Brandon:LabNotes/Project1/2012-5-8: Difference between revisions
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*2. Addition of T7 adapters, (adapter 1, adapter 2) with PCR amplification | *2. Addition of T7 adapters, (adapter 1, adapter 2) with PCR amplification | ||
*3. In vitro transcription with T7, overnight for 16 hours. | *3. In vitro transcription with T7, overnight for 16 hours. | ||
*4. PolyA addition with Terminal Transferase | *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. | |||
Revision as of 06:18, 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 and cDNA synthesis) Blue 5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN -3' 5'- GGG AGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN -3' Orange 5'- CTACATGAG[TTAATACGACTCACTATAGGG]AGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN -3' 5'- GGG AGCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG NNNNNN -3' PolyA tail added with TDT 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'