Matt:LabNotes/2015-5-8

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CA12k_Nov2014_V4 + Suppressor Oligos in vitro Capture Sequencing Analysis[edit]

Check Sequencing Quality[edit]

 /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/src/fastx_quality_stats/fastx_quality_stats -Q33 -i MC20150502-CA12kNov14supp-V4gDNA-1_S33_L001_R1_001.fastq -o MC20150502-CA12kNov2014supp-V4gDNA_qualstats.txt
 /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/src/fastx_quality_stats/fastx_quality_stats -Q33 -i MC20150502-CA12kNov14supp-V4cDNA-2_S34_L001_R1_001.fastq -o MC20150502-CA12kNov2014supp-V4cDNA_qualstats.txt
 /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/scripts/fastq_quality_boxplot_graph.sh -i MC20150502-CA12kNov2014supp-V4gDNA_qualstats.txt -o MC20150502-CA12kNov2014supp-V4gDNA_qualstats.png -t CA12kNov2014supp_V4_gDNA
 /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/scripts/fastq_quality_boxplot_graph.sh -i MC20150502-CA12kNov2014supp-V4cDNA_qualstats.txt -o MC20150502-CA12kNov2014supp-V4cDNA_qualstats.png -t CA12kNov2014supp_V4_cDNA

File:MC20150502-CA12kNov2014supp-V4gDNA qualstats.png File:MC20150502-CA12kNov2014supp-V4cDNA qualstats.png

  • Looks good! No trimming necessary

Mapping Reads to Probelist[edit]

 bowtie2 --phred33 -x CA12k_Nov2014_V4_H1H2 -q MC20150502-CA12kNov14supp-V4gDNA-1_S33_L001_R1_001.fastq > CA12kNov2014supp_V4gDNA_R1_H1H2.sam 2> CA12kNov2014supp_V4gDNA_stderr.txt &
 2620169 reads; of these:
 2620169 (100.00%) were unpaired; of these:
   208445 (7.96%) aligned 0 times
   2411551 (92.04%) aligned exactly 1 time
   173 (0.01%) aligned >1 times
 92.04% overall alignment rate
 bowtie2 --phred33 -x CA12k_Nov2014_V4_H1H2 -q MC20150502-CA12kNov14supp-V4cDNA-2_S34_L001_R1_001.fastq > CA12kNov2014supp_V4cDNA_R1_H1H2.sam 2> CA12kNov2014supp_V4cDNA_stderr.txt &
 2265558 reads; of these:
 2265558 (100.00%) were unpaired; of these:
   133741 (5.90%) aligned 0 times
   2131704 (94.09%) aligned exactly 1 time
   113 (0.00%) aligned >1 times
 94.10% overall alignment rate
 samtools view -bS CA12kNov2014supp_V4gDNA_R1_H1H2.sam | samtools sort - CA12kNov2014supp_V4gDNA_R1_H1H2_sorted
 samtools view -h -F 4 CA12kNov2014supp_V4gDNA_R1_H1H2_sorted.bam > CA12kNov2014supp_V4gDNA_R1_H1H2_sorted_filtered.sam
 samtools view -bS CA12kNov2014supp_V4cDNA_R1_H1H2.sam | samtools sort - CA12kNov2014supp_V4cDNA_R1_H1H2_sorted
 samtools view -h -F 4 CA12kNov2014supp_V4cDNA_R1_H1H2_sorted.bam > CA12kNov2014supp_V4cDNA_R1_H1H2_sorted_filtered.sam

Counting Reads for each Probe[edit]

CountReadsPer_Gene_Probe.pl

  • CA12kNov2014supp_V4gDNA_R1_H1H2_sorted_filtered.sam ->
    • CA12kNov2014supp_V4gDNA_R1_H1H2_sorted_filtered_Genecounts.txt
    • CA12kNov2014supp_V4gDNA_R1_H1H2_sorted_filtered_Probecounts.txt
  • CA12kNov2014supp_V4cDNA_R1_H1H2_sorted_filtered.sam ->
    • CA12kNov2014supp_V4cDNA_R1_H1H2_sorted_filtered_Genecounts.txt
    • CA12kNov2014supp_V4cDNA_R1_H1H2_sorted_filtered_Probecounts.txt

Comparison[edit]

File:20150508 With vs Without Suppressor Scatterplot.JPG

  • Orange line is y=x
  • Blue line is trendline
  • Some of the suppressed genes are labeled
    • Being left of y=x indicates suppressor oligos decreased the number of padlock probes capturing the gene/target

Excel analysis

Design Additional Suppressor Oligos[edit]

  • Seems like suppressor oligos were not effective (enough) for the most highly counted genes
    • Likely due to Tm being between 55-65C and reaction temp being 55C in vitro capture or 60C for DARTFISH
  • Design an extra 5 suppressor oligos
    • Below are the top 10 probes from CA12kNov2014supp_V4gDNA_R1_H1H2_sorted_filtered_Probecounts.txt
Probe (Gene_targetsequence) Count
GNG4_cgacatggtgaaaccccgtctctactaaaaatacaaaaatgagcca 727150
PDE1A_TTTGAacacacacacacacacacacacacacacacacacaTTCTTGTCTC 401898
KCNC2_TTAATAAGAATTTATCggccgggcgcggtggctcacgcctgtaatc 156162
PTPRK_tgcagtggtacaatcatagctcactgcagcctcaaattcctaggctgaag 37244
GNG4_agcctggacgacagaaagagactctgtctcaaaaacaaaaacaaaaac 24799
KCNN3_tccaccaccaaacccagctaatttttgtatttttagtagagatgggg 10877
SLC17A7_cagcctgggcaacatagtgagaccttgtctctacaaagataaaaata 5436
PPARGC1A_TACTTCCCCTAAACCAAGcacacacaccacacacatacatacacacacac 4046
TESPA1_tgtgtcctcacccaaatctcatgtcaaactgtaatccccattgttggagg 3648
  • Design options
    • Feature 1: Design suppressor oligos that anneal to H1 arm (Tm > 55C), has a 2bp gap, and then anneal to whole H2 arm (Tm > 68C)
    • Feature 2: Design suppressor oligos that complement H2 arm and continue up the barcode region
      • Feature 2 makes suppressor oligos specific to V4 probeset
      • Between H2 and barcode region is a poly(A) linker of variable length used make all padlock probes 150bp
Probe (Gene_targetsequence) Count Supp Oligo Left Tm Right Tm Old Supp Tm
GNG4_cgacatggtgaaaccccgtctctactaaaaatacaaaaatgagcca 727150 TGGTGAAACCCCGTCT AA CTACTAAAAATACAAAAATGAGCCATTTTTTTTTCTCGGTCG 58 68 57
PDE1A_TTTGAacacacacacacacacacacacacacacacacacaTTCTTGTCTC 401898 ACACACACACACACAC TT ACACACACACACACATTCTTGTCTCTTTTTCTC 55 68 64
KCNC2_TTAATAAGAATTTATCggccgggcgcggtggctcacgcctgtaatc 156162 GGCCGGGCGC AA GGTGGCTCACGCCTGTAATCTTTTTTTTAGC 55 69 64
PTPRK_tgcagtggtacaatcatagctcactgcagcctcaaattcctaggctgaag 37244 GGTACAATCATAGCTCACTG AA CAGCCTCAAATTCCTAGGCTGAAGTTTTTCTC 56 68 64
GNG4_agcctggacgacagaaagagactctgtctcaaaaacaaaaacaaaaac 24799 GGACGACAGAAAGAGACT AA CTGTCTCAAAAACAAAAACAAAAACTTTTTTTCTCGGTCG 56 68 58