Brandon:LabNotes/Project1/2012-5-8: Difference between revisions
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*adapters will be redesigned for both adaptor 1 and adaptor 2 | *adapters 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" | |||
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After tagementation fragment options: | After tagementation fragment options: | ||
single insertion not able to make a PCRable fragment | -single insertion not able to make a PCRable fragment | ||
-Insertion into bottom strand will just reverse the orientation, orange and/or blue will functional. | |||
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Adaptor 2 | Adaptor 2 barcode (illuminia bPCR adaptor 2) | ||
Blue Orange 3'- GACTCGCCCGACCGTTCCGTCTGGC[bc] | Blue Orange 3'- GACTCGCCCGACCGTTCCGTCTGGC[bc]TAG[AGCATACGGCAGAAGACGAAC] -5' | ||
5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3' | 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3' | ||
Adaptor 1 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' | Adaptor 1 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' |
Revision as of 23:36, 8 May 2012
Nextera modification, Adding T7
- 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 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"
- 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]TAG[AGCATACGGCAGAAGACGAAC] -5' 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3' Adaptor 1 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5' 5'- AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAG -3' 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'