTCCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_7_Universal_Connector_B_D15_ME         GTCTCGTGGGCTCGGCTGTCCCTGTCCAGCTTCAGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_8_Universal_Connector_B_D15_ME         GTCTCGTGGGCTCGGCTGTCCCTGTCCGCGCATTACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_9_Universal_Connector_B_D15_ME         GTCTCGTGGGCTCGGCTGTCCCTGTCCCATAGCCGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_10_Universal_Connector_B_D15_ME         GTCTCGTGGGCTCGGCTGTCCCTGTCCTTCGCGGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_11_Universal_Connector_B_D15_ME         GTCTCGTGGGCTCGGCTGTCCCTGTCCGCGCGAGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_12_Universal_Connector_B_D15_ME         GTCTCGTGGGCTCGGCTGTCCCTGTCCCTATCGCTCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
Read 1 Sequencing Primer (Extended ME A) GCGATCGAGGACGGCAGATGTGTATAAGAGACAG
Read 2 Sequencing Primer (Extended ME B) CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
Index 1 Sequencing Primer (RC of Extended ME B) CTGTCTCTTATACACATCTGAGGCGGAGACGGTG
pMENTS 5Phos/CTGTCTCTTATACACATCT
 
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

Latest revision as of 22:48, 21 September 2015

combinatorial method sequences detail[edit]



  • sequence detail for methods used in above papers


ungrouped sequences are nXTv2 i5/i7 sequences
{sequences added by them}
[moasic end Tn5 binding]
(barcode)

p5_barcoded_transposon
5'- TCGTCGGCAGCGTC{TCCACGC}(XXXXXXXX){GCGATCGAGGACGGC}[AGATGTGTATAAGAGACAG] -3'
                                                   3'- TCTACACATATTCTCTGTC/5Phos/ -5'

p7_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)

p5_barcoded_transposon
NNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'                                      5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNNN -3'
NNNNNNNNNNNNNN        GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5'                                      3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNN -5'


p7_barcoded_transposon
NNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'                                            5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNNN -3'
NNNNNNNNNNNNNN        GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5'                                            3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNN -5'




After tagementation fragment options:
-8 p5 barcodes, 12 p7 barcodes in 8X12 array for 96 unique barcoded fragments.
-Insertion into bottom strand will just reverse the orientation, i5,i7 will be flipped.

p5_barcoded_transposon                                                                        p7_barcoded_transposon
5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'
                                        3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN        GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5'

p7_barcoded_transposon                                                                              p5_barcoded_transposon
5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'
                                              3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN        GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5'

p5_barcoded_transposon                                                                         p5_barcoded_transposon
5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG         NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'
                                        3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN         GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5'

p7_barcoded_transposon                                                                              p7_barcoded_transposon
5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'
                                              3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN        GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5'




After fill in at 72 for 3-5 minutes (gap filling).

p5_barcoded_transposon                                                           p7_barcoded_transposon
5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC -3'
3'- AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG -5'

p7_barcoded_transposon                                                                 p5_barcoded_transposon
5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGCCGTCCTCGATCGC[p5]GCGTGGAGACGCTGCCGACGA -3'
3'- CAGAGCACCCGAGCCGACAGGGACAGG[p7]GTGGCAGAGGCGGAGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5'

p5_barcoded_transposon                                                           p5_barcoded_transposon
5'- TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGCCGTCCTCGATCGC[p5]GCGTGGAGACGCTGCCGACGA -3'
3'- AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACGGCAGGAGCTAGCG[p5]CGCACCTCTGCGACGGCTGCT -5'

p7_barcoded_transposon                                                                 p7_barcoded_transposon
5'- GTCTCGTGGGCTCGGCTGTCCCTGTCC[p7]CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC -3'
3'- CAGAGCACCCGAGCCGACAGGGACAGG[p7]GTGGCAGAGGCGGAGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]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[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC -3'
                                 3'- AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]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[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                                                                                                                   3'- AGCATACGGCAGAAGACGAAC -5
                                                                                                                                                                                          primer_2

Read primers for sequencing:
for index reads, long reads, ~37 or more bp, were used for reading both indexes, for each index primer

       i5_nXTv2_index_read ----->                                                  read_1 ----->                              index_read_1 ----->
    AATGATACGGCGACCACCGAGATCTACAC                             GCGATCGAGGACGGCAGATGTGTATAAGAGACAG               CTGTCTCTTATACACATCTGAGGCGGAGACGGTG
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCTCCACGC[p5]GCGATCGAGGACGGCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN CTGTCTCTTATACACATCTGAGGCGGAGACGGTG[p7]GGACAGGGACAGCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]AGCAGCCGTCGCAGAGGTGCG[p5]CGCTAGCTCCTGCCGTCTACACATATTCTCTGTC NNNNNNNNNNNNN GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC[p7]CCTGTCCCTGTCGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                                               GACAGAGAATATGTGTAGACTCCGCCTCTGCCAC
                                                                                                               <----- read_2   


Sequences used:
Nextera XT v2 used for i5/i7 barcoding
below sequences from Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing

Oligo	Sequence (5'-3')
P5_i5_1_Universal_Connector_A_C15_ME	         TCGTCGGCAGCGTCTCCACGCTATAGCCTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P5_i5_2_Universal_Connector_A_C15_ME	         TCGTCGGCAGCGTCTCCACGCATAGAGGCGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P5_i5_3_Universal_Connector_A_C15_ME	         TCGTCGGCAGCGTCTCCACGCCCTATCCTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P5_i5_4_Universal_Connector_A_C15_ME	         TCGTCGGCAGCGTCTCCACGCGGCTCTGAGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P5_i5_5_Universal_Connector_A_C15_ME	         TCGTCGGCAGCGTCTCCACGCAGGCGAAGGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P5_i5_6_Universal_Connector_A_C15_ME	         TCGTCGGCAGCGTCTCCACGCTAATCTTAGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P5_i5_7_Universal_Connector_A_C15_ME	         TCGTCGGCAGCGTCTCCACGCCAGGACGTGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P5_i5_8_Universal_Connector_A_C15_ME	         TCGTCGGCAGCGTCTCCACGCGTACTGACGCGATCGAGGACGGCAGATGTGTATAAGAGACAG
P7_i7_1_Universal_Connector_B_D15_ME	         GTCTCGTGGGCTCGGCTGTCCCTGTCCCGAGTAATCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_2_Universal_Connector_B_D15_ME	         GTCTCGTGGGCTCGGCTGTCCCTGTCCTCTCCGGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_3_Universal_Connector_B_D15_ME	         GTCTCGTGGGCTCGGCTGTCCCTGTCCAATGAGCGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_4_Universal_Connector_B_D15_ME	         GTCTCGTGGGCTCGGCTGTCCCTGTCCGGAATCTCCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_5_Universal_Connector_B_D15_ME	         GTCTCGTGGGCTCGGCTGTCCCTGTCCTTCTGAATCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_6_Universal_Connector_B_D15_ME	         GTCTCGTGGGCTCGGCTGTCCCTGTCCACGAATTCCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_7_Universal_Connector_B_D15_ME	         GTCTCGTGGGCTCGGCTGTCCCTGTCCAGCTTCAGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_8_Universal_Connector_B_D15_ME	         GTCTCGTGGGCTCGGCTGTCCCTGTCCGCGCATTACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_9_Universal_Connector_B_D15_ME	         GTCTCGTGGGCTCGGCTGTCCCTGTCCCATAGCCGCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_10_Universal_Connector_B_D15_ME	         GTCTCGTGGGCTCGGCTGTCCCTGTCCTTCGCGGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_11_Universal_Connector_B_D15_ME	         GTCTCGTGGGCTCGGCTGTCCCTGTCCGCGCGAGACACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
P7_i7_12_Universal_Connector_B_D15_ME	         GTCTCGTGGGCTCGGCTGTCCCTGTCCCTATCGCTCACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
Read 1 Sequencing Primer (Extended ME A)	 GCGATCGAGGACGGCAGATGTGTATAAGAGACAG
Read 2 Sequencing Primer (Extended ME B)	 CACCGTCTCCGCCTCAGATGTGTATAAGAGACAG
Index 1 Sequencing Primer (RC of Extended ME B) CTGTCTCTTATACACATCTGAGGCGGAGACGGTG
pMENTS	5Phos/CTGTCTCTTATACACATCT

 





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