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==Transposome design and idea== *design a transposon that will be will be able to capture a sequence with single insertions. **current nextera/epicentre transposomes require two insertions to generate a fragment. fragmentation and end labeling is done in one step. **however this results in 50% sample loss at least due to orientations of insertion of the transposomes. Also insertions can be to close to each other or to far from each other. A-----B is a transposon ==== is DNA viable insertion: =====A-----------B=====A-----------B===== =====B-----------A=====B-----------A===== wrong ends: =====B-----------A=====A-----------B===== =====A-----------B=====B-----------A===== to far: =====A-----------B=========================A-----------B===== to close: =====A-----------B=A-----------B===== *Current methods get low coverage, which has much room for improvement, especially with low cell numbers. ===transposome=== *consists of of mosaic ends (ME), followed by illuminia adapters (ILA), followed by T7 RNA Polymerase promoter (T7). Each sequence will be a reverse complement of the other. **Even with one T7, single insertions will provide usable data regardless of orientation. Extension will either be + or - strand. *Thus extension will happen on either side of the insertion during in vitro transcription. With this modification, single insertions will provide usable data. *Extensions will happen on either side too, thus providing twice the amount of data. **reads can be matched together since a 9 bp duplication happens on either side of the insertion, and one read is on + stand and the other is on the - strand. transposon: ME-ILA-<T7----T7>-ILA-ME after insertion: ====[9bp]ME-ILA-<T7----T7>-ILA-ME[9bp]===== ===procedure outline=== *1. design and synthesize transposon sequence *2. PCR primers to make a lot of copies of transposon. possible issue with secondary structure getting in the way of amplification? since nearly identical sequences on on each half of the transposon. *3. add transposon and transpoase together to make transposome complex. *4. Add to open DNA, make sure to use RNase!!, or small amounts of DNA to ensure working correctly. optimize insertion distance? *5. In Vitro Transcription (IVT) do overnight or whatever needed. *only occurs 5' -> 3' on forward and reverse strand. *6. couple options: fragment RNA, provides more even fragment length. add NNNNNNNNN----ILA adaptors to prime addition of ILA adapter to 3 prime end(can prime either way too though), then do fragment size selection. *7. If fragment, then add T4 RNA ligase to add on ILA onto other end of fragments. adds onto 3' prime ends of RNA fragments. *8. make cDNA with ILA primers. will prime 5' -> 3', thus off of 3' ILA primers just added to RNA fragments. *9. PCR amplify with ILA adapters. *10. sequence fragments. paired end? *11. Computational analysis, since it is known that 9 bp duplicated sequences are created on either side of the insertion, then try matching them. With same 9 bp duplicated sequences, one fragment will map to forward strand, one to reverse. Thus can obtain more coverage, covering sequences that are not unique. Paired end reads for each fragment too? then can capture even more sequence. ===Next steps=== *design transposon and test if works (hardest part) *check possible secondary structure issues. *get protocols for specific steps and write outline *Notes pix for reference [[File:0203020117a-use.jpg|600px]] [[File:0203020117c-use.jpg|600px]] [[File:0203020118a-use.jpg|600px]]
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