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

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  5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN  -3'
  5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN  -3'
                             5'- GGG AGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN  -3'
                             5'- GGG AGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN  -3'
Adapters for first/second strand cDNA synthesis (after polyA tailing of RNA frags):
        (second strand synthesis)
      5'- GGGAGCCTCCCTCGCGCCA -3'
  RNA 5'- GGGAGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAA -3'
                                                      3'- V TTTTTTTTTTTTTTTTTT -5' (first strand synthesis)
        (second strand synthesis
      5'- GGGAGCCTTGCCAGCCCGC -3'
  RNA 5'- GGGAGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAA -3'
                                                      3'- V TTTTTTTTTTTTTTTTTT -5' (first strand synthesis)
   
   
  PolyA tail added with TDT, OR added earlier after RNA fragmentation then cDNA synthesis.
  PolyA tail added with TDT, OR added earlier after RNA fragmentation then cDNA synthesis.

Revision as of 07:09, 10 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
    • see T7 information etc
    • first G in GGG is the +1 site
    • at least 5 nucleotides needed before start of sequence to ensure correct binding
  • 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.


Protocol:

  • 1. tagmentation with nextera kit
  • 2. Addition of T7 adapters, (adapter 1, adapter 2) with PCR amplification
  • 3. In vitro transcription with T7, overnight for 16 hours.
  • 4. RNA fragmentation and then possibly A-tail the RNA
  • 5. cDNA synthesis
  • 6. PolyA addition with Terminal Transferase (if haven't A-tailed the RNA)
  • 7. Addition of bPCR lawn adapters with primer mix of sequences for (orange, blue, polyT addition)
  • 8. now ready for input into the sequencer with the combined nextera read primer 1, and nextera read primer 2.


                                                                                                   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]GAGTACATC -5'
                                 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                                 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGT -3'
 Adapter 1          T7 -->

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

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

                                    Blue                                              Orange                                     <-- T7
5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCT[CCCTATAGTGAGTCGTATTAA]CTCATGTAG -3'
3'- GATGTACTC[AATTATGCTGAGTGATATCCC]TCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTACATC -5'
                  T7 -->

RNA synthesized by T7, then RNA fragmentation, RNA A-tailing Ambion PolyA tailing kit, then cDNA synthesis.
OR RNA fragmentation, cDNA synthesis with random hexamers, A-tailing with Terminal transferase

Blue
5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN  -3'
                            5'- GGG AGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN  -3'

Orange
5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN  -3'
                            5'- GGG AGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN  -3'

Adapters for first/second strand cDNA synthesis (after polyA tailing of RNA frags):

       (second strand synthesis)
     5'- GGGAGCCTCCCTCGCGCCA -3'
 RNA 5'- GGGAGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAA -3'
                                                     3'- V TTTTTTTTTTTTTTTTTT -5' (first strand synthesis)
       (second strand synthesis
     5'- GGGAGCCTTGCCAGCCCGC -3'
 RNA 5'- GGGAGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAA -3'
                                                     3'- V TTTTTTTTTTTTTTTTTT -5' (first strand synthesis)

PolyA tail added with TDT, OR added earlier after RNA fragmentation then cDNA synthesis.

Blue
5'- GGGAGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAA -3'

Orange
5'- GGGAGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAA -3' 

lawn 1 adapters added using orange and blue sequences, and lawn 2 adapters added to polyA tail

Blue
                                                                                                               (illuminia bPCR adapter)
                                                                                   3'- V TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                   5'- GGGAGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAA -3'
                                   3'- CCCTCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTT -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTCCCTCGCGCCATCAG -3'
          (illuminia bPCR adapter, sequence modified)  

                                                                                                              (illuminia bPCR adapter)
 Orange                                                                            3'- V TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                   5'- GGGAGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAA -3' 
                                   3'- CCCTCGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTT -5'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTTGCCAGCCCGCTCAGAGATGT -3'
                (illuminia bPCR adapter, sequence modified)  


Now Ready for sequencing:

Blue
                                              (nextera read primer 1)
                                       5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG  -3'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3'
3'- [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
       (illuminia bPCR adapter)                                                                                (illuminia bPCR adapter)

 
 Orange                                   
                                            (nextera read primer 2)
                                       5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG -3'                                        
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3'
3'- [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
       (illuminia bPCR adapter)                                                                                (illuminia bPCR adapter)

Index read primer for both Orange and Blue:                                          5'- AAAAAAAAAAAACTGGC -3'
                                                                                        (index read primer)

  • PCR amplificiation with both bPCR adapters
  • nextera read 1 and nextera read 2 can be combined in the sequencing reaction, then all combinations will be sequenced:
    • blue orange
    • orange blue
    • orange orange
    • blue blue


Primers to order:

Blue
5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGT -3'
     Adapter 1      T7 -->   


Orange
3'- AGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTACATC -5' 
      Adapter 2                 <--   T7 


blue
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTCCCTCGCGCCATCAG -3'
          (illuminia bPCR adapter, sequence modified) 


orange
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTTGCCAGCCCGCTCAGAGATGT -3'
                (illuminia bPCR adapter, sequence modified) 


                        (illuminia bPCR adapter)
3'- V TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
How many A's in polyA are good for priming??


Blue
 (nextera read primer 1)
 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG  -3'


Orange
 (nextera read primer 2)
 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG -3'

Index read primer
 5'- AAAAAAAAAAAACTGGC -3'