Brandon:LabNotes/Project1/2012-5-8: Difference between revisions

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>Bsos
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             5'- CATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGT -3'                            3'- AGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTAC -5'
             5'- CATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGT -3'                            3'- AGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTAC -5'
               Adapter 1      T7 -->                                                                    Adapter 2                <--  T7
               Adapter 1      T7 -->                                                                    Adapter 2                <--  T7
After PCR with Adatper 1/Adapter 2  with T7(and cDNA generation)
                                                                                                      Adapter 2                    <--  T7
                                    Blue                                              Orange        3'- AGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTAC -5'
5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCT[CCCTATAGTGAGTCGTATTAA]CTCATG
3'- GATGTACTC[AATTATGCTGAGTGATATCCC]TCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTAC
                  T7 -->
Orange
   
   
   
   
*If doing single reads then lose out on any insertions that have Orange adapters ONLY in them?, since that is where nextera read primer 2 is.
*If doing single reads then lose out on any insertions that have Orange adapters ONLY in them?, since that is where nextera read primer 2 is.

Revision as of 18:29, 9 May 2012

Nextera modification, Adding T7 after tagmentation

  • T7 would be added by PCR after tagmentation.
  • IVT will be used for amplification and should result in less sample loss and capture of more insertions
  • adapters will be redesigned for both adapter 1 and adapter 2
  • Efficiency will be high in terms of capture of insertions, but not 100% since PCR is not 100% efficient
  • information taken from "nextera Detailed Nextera Protocol.pdf", and "nextera sample prep protocol 307pl0611.pdf"
  • flowcell sequences present on LC Sciences DMR probes design


  • Current sequences and method used by nextera protocol:
Current nextera sequences

5'-G[CCTCCCTCGCGCCATCAG][AGATGTGTATAAGAGACAG]-3'
          Blue                  ME

5'-G[CCTTGCCAGCCCGCTCAG][AGATGTGTATAAGAGACAG]-3' 
          Orange                ME


Assumed nextera transposons for transposon loading or before restriction digest after loading.

Blue
5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'
                   3'- TCTACACATATTCTCTGTC/5phos/ -5'

Orange
5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG -3' 
                   3'- TCTACACATATTCTCTGTC/5phos/ -5'


After tagementation fragment options:
-single insertion not able to make a PCRable fragment
-Insertion into bottom strand will just reverse the orientation, orange and/or blue will functional.


Orange                                                        Blue
5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- CTGTCTCTTATACACATCT -3'
                   3'- TCTACACATATTCTCTGTC ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5'

Blue                                                          Orange
5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- CTGTCTCTTATACACATCT -3'
                   3'- TCTACACATATTCTCTGTC ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'

Orange                                                        Orange
5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- CTGTCTCTTATACACATCT -3'
                   3'- TCTACACATATTCTCTGTC ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'

Blue                                                          Blue
5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- CTGTCTCTTATACACATCT -3'
                   3'- TCTACACATATTCTCTGTC ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5'


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

Orange                                                        Blue
5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGC -3'
3'- CGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5'

Blue                                                          Orange
5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'

Orange                                                        Orange
5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
3'- CGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'

Blue                                                          Blue
5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGC -3'
3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5' 


Now PCRable, adding on adapters.


                                                                                                   Adapter 2                  barcode (illuminia bPCR adaptor 2)
                              Blue                                                  Orange         3'- GACTCGCCCGACCGTTCCGTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                              5'- GCCT CCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                              3'- CGGA GGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]CCCTCGCGCCATCAG -3'
Adapter 1  (illuminia bPCR adaptor)

Amplification with primer 1 and primer 2.


                             Blue                                                 Orange                          (primer 2)     3'- AGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG[bc]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGTCTGGC[bc]TAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGA -3'  (primer 1)

 
Nextera read primers for sequencing:

                             Blue                                                 Orange
                                                                              3'- GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'  (nextera read primer 2)
5'- AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG[bc]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGTCTGGC[bc]TAGAGCATACGGCAGAAGACGAAC -5'
   (nextera read primer 1)    5'-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG-3'
                                             (nextera index read primer)      5'- CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG -3'



After single insertion

            Orange                                 Blue
NNNNN------ CTGTCTCTTATACACATCT -3'            5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'            3'- TCTACACATATTCTCTGTC ------NNNN

  • modification and addition of T7 with in place of adapter sequences
  • ME sequence + Blue or orange sequence = length of nextera read primer 1, nextera read primer 2.
  • Adapter 1 to Blue primer, Adapter 2 to Orange primer. Bother will have T7 on them, thus both ends will always be tagged.
    • flipped orientations results in the reversed strandedness of T7 synthesis direction.


                                                                                                    Adapter 2                  barcode (illuminia bPCR adaptor 2)
                              Blue                                                   Orange         3'- GACTCGCCCGACCGTTCCGTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                              5'- GCCT CCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                              3'- CGGA GGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]CCCTCGCGCCATCAG -3'
Adapter 1  (illuminia bPCR adapter)


                                                                                                     Adapter 2                     <--   T7
                                 Blue                                                 Orange        3'- AGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTAC -5'
                                 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                                 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGT -3'
 Adapter 1          T7 -->

            5'- CATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGT -3'                             3'- AGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTAC -5'
              Adapter 1      T7 -->                                                                     Adapter 2                 <--   T7

After PCR with Adatper 1/Adapter 2  with T7(and cDNA generation)

                                                                                                     Adapter 2                     <--   T7
                                    Blue                                              Orange        3'- AGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTAC -5'
5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCT[CCCTATAGTGAGTCGTATTAA]CTCATG
3'- GATGTACTC[AATTATGCTGAGTGATATCCC]TCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTAC
                  T7 -->

Orange








  • If doing single reads then lose out on any insertions that have Orange adapters ONLY in them?, since that is where nextera read primer 2 is.