Brandon:Protocols/Nextera XT V2 sequences and outline

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Revision as of 18:58, 4 August 2015 by >Bsos (→‎Current sequences and method used by nextera protocol:)
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Current sequences and method used by nextera protocol:[edit]

Current nextera sequences

i5 adapter
5’- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'
              3'- TCTACACATATTCTCTGTC/5Phos/ -5'

i7 adapter
5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'
               3'- TCTACACATATTCTCTGTC/5Phos/ -5'




After single insertion (Tn5 have only i5 or i7 bound, fragments, 9 base pair gap)

i5 adapter                                               i5 adapter
NNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'            5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNN
NNNNNNNNNNNNNN        GACAGAGAATATGTGTAGACTGCGACGGCTGCT -5'            3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNN


i7 adapter                                               i7 adapter
NNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'            5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG NNNNNNNNNNNNN
NNNNNNNNNNNNNN        GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'            3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNN 




After tagementation fragment options:
-single insertion not able to make a PCRable fragment
-Insertion into bottom strand will just reverse the orientation, i5,i7 will be flipped.

i5 adapter                                                          i7 adapter
5’- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG       NNNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'
              3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNNN       GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

i7 adapter                                                           i5 adapter
5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG       NNNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'
               3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNNN       GACAGAGAATATGTGTAGACTGCGACGGCTGCT -5'

i5 adapter                                                          i5 adapter  
5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG       NNNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'
              3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNNN       GACAGAGAATATGTGTAGACTGCGACGGCTGCT -5'

i7 adapter                                                           i7 adapter  
5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG       NNNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'
               3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNNN       GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'



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

i5 adapter                                                       i7 adapter
5’- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

i7 adapter                                                       i5 adapter
5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTGACGCTGCCGACGA -3'
3'- CAGAGCACCCGAGCCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT -5'

i5 adapter                                                       i5 adapter  
5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTGACGCTGCCGACGA -3'
3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT -5'

i7 adapter                                                       i7 adapter  
5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
3'- CAGAGCACCCGAGCCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' 



Now PCRable, adding on dual barcodes.

i5 adapter                                                      
5’ AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3'
                                5’- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
                                3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                                                 GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC
                                                                                                                  i7 adapter

Amplification with primer 1 and primer 2 (optional)

    (primer 1)
5'- AATGATACGGCGACCACCGA -3'    i5 adapter
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                                                     i7 adapter     3'- AGCATACGGCAGAAGACGAAC -5'
                                                                                                                                               (primer 2)
 

Nextera read primers for sequencing:
 
             i5 index read ----->                        read1  ----->                                       i7 index read ----->
    AATGATACGGCGACCACCGAGATCTACAC    TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG                         CTGTCTCTTATACACATCTCCGAGCCCACGAGAC
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                               GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG
                                                                                               <----- read2
num.	i5 barcode	i5 name	i5 smplsheet	i5 full sequence
1	CTCTCTAT	S502	CTCTCTAT	AATGATACGGCGACCACCGAGATCTACACCTCTCTATTCGTCGGCAGCGTC
2	TATCCTCT	S503	TATCCTCT	AATGATACGGCGACCACCGAGATCTACACTATCCTCTTCGTCGGCAGCGTC
3	GTAAGGAG	S505	GTAAGGAG	AATGATACGGCGACCACCGAGATCTACACGTAAGGAGTCGTCGGCAGCGTC
4	ACTGCATA	S506	ACTGCATA	AATGATACGGCGACCACCGAGATCTACACACTGCATATCGTCGGCAGCGTC
5	AAGGAGTA	S507	AAGGAGTA	AATGATACGGCGACCACCGAGATCTACACAAGGAGTATCGTCGGCAGCGTC
6	CTAAGCCT	S508	CTAAGCCT	AATGATACGGCGACCACCGAGATCTACACCTAAGCCTTCGTCGGCAGCGTC
7	CGTCTAAT	S510	CGTCTAAT	AATGATACGGCGACCACCGAGATCTACACCGTCTAATTCGTCGGCAGCGTC
8	TCTCTCCG	S511	TCTCTCCG	AATGATACGGCGACCACCGAGATCTACACTCTCTCCGTCGTCGGCAGCGTC
9	TCGACTAG	S513	TCGACTAG	AATGATACGGCGACCACCGAGATCTACACTCGACTAGTCGTCGGCAGCGTC
10	TTCTAGCT	S515	TTCTAGCT	AATGATACGGCGACCACCGAGATCTACACTTCTAGCTTCGTCGGCAGCGTC
11	CCTAGAGT	S516	CCTAGAGT	AATGATACGGCGACCACCGAGATCTACACCCTAGAGTTCGTCGGCAGCGTC
12	GCGTAAGA	S517	GCGTAAGA	AATGATACGGCGACCACCGAGATCTACACGCGTAAGATCGTCGGCAGCGTC
13	CTATTAAG	S518	CTATTAAG	AATGATACGGCGACCACCGAGATCTACACCTATTAAGTCGTCGGCAGCGTC
14	AAGGCTAT	S520	AAGGCTAT	AATGATACGGCGACCACCGAGATCTACACAAGGCTATTCGTCGGCAGCGTC
15	GAGCCTTA	S521	GAGCCTTA	AATGATACGGCGACCACCGAGATCTACACGAGCCTTATCGTCGGCAGCGTC
16	TTATGCGA	S522	TTATGCGA	AATGATACGGCGACCACCGAGATCTACACTTATGCGATCGTCGGCAGCGTC
				
				
num.	i7 barcode	i7 name	i7 smplsheet	i7 full sequence
1	TCGCCTTA	N701	TAAGGCGA	CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG
2	CTAGTACG	N702	CGTACTAG	CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGG
3	TTCTGCCT	N703	AGGCAGAA	CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGG
4	GCTCAGGA	N704	TCCTGAGC	CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGG
5	AGGAGTCC	N705	GGACTCCT	CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGG
6	CATGCCTA	N706	TAGGCATG	CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGG
7	GTAGAGAG	N707	CTCTCTAC	CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGG
8	CAGCCTCG	N710	CGAGGCTG	CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGG
9	TGCCTCTT	N711	AAGAGGCA	CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGG
10	TCCTCTAC	N712	GTAGAGGA	CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGG
11	TCATGAGC	N714	GCTCATGA	CAAGCAGAAGACGGCATACGAGATTCATGAGCGTCTCGTGGGCTCGG
12	CCTGAGAT	N715	ATCTCAGG	CAAGCAGAAGACGGCATACGAGATCCTGAGATGTCTCGTGGGCTCGG
13	TAGCGAGT	N716	ACTCGCTA	CAAGCAGAAGACGGCATACGAGATTAGCGAGTGTCTCGTGGGCTCGG
14	GTAGCTCC	N718	GGAGCTAC	CAAGCAGAAGACGGCATACGAGATGTAGCTCCGTCTCGTGGGCTCGG
15	TACTACGC	N719	GCGTAGTA	CAAGCAGAAGACGGCATACGAGATTACTACGCGTCTCGTGGGCTCGG
16	AGGCTCCG	N720	CGGAGCCT	CAAGCAGAAGACGGCATACGAGATAGGCTCCGGTCTCGTGGGCTCGG
17	GCAGCGTA	N721	TACGCTGC	CAAGCAGAAGACGGCATACGAGATGCAGCGTAGTCTCGTGGGCTCGG
18	CTGCGCAT	N722	ATGCGCAG	CAAGCAGAAGACGGCATACGAGATCTGCGCATGTCTCGTGGGCTCGG
19	GAGCGCTA	N723	TAGCGCTC	CAAGCAGAAGACGGCATACGAGATGAGCGCTAGTCTCGTGGGCTCGG
20	CGCTCAGT	N724	ACTGAGCG	CAAGCAGAAGACGGCATACGAGATCGCTCAGTGTCTCGTGGGCTCGG
21	GTCTTAGG	N726	CCTAAGAC	CAAGCAGAAGACGGCATACGAGATGTCTTAGGGTCTCGTGGGCTCGG
22	ACTGATCG	N727	CGATCAGT	CAAGCAGAAGACGGCATACGAGATACTGATCGGTCTCGTGGGCTCGG
23	TAGCTGCA	N728	TGCAGCTA	CAAGCAGAAGACGGCATACGAGATTAGCTGCAGTCTCGTGGGCTCGG
24	GACGTCGA	N729	TCGACGTC	CAAGCAGAAGACGGCATACGAGATGACGTCGAGTCTCGTGGGCTCGG