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

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*T7 would be added by PCR 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
*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
*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
*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"
*information taken from "nextera Detailed Nextera Protocol.pdf", and "nextera sample prep protocol 307pl0611.pdf"
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*Current sequences and method used by nextera protocol:
===Current sequences and method used by nextera protocol:===
  Current nextera sequences
  Current nextera sequences
   
   
Line 71: Line 71:
   
   
   
   
  Now PCRable, adding on adapters.
  Now PCRable, adding on adaptors.
   
   
   
   
                                                                                                     Adapter 2                  barcode (illuminia bPCR adaptor 2)
                                                                                                     Adaptor 2                  barcode (illuminia bPCR adaptor 2)
                               Blue                                                  Orange        3'- GACTCGCCCGACCGTTCCGTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                               Blue                                                  Orange        3'- GACTCGCCCGACCGTTCCGTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                               5'- GCCT CCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                               5'- GCCT CCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                               3'- CGGA GGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
                               3'- CGGA GGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]CCCTCGCGCCATCAG -3'
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]CCCTCGCGCCATCAG -3'
  Adapter 1  (illuminia bPCR adaptor)
  Adaptor 1  (illuminia bPCR adaptor)
   
   
  Amplification with primer 1 and primer 2.
  Amplification with primer 1 and primer 2.
Line 108: Line 108:
   
   


*modification and addition of T7 with in place of adapter sequences
 
 
===modification of Shendure protocol with ME-t7===
 
*modification and addition of T7 in place of adaptor sequences
**see [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-2-6 T7 information etc]
**see [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-2-6 T7 information etc]
**first G in GGG is the +1 site
**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.
*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.
*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.
**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 (ME-T7-RC)
            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'
        (ME-T7-RC) T7 comp -->
Fill-in and ligation to append ME-T7 adaptors. (shendure used T4 DNAP and ampligase)
  Orange                                                                    Blue                    <-- T7 Comp
            5'- GCCTTGCCAGCCCGCTCAG AGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCT[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3'
                X XXXX XXXXXX XXXXX                                                            XXXXXXXXXX X X XX X     
3'-GTACTCT[AATTATGCTGAGTGATATCCCTCT]TCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGA GACTACCGCGCTCCCTCCG -5'
                T7 comp -->
Denature, anneal with T7 primer for second strand synthesis (taq polymerase):
Can also try IVT without second strand synthesis, (reports possibly saying T7 can initiate
without needing a double stranded template).
  Orange                                                                              <-- T7 Comp
5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCT[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3'
                                                                            3'-  [AGAGGGATATCACTCAGCATAATT]AGAGTAC -5'  (T7prmr-sss)
                                                                              Blue
5'- CATGAGA[TTAATACGACTCACTATAGGGAGA] -3'  (T7prmr-sss)
3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]TCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5'
                T7 comp -->
After second strand synthesis:
    Orange                                                                              <-- T7 Comp
5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCT[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3'
3'- CGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGA[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5'
                                                                              Blue
5'- CATGAGA[TTAATACGACTCACTATAGGGAGA]AGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGC -3'
3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]TCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5'
                  T7 comp -->
After RNA generation from IVT. addition of lawn adapters (then fragmentation and polyA addition???):
Can also try adding adapter to 3'OH end with T4 RNA ligase.
primers for ends can also be used, same primers used in nextera.
(could update with polyA addition sometime, which will probably be done instead)
      (2nd strand synthesis)(bPCR adaptor)
  5'- AATGATACGGCGACCACCGA -3'  (primer 1, from nextera)
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGAAGATGTGTATAAGAGACAG -3'                    Blue
                              RNA  5'- GGGAGAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGC -3'          (bPCR adaptor)
                                                                                              3'-  TAGAGACTACCGCGCTCCCTCCGCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                                                    3'- AGCATACGGCAGAAGACGAAC -5'
                                                                                                    (first strand synthesis-B)      (primer 2, from nextera) 
    (2nd strand synthesis)(bPCR adaptor)
  5'- AATGATACGGCGACCACCGA -3'  (primer 1, from nextera)
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGAAGATGTGTATAAGAGACAG -3'                  Orange
                              RNA  5-' GGGAGAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'          (bPCR adaptor)
                                                                                              3'-  TAGAGACTCGCCCGACCGTTCCGCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                                                    3'- AGCATACGGCAGAAGACGAAC -5'
                                                                                                    (first strand synthesis-O)      (primer 2, from nextera)
now ready for sequencing:
                                                                                      Blue
                                      (read 1 primer)
                                    5'- GGGAGAAGATGTGTATAAGAGACAG -3'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGCGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3'
3'- [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCTTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCGCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                3'- GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCC -5'
                                                                                      (read primer 2)
                                                                                                    5'- TGATGGCGCGAGGGAGGCGACCG -3' (index read primer 1)
                                                                                    Orange
                                      (read 1 primer)
                                    5'- GGGAGAAGATGTGTATAAGAGACAG -3'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCCGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3'
3'- [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCTTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGGCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'         
                                                                                    GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG
                                                                                      (read primer 2)
                                                                                                    5'- CTGAGCGGGCTGGCAAGGCCGACCG -3' (index read primer 2)
===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.


   
   
                                                                                                     Adapter 2                  barcode (illuminia bPCR adaptor 2)
                                                                                                     Adaptor 2                  barcode (illuminia bPCR adaptor 2)
                               Blue                                                  Orange        3'- GACTCGCCCGACCGTTCCGTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                               Blue                                                  Orange        3'- GACTCGCCCGACCGTTCCGTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                               5'- GCCT CCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                               5'- GCCT CCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                               3'- CGGA GGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
                               3'- CGGA GGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]CCCTCGCGCCATCAG -3'
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]CCCTCGCGCCATCAG -3'
  Adapter 1  (illuminia bPCR adapter)
  Adaptor 1  (illuminia bPCR adaptor)
   
   
   
   
                                                                                                       Adapter 2                    <--  T7
                                                                                                       Adaptor 2                    <--  T7
                                   Blue                                                Orange        3'- AGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTACATC -5'
                                   Blue                                                Orange        3'- AGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTACATC -5'
                                   5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                                   5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                                   3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
                                   3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
  5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGT -3'
  5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGT -3'
   Adapter 1          T7 -->
   Adaptor 1          T7 -->
   
   
             5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGT -3'                            3'- AGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTACATC -5'
             5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGT -3'                            3'- AGACTCGCCCGACCGTTCCGA[GGGATATCACTCAGCATAATT]GAGTACATC -5'
               Adapter 1      T7 -->                                                                    Adapter 2                <--  T7
               Adaptor 1      T7 -->                                                                    Adaptor 2                <--  T7
   
   
  After PCR with Adatper 1/Adapter 2  with T7(and cDNA generation)
  After PCR with Adatper 1/Adaptor 2  with T7(and cDNA generation)
   
   
                                     Blue                                              Orange                                    <-- T7
                                     Blue                                              Orange                                    <-- T7
Line 141: Line 305:
                   T7 -->
                   T7 -->
   
   
  RNA synthesized by T7, then RNA fragmentation and cDNA synthesis)
  RNA synthesized by T7, then RNA fragmentation, RNA A-tailing [http://products.invitrogen.com/ivgn/product/AM1350 Ambion PolyA tailing kit], then cDNA synthesis.
   
   
  Blue
  Blue
  5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN  -3'
  5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN  -3'
                            5'- GGG AGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN  -3'
                        RNA  5'- GGG AGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN  -3'
   
   
  Orange
  Orange
  5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN  -3'
  5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN  -3'
                            5'- GGG AGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN  -3'
                        RNA  5'- GGG AGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN  -3'
   
   
  PolyA tail added with TDT
  Adaptors for first/second strand cDNA synthesis (after polyA tailing of RNA frags???):
   
   
  Blue
  Blue
  5'- GGGAGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAA -3'
        (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)
 
   
   
  Orange
  lawn 1 adaptors added using orange and blue sequences, and lawn 2 adaptors added to polyA tail
5'- GGGAGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAA -3'
   
   
lawn 1 adapters added using orange and blue sequences, and lawn 2 adapters added to polyA tail
  Blue
  Blue
                                                                                                                 (illuminia bPCR adapter)
                                                                                                                 (illuminia bPCR adaptor)
                                                                                     3'- V TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                     3'- V TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                     5'- GGGAGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAA -3'
                                     5'- GGGAGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAA -3'
                                     3'- CCCTCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTT -5'
                                     3'- CCCTCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTT -5'
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTCCCTCGCGCCATCAG -3'
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTCCCTCGCGCCATCAG -3'
           (illuminia bPCR adapter, sequence modified)   
           (illuminia bPCR adaptor, sequence modified)   
 
                                                                                                               (illuminia bPCR adapter)
                                                                                                               (illuminia bPCR adaptor)
   Orange                                                                            3'- V TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
   Orange                                                                            3'- V TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                     5'- GGGAGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAA -3'  
                                     5'- GGGAGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAA -3'  
                                     3'- CCCTCGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTT -5'
                                     3'- CCCTCGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTT -5'
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTTGCCAGCCCGCTCAGAGATGT -3'
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTTGCCAGCCCGCTCAGAGATGT -3'
                 (illuminia bPCR adapter, sequence modified)   
                 (illuminia bPCR adaptor, sequence modified)   
   
   
   
   
  Now Ready for sequencing:
  Now Ready for sequencing:
 
  Blue
  Blue
                                               (nextera read primer 1)
                                               (nextera read primer 1)
Line 184: Line 356:
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3'
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3'
  3'- [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
  3'- [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
         (illuminia bPCR adapter)                                                                                (illuminia bPCR adapter)
         (illuminia bPCR adaptor)                                                                                (illuminia bPCR adaptor)
   
   
    
    
Line 192: Line 364:
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3'
  5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN AAAAAAAAAAAAGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3'
  3'- [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
  3'- [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNN TTTTTTTTTTTTCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
         (illuminia bPCR adapter)                                                                                (illuminia bPCR adapter)
         (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 adapters
*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:
*nextera read 1 and nextera read 2 can be combined in the sequencing reaction, then all combinations will be sequenced:
**blue orange
**blue orange
Line 206: Line 378:




Primers to order:
===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
  (ME-T7-RC)  5'- CTGTCTCTTATACACATCT[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3'
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGCCTTGCCAGCCCGCTCAGAGATGT -3'
                (illuminia bPCR adapter, sequence modified)
   
   
   
   
                        (illuminia bPCR adapter)
  (T7prmr-sss) 5'- CATGAGA[TTAATACGACTCACTATAGGGAGA] -3'
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'

Latest revision as of 22:53, 16 May 2012

Nextera modification, Adding T7 after tagmentation[edit]

  • 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:[edit]

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 of Shendure protocol with ME-t7[edit]

  • 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 (ME-T7-RC)
            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'
        (ME-T7-RC) T7 comp -->


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

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


Denature, anneal with T7 primer for second strand synthesis (taq polymerase):
Can also try IVT without second strand synthesis, (reports possibly saying T7 can initiate 
without needing a double stranded template).

  Orange                                                                              <-- T7 Comp
5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCT[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3'
                                                                            3'-  [AGAGGGATATCACTCAGCATAATT]AGAGTAC -5'  (T7prmr-sss)

                                                                             Blue
5'- CATGAGA[TTAATACGACTCACTATAGGGAGA] -3'  (T7prmr-sss)
3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]TCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5'
                T7 comp -->



After second strand synthesis:

   Orange                                                                               <-- T7 Comp
5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCT[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3'
3'- CGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGA[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5'

                                                                             Blue
5'- CATGAGA[TTAATACGACTCACTATAGGGAGA]AGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGC -3'
3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]TCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCG -5'
                  T7 comp -->


After RNA generation from IVT. addition of lawn adapters (then fragmentation and polyA addition???):
Can also try adding adapter to 3'OH end with T4 RNA ligase.
primers for ends can also be used, same primers used in nextera.
(could update with polyA addition sometime, which will probably be done instead)

      (2nd strand synthesis)(bPCR adaptor)
 5'- AATGATACGGCGACCACCGA -3'  (primer 1, from nextera)
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGAAGATGTGTATAAGAGACAG -3'                    Blue
                              RNA  5'- GGGAGAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGC -3'           (bPCR adaptor)
                                                                                              3'-  TAGAGACTACCGCGCTCCCTCCGCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                                                   3'- AGCATACGGCAGAAGACGAAC -5'
                                                                                                   (first strand synthesis-B)       (primer 2, from nextera)   

    (2nd strand synthesis)(bPCR adaptor)
 5'- AATGATACGGCGACCACCGA -3'  (primer 1, from nextera)
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGAAGATGTGTATAAGAGACAG -3'                   Orange
                              RNA  5-' GGGAGAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'          (bPCR adaptor)
                                                                                             3'-   TAGAGACTCGCCCGACCGTTCCGCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                                                   3'- AGCATACGGCAGAAGACGAAC -5'
                                                                                                   (first strand synthesis-O)       (primer 2, from nextera) 




now ready for sequencing:

                                                                                      Blue
                                     (read 1 primer)
                                   5'- GGGAGAAGATGTGTATAAGAGACAG -3' 
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGCGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3'
3'- [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCTTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCGCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                3'- GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCC -5'
                                                                                     (read primer 2)
                                                                                                    5'- TGATGGCGCGAGGGAGGCGACCG -3' (index read primer 1)


                                                                                    Orange
                                      (read 1 primer)
                                   5'- GGGAGAAGATGTGTATAAGAGACAG -3' 
5'- [AATGATACGGCGACCACCGAGATCTACACGCCT]GGGAGAAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCCGACCG[bc][ATCTCGTATGCCGTCTTCTGCTTG] -3'
3'- [TTACTATGCCGCTGGTGGCTCTAGATGTGCGGA]CCCTCTTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCGGCTGGC[bc][TAGAGCATACGGCAGAAGACGAAC] -5'           
                                                                                    GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG
                                                                                     (read primer 2)
                                                                                                   5'- CTGAGCGGGCTGGCAAGGCCGACCG -3' (index read primer 2)

Method using PCR for addition of T7 polymerase[edit]

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:[edit]

(ME-T7-RC)   5'- CTGTCTCTTATACACATCT[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3'


(T7prmr-sss) 5'- CATGAGA[TTAATACGACTCACTATAGGGAGA] -3'