Matt:LabNotes/2016-4-18: Difference between revisions
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=Convert Probes to Oligos to Order= | =Convert Probes to Oligos to Order= | ||
*Add | *Probes: [[Matt:LabNotes/2016-4-1|Human Brain]], [[Matt:LabNotes/2016-4-6|Mouse Brain]], [[Matt:LabNotes/2016-4-7|Mouse Embryo]], [[Matt:LabNotes/2016-4-8|Control Genes]] | ||
*Add Barcodes and Amplification Primers | |||
*Files in Dropbox/GradZhangLab/CA12k_Apr2016 | *Files in Dropbox/GradZhangLab/CA12k_Apr2016 | ||
*dcProbe_Sequences.txt | *dcProbe_Sequences.txt | ||
**Cycle \t Color \t 20nt Sequence | **header: "Cycle \t Color \t 20nt Sequence" | ||
**21 (7x3) total | **21 (7x3) total | ||
==Make Barcode Sequence File== | |||
*eg. B01220 ATCGGGAT... | |||
*Use MakeBarcodeSequenceFile.py in Dropbox/PythonScripts/ to make 3 'on' barcodes | *Use MakeBarcodeSequenceFile.py in Dropbox/PythonScripts/ to make 3 'on' barcodes | ||
**decoding_linkers_5-cycles.txt | **decoding_linkers_5-cycles.txt | ||
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**decoding_linkers_6-cycles_4-on.txt | **decoding_linkers_6-cycles_4-on.txt | ||
==Combine Human and Mouse Control/Housekeeping Genes== | |||
*CA12k_Apr2016_ControlGenes_OutputFile.py | *CA12k_Apr2016_ControlGenes_OutputFile.py | ||
***First need to make alter gene names so human and mouse are separate '''and''' barcodes are assigned appropriately | ***First need to make alter gene names so human and mouse are separate '''and''' barcodes are assigned appropriately | ||
**Mouse | **Mouse | ||
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***Add "h-" in front of each gene name | ***Add "h-" in front of each gene name | ||
==Add 3 'on' barcodes to probes== | |||
*Mouse Brain has only 2 'off' there 5 cycles is all that is needed to decode | |||
*Others have 3 'off' and require 6 cycles of decoding | |||
perl Probe2padlockFISSEQ_Apr2016_HumanBrain_V4.pl > outputFile_0gap_FULL_HumanBrain_V4.txt | perl Probe2padlockFISSEQ_Apr2016_HumanBrain_V4.pl > outputFile_0gap_FULL_HumanBrain_V4.txt | ||
perl Probe2padlockFISSEQ_Apr2016_MouseBrain_V7.pl > outputFile_0gap_FULL_MouseBrain_V7.txt | perl Probe2padlockFISSEQ_Apr2016_MouseBrain_V7.pl > outputFile_0gap_FULL_MouseBrain_V7.txt | ||
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perl Probe2padlockFISSEQ_Apr2016_ControlGenes_V8.pl > outputFile_0gap_FULL_ControlGenes_V8.txt | perl Probe2padlockFISSEQ_Apr2016_ControlGenes_V8.pl > outputFile_0gap_FULL_ControlGenes_V8.txt | ||
*6 cycle: 4 'on' 2 'off' barcodes | ==Add 4 'on barcodes to probes== | ||
*All probesets are 6 cycle: 4 'on' 2 'off' barcodes | |||
perl Probe2padlockFISSEQ_Apr2016_HumanBrain_6cycle_V4.pl > outputFile_0gap_FULL200_HumanBrain_V4.txt | perl Probe2padlockFISSEQ_Apr2016_HumanBrain_6cycle_V4.pl > outputFile_0gap_FULL200_HumanBrain_V4.txt | ||
perl Probe2padlockFISSEQ_Apr2016_MouseBrain_6cycle_V7.pl > outputFile_0gap_FULL200_MouseBrain_V7.txt | perl Probe2padlockFISSEQ_Apr2016_MouseBrain_6cycle_V7.pl > outputFile_0gap_FULL200_MouseBrain_V7.txt | ||
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perl padlock2oligo_order.pl ("CA12k_Apr2016_OligosOrder.txt") | perl padlock2oligo_order.pl ("CA12k_Apr2016_OligosOrder.txt") | ||
==3 'on' 180bp oligos vs 4 'on' 200bp oligos== | ==3 'on' 180bp oligos vs 4 'on' 200bp oligos== | ||
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*Conclusions: Go with 200bp | *Conclusions: Go with 200bp | ||
==Modify Certain Genes so Every Probe Unique Barcode== | |||
*Need to modify outputFile_0gap_filtered gene names so that some probes have unique barcodes so decoding can be traced back to probe instead of gene | *Need to modify outputFile_0gap_filtered gene names so that some probes have unique barcodes so decoding can be traced back to probe instead of gene | ||
**outputFile_0gap_filtered.txt -> outputFile_0gap_mod.txt | **outputFile_0gap_filtered.txt -> outputFile_0gap_mod.txt | ||
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*Remove 2,909 probes from Human Control Genes to get to 12k oligos | *Remove 2,909 probes from Human Control Genes to get to 12k oligos | ||
perl padlock2oligo_order.pl ("CA12k_Apr2016_OligosOrder.txt") | perl padlock2oligo_order.pl ("CA12k_Apr2016_OligosOrder.txt") | ||
==Final Summary== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="font-size:11pt" valign="bottom" | |||
| width="51" height="14" | Probeset | |||
| width="51" | Amplification | |||
| width="51" | # Probes | |||
| width="51" | # Barcodes | |||
| width="51" | # Genes | |||
|- style="font-size:11pt" valign="bottom" | |||
| height="14" | Human Brain | |||
| V4 | |||
| align="right" | 4,978 | |||
| align="right" | 391 | |||
| align="right" | 368 | |||
|- style="font-size:11pt" valign="bottom" | |||
| height="14" | Mouse Brain | |||
| V7 | |||
| align="right" | 1,808 | |||
| align="right" | 173 | |||
| align="right" | 150 | |||
|- style="font-size:11pt" valign="bottom" | |||
| height="14" | Mouse Embryo | |||
| V6 | |||
| align="right" | 2,469 | |||
| align="right" | 251 | |||
| align="right" | 220 | |||
|- style="font-size:11pt" valign="bottom" | |||
| height="14" | Control Genes | |||
| V8 | |||
| 85 mouse, 2660 human | |||
| 85 mouse, 245 human | |||
| 33 mouse, 171 human | |||
|} |
Revision as of 22:17, 21 April 2016
Convert Probes to Oligos to Order
- Probes: Human Brain, Mouse Brain, Mouse Embryo, Control Genes
- Add Barcodes and Amplification Primers
- Files in Dropbox/GradZhangLab/CA12k_Apr2016
- dcProbe_Sequences.txt
- header: "Cycle \t Color \t 20nt Sequence"
- 21 (7x3) total
Make Barcode Sequence File
- eg. B01220 ATCGGGAT...
- Use MakeBarcodeSequenceFile.py in Dropbox/PythonScripts/ to make 3 'on' barcodes
- decoding_linkers_5-cycles.txt
- decoding_linkers_6-cycles.txt
- Use Make4onBarcodeSequenceFile.py to make 4 'on' barcodes
- decoding_linkers_6-cycles_4-on.txt
Combine Human and Mouse Control/Housekeeping Genes
- CA12k_Apr2016_ControlGenes_OutputFile.py
- First need to make alter gene names so human and mouse are separate and barcodes are assigned appropriately
- Mouse
- Since only 85 probes each probe gets own barcode by adding #1-85 to end of gene name (eg. m-Rpl8-85_0)
- Add "m-" in front of each gene name
- Human
- Since 5,569 probes too many for unique barcodes
- Instead, each gene gets 2 barcodes. One barcode is for highest score probe for that gene. Second barcode is for the other probes of that gene.
- 191 genes x 2 = 382 barcodes
- Add "h-" in front of each gene name
- Since 5,569 probes too many for unique barcodes
Add 3 'on' barcodes to probes
- Mouse Brain has only 2 'off' there 5 cycles is all that is needed to decode
- Others have 3 'off' and require 6 cycles of decoding
perl Probe2padlockFISSEQ_Apr2016_HumanBrain_V4.pl > outputFile_0gap_FULL_HumanBrain_V4.txt perl Probe2padlockFISSEQ_Apr2016_MouseBrain_V7.pl > outputFile_0gap_FULL_MouseBrain_V7.txt perl Probe2padlockFISSEQ_Apr2016_MouseEmbryo_V6.pl > outputFile_0gap_FULL_MouseEmbryo_V6.txt perl Probe2padlockFISSEQ_Apr2016_ControlGenes_V8.pl > outputFile_0gap_FULL_ControlGenes_V8.txt
Add 4 'on barcodes to probes
- All probesets are 6 cycle: 4 'on' 2 'off' barcodes
perl Probe2padlockFISSEQ_Apr2016_HumanBrain_6cycle_V4.pl > outputFile_0gap_FULL200_HumanBrain_V4.txt perl Probe2padlockFISSEQ_Apr2016_MouseBrain_6cycle_V7.pl > outputFile_0gap_FULL200_MouseBrain_V7.txt perl Probe2padlockFISSEQ_Apr2016_MouseEmbryo_6cycle_V6.pl > outputFile_0gap_FULL200_MouseEmbryo_V6.txt perl Probe2padlockFISSEQ_Apr2016_ControlGenes_6cycle_V8.pl > outputFile_0gap_FULL200_ControlGenes_V8.txt
perl padlock2oligo_order.pl ("CA12k_Apr2016_OligosOrder.txt")
3 'on' 180bp oligos vs 4 'on' 200bp oligos
- Going with 180bp will increase % perfect oligos by 10%
- CustomArray Error Rate per Base = 0.5-1.0%
- % perfect for 180bp = 0.995^180 = 40.6%
- % perfect for 200bp = 0.995^200 = 36.7%
- Going with 180bp will decrease risk of too many truncated oligos such that the pool will not amplify
- Going with 200bp will leave much more unused barcodes
- HumanBrain: 23% to 69% unused
- MouseBrain: 38% (5cycles) to 87% unused
- MouseEmbryo: 55% to 81%
- 180bp will improve rolony generation
- Possibly more accurate padlock probes
- Base substitution is much less likely than deletion
- Possibly more rolonies
- Possibly more accurate padlock probes
- 200bp will improve decoding accuracy
- Less likely for incorrectly decoded rolony to be assigned to used barcode
- Conclusions: Go with 200bp
Modify Certain Genes so Every Probe Unique Barcode
- Need to modify outputFile_0gap_filtered gene names so that some probes have unique barcodes so decoding can be traced back to probe instead of gene
- outputFile_0gap_filtered.txt -> outputFile_0gap_mod.txt
- Select certain genes to modify with "-p#" eg."GAD1-p5"
- HumanBrain genes modified: SLC17A7, GNG4, PDE9A
- MouseBrain genes modified: Gad1, Vip, Pvalb
- MouseEmbryo genes modified: Myf5, Col1a1, Foxa1
- outputFile_0gap_filtered.txt -> outputFile_0gap_mod.txt
- 6 cycle: 4 'on' 2 'off' barcodes
perl Probe2padlockFISSEQ_Apr2016_HumanBrain_6cycle_V4.pl > outputFile_0gap_FULL200_HumanBrain_V4.txt perl Probe2padlockFISSEQ_Apr2016_MouseBrain_6cycle_V7.pl > outputFile_0gap_FULL200_MouseBrain_V7.txt perl Probe2padlockFISSEQ_Apr2016_MouseEmbryo_6cycle_V6.pl > outputFile_0gap_FULL200_MouseEmbryo_V6.txt perl Probe2padlockFISSEQ_Apr2016_ControlGenes_6cycle_V8.pl > outputFile_0gap_FULL200_ControlGenes_V8.txt
- Remove 2,909 probes from Human Control Genes to get to 12k oligos
perl padlock2oligo_order.pl ("CA12k_Apr2016_OligosOrder.txt")
Final Summary
Probeset | Amplification | # Probes | # Barcodes | # Genes |
Human Brain | V4 | 4,978 | 391 | 368 |
Mouse Brain | V7 | 1,808 | 173 | 150 |
Mouse Embryo | V6 | 2,469 | 251 | 220 |
Control Genes | V8 | 85 mouse, 2660 human | 85 mouse, 245 human | 33 mouse, 171 human |