Brandon:LabNotes/Project1/2015-8-3: Difference between revisions

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>Bsos
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*Thus will redesign THS-seq using Nextera XT V2 sequencing/index/flowcell/amplification primers.
*Thus will redesign THS-seq using [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/Nextera_XT_V2_sequences_and_outline Nextera XT V2 sequencing/index/flowcell/amplification primers.]




Line 45: Line 45:
   
   
   
   
  5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot)
  5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-T7tspn-bot)
   
   
   '''T7 description:'''
   '''T7 description:'''
Line 57: Line 57:
   
   
         5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’      (T7-nXTv2-1-top)
         5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’      (T7-nXTv2-1-top)
                                                     3'- TCTACACATATTCTCTGTC [Phos]-5' (T7tspn-bot)
                                                     3'- TCTACACATATTCTCTGTC [Phos]-5' (P-T7tspn-bot)
   
   
   
   
Line 104: Line 104:
  '''Extension with BST or klenow exo- or DNAP1 or Taq2x (been using taq2x)'''
  '''Extension with BST or klenow exo- or DNAP1 or Taq2x (been using taq2x)'''
   
   
  DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'                (Tm=70, anneal@64C) (sss_nXTv2_prmr)
  DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'                (Tm=70, anneal@64C) (P-sss_nXTv2_prmr)
  cDNA      3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'
  cDNA      3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'
   
   
Line 110: Line 110:
  '''Now have double stranded DNA, which should be long.'''
  '''Now have double stranded DNA, which should be long.'''
   
   
  DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' (Tm=70, anneal@64C) (sss_nXTv2_prmr)
  DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' (Tm=70, anneal@64C) (P-sss_nXTv2_prmr)
  cDNA      3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'
  cDNA      3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'


Line 117: Line 117:
          
          
     5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'        (nXTv2_i7_top)
     5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'        (nXTv2_i7_top)
                   3'- TCTACACATATTCTCTGTC/5Phos/ -5' (nXTv2_i7_bot)
                   3'- TCTACACATATTCTCTGTC/5Phos/ -5' (P-nXTv2_i7_bot)
   
   
   
   
     5'- /5Phos/CTGTCTCTTATACACATCT -3'                (nXTv2_i7_bot)
     5'- /5Phos/CTGTCTCTTATACACATCT -3'                (P-nXTv2_i7_bot)
           3’- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (nXTv2_i7_top)
           3’- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (nXTv2_i7_top)
                
                
Line 143: Line 143:
   
   
  5'- AATGATACGGCGACCACCGA -3'                                  (i5_PCR_ILAdptr, Tm=59C)
  5'- AATGATACGGCGACCACCGA -3'                                  (i5_PCR_ILAdptr, Tm=59C)
  5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGC -3'    (i5_shrt, comp region Tm=60C, whole=76C)
  5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGC -3'    (i5XX_T7-1_shrt, comp region Tm=60C, whole=76C)
  5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTC -3' (i5_long, comp region Tm=65C, whole=77C)
  5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTC -3' (i5XX_T7-1_long, comp region Tm=65C, whole=77C)
       5’- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3' (i5_orig, comp region Tm=56C, whole=75C)
       5’- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3' (i5XX_orig, comp region Tm=56C, whole=75C)
                                       XXXXXX
                                       XXXXXX
                       DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
                       DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
                       cDNA        3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                       cDNA        3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                              (i7_orig, comp region Tm=57C, whole=73C)  3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                            (i7XX_orig, comp region Tm=57C, whole=73C)  3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                              (i7_long, comp region Tm=59C, whole=73C) 3'- GAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                            (i7XX_long, comp region Tm=59C, whole=73C) 3'- GAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                              (i5_PCR_ILAdptr, Tm=58C)                                         3'- AGCATACGGCAGAAGACGAAC -5'
                                                            (i5_PCR_ILAdptr, Tm=58C)                                           3'- AGCATACGGCAGAAGACGAAC -5'
   
   
  '''Read Primers:'''
  '''Read Primers:'''
Line 161: Line 161:
  5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
  5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
  3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
  3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                       3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                       3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                           <----- read2 nXT
                                                                                           <----- read2 nXT
primers to order:
(T7-nXTv2_i5-1-top) 5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’
(T7-nXTv2_i5-2-top) 5'- CATGGAATTAATACGACTCACTATAGGGAGACTCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’
(P-T7tspn-bot)      5'- /5Phos/CTGTCTCTTATACACATCT -3'
(P-sss_nXTv2_prmr)  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'
(nXTv2_i7_top)      5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'       
(P-nXTv2_i7_bot)    5'- /5Phos/CTGTCTCTTATACACATCT -3'
(i5_PCR_ILAdptr)    5'- AATGATACGGCGACCACCGA -3'
(i5_PCR_ILAdptr)    5'- CAAGCAGAAGACGGCATACGA -3'
(i502_T7-1_shrt)    5'- AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGC -3'
(i503_T7-1_shrt)    5'- AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGC -3'
(i505_T7-1_shrt)    5'- AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGC -3'
(i506_T7-1_shrt)    5'- AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGC -3'
(i502_T7-1_long)    5'- AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGCGTC -3'
(i503_T7-1_long)    5'- AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGCGTC -3'
(i505_T7-1_long)    5'- AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGCGTC -3'
(i506_T7-1_long)    5'- AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGCGTC -3'
(i701_long)        5'- CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAG -3'
(i702_long)        5'- CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAG -3'
(i703_long)        5'- CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAG -3'
(i704_long)        5'- CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAG -3'
'''original Nextera XT V2 i5/i7 barcode sequencers and primers'''
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

Revision as of 01:56, 5 August 2015

Update of THS-seq protocol sequences to use Nextera XT V2 sequences



  • Called illumina and they said it is either sequencer not being cleaned right, or concentrations are off, or sequences in library generation protocol are not compatible. Considering currently old nextera sequences are being used from 5 years ago, and all current systems illumina sequencing systems do not use them, then maybe that is the problem.



Sequences information, Using T7-nXTv2_i5-1-top and T7-nXTv2_i5-2-top

Sequences:
ORIGINAL T7tspn-top2: (69 bp)
5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][TCCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’ (T7tspn-top2)
      spcr      T7 forward    required spcr   read sequence  spcr       ME 

NEXTERA XT V2 R1 TOP (64 bp)
5'- [CATGG][AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’            (T7-nXTv2_i5-1-top)
     \---/  \-/\---------------------/ \--------------------------------/
     spcr T7 rec.  T7 req. forward         read seq.  \-----------------/
                                                              ME  
                                                                                 
NEXTERA XT V2 R1 TOP + EXTRA (69 bp)
5'- [CATGG][AATTAATACGACTCACTATAGGGAGA][CTCCT][TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’    (T7-nXTv2_i5-2-top)    
     \---/  \-/\---------------------/  \---/  \--------------------------------/
     spcr T7 rec.  T7 req. forward      spcr      read seq.   \-----------------/
                                                                     ME  


5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-T7tspn-bot)

 T7 description:
                                       +1
         [CATGG][AAT][TAATACGACTCACTATA|G|GGAGA]
[end sequence]   rec.   T7 req -->        [front sequence for optimal binding]



After Annealing:

       5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’       (T7-nXTv2-1-top)
                                                    3'- TCTACACATATTCTCTGTC [Phos]-5' (P-T7tspn-bot)


5'- [phos] CTGTCTCTTATACACATCT -3'
      3’-  GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5’


Fragments created after transposition:

5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG    NNNNNNNNNN--------- 5P/CTGTCTCTTATACACATCT -3'
                                             3'- TCTACACATATTCTCTGTC/5P ---------NNNNNNNNNN    GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5’


Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom:

5'- NNN--------- 5P/CTGTCTCTTATACACATCT -3'           5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG    NNNNNNNNNNNN -3'
3'- NNNNNNNNNNNN    GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5’           3'- TCTACACATATTCTCTGTC/5P ---------NNN -5'


After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also)

5'- CATGG[AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN -3'
3'- GTACC[TTAATTATGCTGAGTGATATCCCTCT]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC (NNNNNNNN)NNN -5'

5'- NNN(NNNNNNNNN) CTGTCTCTTATACACATCTGACGCTGCCGACGA[TCTCCCTATAGTGAGTCGTATTAATT]CCATG -3'
3'- NNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT[AGAGGGATATCACTCAGCATAATTAA]GGTAC -5’   



After IVT, All RNA strands will have same 5' end. RNA is also purified after IVT.
First G in GGG is the +1 site for the beginning of RNA synthesis for T7.
for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator)
All Tm's calculated using, "taq DNA polymerase", "taq 2X Mastermix"

RNA 5’- GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN -3'



After single strand cDNA synthesis to form cDNA/RNA hybrid

RNA  5’- GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' 
cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'


After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer.
Extension with BST or klenow exo- or DNAP1 or Taq2x (been using taq2x)

DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'                 (Tm=70, anneal@64C) (P-sss_nXTv2_prmr)
cDNA       3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'


Now have double stranded DNA, which should be long.

DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' (Tm=70, anneal@64C) (P-sss_nXTv2_prmr)
cDNA       3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'
Custom Nextera i7 transposome
   i7 adapter
       
   5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'        (nXTv2_i7_top)
                  3'- TCTACACATATTCTCTGTC/5Phos/ -5' (P-nXTv2_i7_bot)


   5'- /5Phos/CTGTCTCTTATACACATCT -3'                (P-nXTv2_i7_bot)
          3’- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (nXTv2_i7_top)
              

After custom nextera tagmentation:

DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCT -3'
cDNA       3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'



After fill in:

DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
cDNA       3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'


Amplification (to add i5/i7 barcodes):
for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator)
All Tm's calculated using, "taq DNA polymerase", "taq 2X Mastermix"


5'- AATGATACGGCGACCACCGA -3'                                  (i5_PCR_ILAdptr, Tm=59C)
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGC -3'    (i5XX_T7-1_shrt, comp region Tm=60C, whole=76C)
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTC -3' (i5XX_T7-1_long, comp region Tm=65C, whole=77C)
      5’- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3' (i5XX_orig, comp region Tm=56C, whole=75C)
                                     XXXXXX
                     DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
                     cDNA        3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                            (i7XX_orig, comp region Tm=57C, whole=73C)   3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                            (i7XX_long, comp region Tm=59C, whole=73C) 3'- GAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                            (i5_PCR_ILAdptr, Tm=58C)                                           3'- AGCATACGGCAGAAGACGAAC -5'

Read Primers:
Using i5_shrt or i5_long and i7_long
 

         nXT i5 index read ----->                           nXT read1 ----->                        nXT i7 index read ----->
5'- AATGATACGGCGACCACCGAGATCTACAC -3'  5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'      5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                      3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'  
                                                                                          <----- read2 nXT
primers to order:


(T7-nXTv2_i5-1-top) 5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’
(T7-nXTv2_i5-2-top) 5'- CATGGAATTAATACGACTCACTATAGGGAGACTCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’
(P-T7tspn-bot)      5'- /5Phos/CTGTCTCTTATACACATCT -3'
(P-sss_nXTv2_prmr)  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'
(nXTv2_i7_top)      5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'        
(P-nXTv2_i7_bot)    5'- /5Phos/CTGTCTCTTATACACATCT -3'
(i5_PCR_ILAdptr)    5'- AATGATACGGCGACCACCGA -3'
(i5_PCR_ILAdptr)    5'- CAAGCAGAAGACGGCATACGA -3'
(i502_T7-1_shrt)    5'- AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGC -3'
(i503_T7-1_shrt)    5'- AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGC -3'
(i505_T7-1_shrt)    5'- AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGC -3'
(i506_T7-1_shrt)    5'- AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGC -3'
(i502_T7-1_long)    5'- AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGCGTC -3'
(i503_T7-1_long)    5'- AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGCGTC -3'
(i505_T7-1_long)    5'- AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGCGTC -3'
(i506_T7-1_long)    5'- AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGCGTC -3'
(i701_long)         5'- CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAG -3'
(i702_long)         5'- CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAG -3'
(i703_long)         5'- CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAG -3'
(i704_long)         5'- CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAG -3'


original Nextera XT V2 i5/i7 barcode sequencers and primers

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