Brandon:LabNotes/Project1/2015-9-18

From ZhangLabWiki
Jump to navigation Jump to search

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