Matt:LabNotes/2013-10-15

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Revision as of 01:28, 16 October 2013 by >Mzcai (→‎Count Nuclei App)
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V4S1 Mock Reads with Novoalign Full Reference

Error 1% substitution only

  • Align
 novoalign -d ../mimicerror/V4S1_fullrevcomp.ndx -f v4s1_mockseq_1s.fq -F STDFQ -r ALL -o SAM -o FULLNW > v4s1_mockseq_1s_novoalign_fullrevcomp.sam &
 #     Read Sequences:  2959000
 #            Aligned:  2953594
 #   Unique Alignment:  2949594
 #   Gapped Alignment:     2816
 #     Quality Filter:     5048
 # Homopolymer Filter:        0
 #       Elapsed Time: 67.870 (secs.)
 #           CPU Time: 11.91 (min.)
 # Done at Tue Oct 15 11:22:00 2013
  • Samtools
 samtools view -bS v4s1_mockseq_1s_novoalign_fullrevcomp.sam | samtools sort - v4s1_mockseq_1s_novoalign_fullrevcomp_sorted
 samtools view -h -F 4 -q 70 v4s1_mockseq_1s_novoalign_fullrevcomp_sorted.bam > v4s1_mockseq_1s_novoalign_fullrevcomp_sorted_filtered.sam
 samtools calmd -eS v4s1_mockseq_1s_novoalign_fullrevcomp_sorted_filtered.sam /home/mzcai/DansProbes/V4S1_fullrevcomp.fa > v4s1_mockseq_1s_novoalign_fullrevcomp_sf=.sam
  • CountErrorNovoalignFullRef_EachBase.pl
    • Bases 10-30 Average
      • Del: 8.91*10^-5 %
      • Ins: 0.001545 %
      • Sub: 0.985943 %

File:V4S1mock sub1 Novoalign FullRef ErrorPerBase.JPG

  • CountErrorNovoalignFullRefFilter_EachBase.pl
    • Bases 10-30 Average
      • Del: 0 %
      • Ins: 6.48*10^-6 %
      • Sub: 0.986033 %

File:V4S1mock sub1 Novoalign FullRef Filter ErrorPerBase.JPG

Low substitution High deletion (1.01% Del, 0.26% Sub, 0.05% Ins)

  • Align
 novoalign -d ../mimicerror/V4S1_fullrevcomp.ndx -f v4s1_mockseq_error_losubhidel.fq -F STDFQ -r ALL -o SAM -o FULLNW > v4s1_mockseq_losubhidel_novoalign_fullrevcomp.sam &
 #     Read Sequences:  2959000
 #            Aligned:  2933687
 #   Unique Alignment:  2929681
 #   Gapped Alignment:  1256873
 #     Quality Filter:     4897
 # Homopolymer Filter:        0
 #       Elapsed Time: 96.893 (secs.)
 #           CPU Time: 19.85 (min.)
 # Done at Tue Oct 15 13:17:16 2013
  • Samtools
 samtools view -bS v4s1_mockseq_losubhidel_novoalign_fullrevcomp.sam | samtools sort - v4s1_mockseq_losubhidel_novoalign_fullrevcomp_sorted
 samtools view -h -F 4 -q 70 v4s1_mockseq_losubhidel_novoalign_fullrevcomp_sorted.bam > v4s1_mockseq_losubhidel_novoalign_fullrevcomp_sorted_filtered.sam
 samtools calmd -eS v4s1_mockseq_losubhidel_novoalign_fullrevcomp_sorted_filtered.sam /home/mzcai/DansProbes/V4S1_fullrevcomp.fa > v4s1_mockseq_losubhidel_novoalign_fullrevcomp_sf=.sam
  • CountErrorNovoalignFullRef_EachBase.pl
    • Bases 10-30 Average
      • Del: 0.935%
      • Ins: 0.285 %
      • Sub: 0.106 %

File:V4S1mock losubhidel Novoalign FullRef ErrorPerBase.JPG

  • CountErrorNovoalignFullRefFilter_EachBase.pl
    • Bases 10-30 Average
      • Del: 0.932 %
      • Ins: 0.229 %
      • Sub: 0.107 %

File:V4S1mock losubhidel Novoalign FullRef Filter ErrorPerBase.JPG

Count Nuclei App

  • The reason the 16-bit images (converted from 8-bit) didn't work yesterday was because they were the cropped images (not raw)
  • Count Primary Rolonies from Hosuk:LabNotes/2013-10-9
    • File: Series021_20xobj-Zstack-2kx2k_MaxProject_Cy5-16bit
    • Min width: 0.7um; Max width: 2um; Intensity above local bg: 50
    • 42,019 Rolonies

[[]]

  • Count ACTB Target regions on Primary Rolonies
    • File: Series021_20xobj-Zstack-2kx2k_MaxProject_ATTO488-16bit
    • Min width: 0.7um; Max width: 2um; Intensity above local bg: 40
    • 312 Rolonies

[[]]