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 of Shendure protocol with ME-t7
- 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) 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): Orange <-- T7 Comp 5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCT[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3' 3'- [AGAGGGATATCACTCAGCATAATT]AGAGTAC -5' Blue 5'- CATGAGA[TTAATACGACTCACTATAGGGAGA] -3' 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???): primers for ends can also be used, same primers used in nextera. (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.
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'