Matt:LabNotes/2016-4-18: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
mNo edit summary
>Mzcai
mNo edit summary
Line 1: Line 1:
=Convert Probes to Oligos to Order=
=Convert Probes to Oligos to Order=
*Add barcodes
*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
Line 13: Line 17:
**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
**Combine human and mouse control genes
***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
Line 25: Line 29:
***Add "h-" in front of each gene name
***Add "h-" in front of each gene name


*3 'on' 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_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
Line 31: Line 37:
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
Line 38: Line 45:


perl padlock2oligo_order.pl ("CA12k_Apr2016_OligosOrder.txt")
perl padlock2oligo_order.pl ("CA12k_Apr2016_OligosOrder.txt")
*Need to modify outputFile_0gap_FULL_* so that some genes, exons, and probes have multiple barcodes
**Modify outputFile_0gap_FULL_MouseEmbryo_V6.txt ->outputFile_0gap_FULLmod_MouseEmbryo_V6.txt
***Acta2: each probe has different barcode
  Acta2 Acta2_0 chr19:34241431-34241432 AATCACTTCAACAGAGGAAGGTCCA 64 TATATTCCCAGGCATGGATGGCATC 64 2.715838 C TATATTCCCAGGCATGGATGGCATCAATCACTTCAACAGAGGAAGGTCCA B010303 GTCATATCGGTCACTGTTAATCACTTCAACAGAGGAAGGTCCACTTCAGCTTCCCGATATCCGACGGTAGTGT*TCTTGCGTGCGATACGGAGTAACTGCGACCGTCGGCTTACCATGCGATTAACCGCGACTG*AAATATATTCCCAGGCATGGATGGCATCGATCAGGATACACACTACCC
  Acta2 Acta2_0 chr19:34241664-34241665 GCCTCCAGTTCCTTTCCAAATCAT 64 CAACTGTGAACGTTTTGTGGATCAGC 63 2.761038 C CAACTGTGAACGTTTTGTGGATCAGCGCCTCCAGTTCCTTTCCAAATCAT B020023 GTCATATCGGTCACTGTTGCCTCCAGTTCCTTTCCAAATCATCTTCAGCTTCCCGATATCCGACGGTAGTGT*AACGGTATTCGGTCGTCATCTACCATTCGCGTGCGATTCCCATGCGATTAACCGCGACTG*AAACAACTGTGAACGTTTTGTGGATCAGCGATCAGGATACACACTACCC
  Acta2 Acta2_1 chr19:34243507-34243508 CATCATGAAGTGTGATATTGACATCA 59 GCATCCACGAAACCACCTATAACAG 64 2.768782 C GCATCCACGAAACCACCTATAACAGCATCATGAAGTGTGATATTGACATCA B030031 GTCATATCGGTCACTGTTCATCATGAAGTGTGATATTGACATCACTTCAGCTTCCCGATATCCGACGGTAGTGT*CTGGTTCGGGCGTACCTAACCAGGGATCGGTCGAGTACGCGAGTGTCGCGCAACTTAGCG*AAGCATCCACGAAACCACCTATAACAGGATCAGGATACACACTACCC
  Acta2 Acta2_2 chr19:34245117-34245118 CTGTGCTATGTAGCTCTGGACTTTG 63 GATTGTCCGTGACATCAAGGAGAAG 64 2.760554 C GATTGTCCGTGACATCAAGGAGAAGCTGTGCTATGTAGCTCTGGACTTTG B300032 GTCATATCGGTCACTGTTCTGTGCTATGTAGCTCTGGACTTTGCTTCAGCTTCCCGATATCCGACGGTAGTGT*ACGCTACGGAGTACGCCACTCAGGGATCGGTCGAGTACGCACGTCTGCGTACCGGCTTAG*AAAGATTGTCCGTGACATCAAGGAGAAGGATCAGGATACACACTACCC
  Acta2 Acta2_3 chr19:34246264-34246265 TGTGACTCACAACGTGCCTATCTAT 64 GTATTGTGCTGGACTCTGGAGATGG 63 2.744643 C GTATTGTGCTGGACTCTGGAGATGGTGTGACTCACAACGTGCCTATCTAT B210020 GTCATATCGGTCACTGTTTGTGACTCACAACGTGCCTATCTATCTTCAGCTTCCCGATATCCGACGGTAGTGT*CGTATCGGTAGTCGCAACGCTCTTGCGTGCGATACGGAGTTACCATTCGCGTGCGATTCC*AAAGTATTGTGCTGGACTCTGGAGATGGGATCAGGATACACACTACCC
  Acta2 Acta2_4 chr19:34246758-34246759 CCATGTATGTGGCTATTCAGGCTG 64 TATGTTTGAGACCTTCAATGTCCCCG 63 2.740051 C TATGTTTGAGACCTTCAATGTCCCCGCCATGTATGTGGCTATTCAGGCTG B110020 GTCATATCGGTCACTGTTCCATGTATGTGGCTATTCAGGCTGCTTCAGCTTCCCGATATCCGACGGTAGTGT*TGTATCGCGCTCGATTGGCATCTTGCGTGCGATACGGAGTTACCATTCGCGTGCGATTCC*AAATATGTTTGAGACCTTCAATGTCCCCGGATCAGGATACACACTACCC
  Acta2 Acta2_5 chr19:34248545-34248546 CCCTGAAGAGCATCCGACACTG 63 CTTTCTATAACGAGCTTCGTGTGGC 64 2.661623 C CTTTCTATAACGAGCTTCGTGTGGCCCCTGAAGAGCATCCGACACTG B023100 GTCATATCGGTCACTGTTCCCTGAAGAGCATCCGACACTGCTTCAGCTTCCCGATATCCGACGGTAGTGT*AACGGTATTCGGTCGTCATCGACGAACGGTCGAGATTTACGAATTGTCCGCGCTCTACGA*AAAAAACTTTCTATAACGAGCTTCGTGTGGCGATCAGGATACACACTACCC
  Acta2 Acta2_6 chr19:34251822-34251823 GATAGAACACGGCATCATCACCAAC 64 AGGGATCCTGACGCTGAAGTATCC 64 2.693671 C AGGGATCCTGACGCTGAAGTATCCGATAGAACACGGCATCATCACCAAC B110030 GTCATATCGGTCACTGTTGATAGAACACGGCATCATCACCAACCTTCAGCTTCCCGATATCCGACGGTAGTGT*TGTATCGCGCTCGATTGGCATCTTGCGTGCGATACGGAGTCAGGGATCGGTCGAGTACGC*AAAAAGGGATCCTGACGCTGAAGTATCCGATCAGGATACACACTACCC
  Acta2 Acta2_7 chr19:34252601-34252602 GTGTGAAGAGGAAGACAGCACAG 64 CTGAGACGCTGCTCCAGCTAT 64 2.541243 C CTGAGACGCTGCTCCAGCTATGTGTGAAGAGGAAGACAGCACAG B210002 GTCATATCGGTCACTGTTGTGTGAAGAGGAAGACAGCACAGCTTCAGCTTCCCGATATCCGACGGTAGTGT*CGTATCGGTAGTCGCAACGCTCTTGCGTGCGATACGGAGTACGTCTGCGTACCGGCTTAG*AAAAAAAAACTGAGACGCTGCTCCAGCTATGATCAGGATACACACTACCC


==3 'on' 180bp oligos vs 4 'on' 200bp oligos==
==3 'on' 180bp oligos vs 4 'on' 200bp oligos==
Line 73: Line 67:
*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
Line 87: Line 81:
*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

  • 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

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
  • 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
  • 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