Brandon:LabNotes/Project1/2012-5-8

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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
  • adaptors will be redesigned for both adaptor 1 and adaptor 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 adaptors.


                                                                                                   Adaptor 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'
Adaptor 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 in place of adaptor 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.
  • Adaptor 1 to Blue primer, Adaptor 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. Partially denature at 72C (5-10C higher than the Tm for ME sequences)
  • 3. Anneal a ME-T7 adaptor. make it specific for orange/blue too?
  • 4. Gap filling and ligation. (T4 DNAP/Ampligase as in Shendure protocol)
  • 5. IVT and conversion to sequencing libraries.

Summary:

T7 description:
                                                       +1
                            CATGAGA[TTAATACGACTCACTATA|G|GGAGA]
 [end sequence for optimal binding]         T7 -->      [front sequence for optimal binding]



After Nextera tagmentation:

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'



Partially denaturing at 72C, higher temp than Tm for ME sequence:

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

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

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

Blue                                                          Blue
5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN--------- -3'
                                       3'- ---------NNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5'
 
 
Anneal ME-T7 adaptor (add more sequence specific to orange/blue?)

Orange                                                                     Blue                     <-- T7 Comp
            5'- GCCTTGCCAGCCCGCTCAG AGATGTGTATAAGAGACAG NNNNNNNNN--------- CTGTCTCTTATACACATCT[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3'
                X XXXX XXXXXX XXXXX                                                            XXXXXXXXXX X X XX X      
3'-GTACTCT[AATTATGCTGAGTGATATCCCTCT]TCTACACATATTCTCTGTC ---------NNNNNNNNN GACAGAGAATATGTGTAGA GACTACCGCGCTCCCTCCG -5'
                T7 comp -->

Fill-in and ligation to append ME-T7 adaptors. (shendure used T4 DNAP and ampligase)




                                                       +1
                             CATGAGA[TTAATACGACTCACTATA|G|GGAGA]
[end sequence for optimal binding]         T7 -->          [front sequence for optimal binding]




Method using PCR for addition of T7 polymerase.


Protocol:

  • 1. tagmentation with nextera kit
  • 2. Addition of T7 adaptors, (adaptor 1, adaptor 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 adaptors 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.


                                                                                                   Adaptor 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'
Adaptor 1  (illuminia bPCR adaptor)


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

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

After PCR with Adatper 1/Adaptor 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.


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

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

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

Blue
       (second strand synthesis)
     5'- GGGAGCCTCCCTCGCGCCA -3'
 RNA 5'- GGGAGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
                                                     3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5' (first strand synthesis)
Orange
        (second strand synthesis)
     5'- GGGAGCCTTGCCAGCCCGC -3'
 RNA 5'- GGGAGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
                                                     3'- V TTTTTTTTTTTTTTTTTTTTTTTT -5' (first strand synthesis)

 

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

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

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

 
 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 adaptor)                                                                                (illuminia bPCR adaptor)

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

  • PCR amplificiation with both bPCR adaptors
  • 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'
     Adaptor 1      T7 -->   


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


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


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


                        (illuminia bPCR adaptor)
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'