Matt:LabNotes/2016-4-18
Convert Probes to Oligos to Order
- Add barcodes
- Files in Dropbox/GradZhangLab/CA12k_Apr2016
- dcProbe_Sequences.txt
- Cycle \t Color \t 20nt Sequence
- 21 (7x3) total
- 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
- 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
- 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
- Combine human and mouse control genes
- 3 'on' barcodes
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
- 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")
- 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
- Modify outputFile_0gap_FULL_MouseEmbryo_V6.txt ->outputFile_0gap_FULLmod_MouseEmbryo_V6.txt
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
- 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
- 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")