Matt:LabNotes/2016-4-18
Convert Probes to Oligos to Order[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
- 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[edit]
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 |
- Note: Added 1,000 more human control gene probes to make 13,000 probes for Agilent order "Agi13k_Apr2016_DARTFISHOligosOrder.txt"