Matt:LabNotes/2015-7-12

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CA12k_Nov2014_V4 + Suppressor Oligos v2 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 MC20150522-CA12kNov14suppv2-cDNAdT-2_S6_L001_R1_001.fastq -o MC20150522-CA12kNov14suppv2-cDNAdT_qualstats.txt
 /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/src/fastx_quality_stats/fastx_quality_stats -Q33 -i MC20150522-CA12kNov14suppv2-cDNARan-3_S11_L001_R1_001.fastq -o MC20150522-CA12kNov14suppv2-cDNARan_qualstats.txt
 /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/src/fastx_quality_stats/fastx_quality_stats -Q33 -i MC20150522-CA12kNov14suppv2-gDNA-1_S5_L001_R1_001.fastq -o MC20150522-CA12kNov14suppv2-gDNA_qualstats.txt
 /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/src/fastx_quality_stats/fastx_quality_stats -Q33 -i MC20150522-CA12kNov14suppv2-NegCtrl-1_S12_L001_R1_001.fastq -o MC20150522-CA12kNov14suppv2-NegCtrl_qualstats.txt
 /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/scripts/fastq_quality_boxplot_graph.sh -i MC20150522-CA12kNov14suppv2-cDNAdT_qualstats.txt -o MC20150522-CA12kNov14suppv2-cDNAdT_qualstats.png -t CA12kNov2014supp_V4_cDNAdT
 /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/scripts/fastq_quality_boxplot_graph.sh -i MC20150522-CA12kNov14suppv2-cDNARan_qualstats.txt -o MC20150522-CA12kNov14suppv2-cDNARan_qualstats.png -t CA12kNov2014supp_V4_cDNARan
 /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/scripts/fastq_quality_boxplot_graph.sh -i MC20150522-CA12kNov14suppv2-gDNA_qualstats.txt -o MC20150522-CA12kNov14suppv2-gDNA_qualstats.png -t CA12kNov2014supp_V4_gDNA
 /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/scripts/fastq_quality_boxplot_graph.sh -i MC20150522-CA12kNov14suppv2-NegCtrl_qualstats.txt -o MC20150522-CA12kNov14suppv2-NegCtrl_qualstats.png -t CA12kNov2014supp_V4_NegCtrl

File:MC20150522-CA12kNov14suppv2-cDNAdT qualstats.png File:MC20150522-CA12kNov14suppv2-cDNARan qualstats.png File:MC20150522-CA12kNov14suppv2-gDNA qualstats.png File:MC20150522-CA12kNov14suppv2-NegCtrl qualstats.png

  • All have acceptable quality scores
  • NegCtrl is slightly worse than the other

Mapping Reads to Probelist[edit]

 bowtie2 --phred33 -x /media/LTS_15T/MC_LTS/InSitu_MiSeq_150312_Analysis/CA12k_Nov2014_V4_H1H2 -q MC20150522-CA12kNov14suppv2-cDNAdT-2_S6_L001_R1_001.fastq > MC20150522-CA12kNov14suppv2-cDNAdT_H1H2.sam 2> MC20150522-CA12kNov14suppv2-cDNAdT_stderr.txt &
 352826 reads; of these:
 352826 (100.00%) were unpaired; of these:
   79183 (22.44%) aligned 0 times
   272540 (77.24%) aligned exactly 1 time
   1103 (0.31%) aligned >1 times
 77.56% overall alignment rate
 bowtie2 --phred33 -x /media/LTS_15T/MC_LTS/InSitu_MiSeq_150312_Analysis/CA12k_Nov2014_V4_H1H2 -q MC20150522-CA12kNov14suppv2-cDNARan-3_S11_L001_R1_001.fastq > MC20150522-CA12kNov14suppv2-cDNARan_H1H2.sam 2> MC20150522-CA12kNov14suppv2-cDNARan_stderr.txt &
 2223465 reads; of these:
 2223465 (100.00%) were unpaired; of these:
   168135 (7.56%) aligned 0 times
   2054429 (92.40%) aligned exactly 1 time
   901 (0.04%) aligned >1 times
 92.44% overall alignment rate
 bowtie2 --phred33 -x /media/LTS_15T/MC_LTS/InSitu_MiSeq_150312_Analysis/CA12k_Nov2014_V4_H1H2 -q MC20150522-CA12kNov14suppv2-gDNA-1_S5_L001_R1_001.fastq > MC20150522-CA12kNov14suppv2-gDNA_H1H2.sam 2> MC20150522-CA12kNov14suppv2-gDNA_stderr.txt &
 267387 reads; of these:
 267387 (100.00%) were unpaired; of these:
   28205 (10.55%) aligned 0 times
   239156 (89.44%) aligned exactly 1 time
   26 (0.01%) aligned >1 times
 89.45% overall alignment rate
 bowtie2 --phred33 -x /media/LTS_15T/MC_LTS/InSitu_MiSeq_150312_Analysis/CA12k_Nov2014_V4_H1H2 -q MC20150522-CA12kNov14suppv2-NegCtrl-1_S12_L001_R1_001.fastq > MC20150522-CA12kNov14suppv2-NegCtrl_H1H2.sam 2> MC20150522-CA12kNov14suppv2-NegCtrl_stderr.txt &
 2530125 reads; of these:
 2530125 (100.00%) were unpaired; of these:
   1817218 (71.82%) aligned 0 times
   675182 (26.69%) aligned exactly 1 time
   37725 (1.49%) aligned >1 times
 28.18% overall alignment rate
 samtools view -bS MC20150522-CA12kNov14suppv2-cDNAdT_H1H2.sam | samtools sort - CA12kNov14suppv2-cDNAdT_H1H2_sorted
 samtools view -h -F 4 CA12kNov14suppv2-cDNAdT_H1H2_sorted.bam > CA12kNov14suppv2-cDNAdT_H1H2_sorted_filtered.sam
 samtools view -bS MC20150522-CA12kNov14suppv2-cDNARan_H1H2.sam | samtools sort - CA12kNov14suppv2-cDNARan_H1H2_sorted
 samtools view -h -F 4 CA12kNov14suppv2-cDNARan_H1H2_sorted.bam > CA12kNov14suppv2-cDNARan_H1H2_sorted_filtered.sam
 samtools view -bS MC20150522-CA12kNov14suppv2-gDNA_H1H2.sam | samtools sort - CA12kNov14suppv2-gDNA_H1H2_sorted
 samtools view -h -F 4 CA12kNov14suppv2-gDNA_H1H2_sorted.bam > CA12kNov14suppv2-gDNA_H1H2_sorted_filtered.sam
 samtools view -bS MC20150522-CA12kNov14suppv2-NegCtrl_H1H2.sam | samtools sort - CA12kNov14suppv2-NegCtrl_H1H2_sorted
 samtools view -h -F 4 CA12kNov14suppv2-NegCtrl_H1H2_sorted.bam > CA12kNov14suppv2-NegCtrl_H1H2_sorted_filtered.sam

Counting Reads for each Probe[edit]

CountReadsPer_Gene_Probe.pl

  • CA12kNov14suppv2-cDNAdT_H1H2_sorted_filtered.sam ->
    • CA12kNov14suppv2-cDNAdT_H1H2_sorted_filtered_Genecounts.txt
    • CA12kNov14suppv2-cDNAdT_H1H2_sorted_filtered_Probecounts.txt
  • CA12kNov14suppv2-cDNARan_H1H2_sorted_filtered.sam ->
    • CA12kNov14suppv2-cDNARan_H1H2_sorted_filtered_Genecounts.txt
    • CA12kNov14suppv2-cDNARan_H1H2_sorted_filtered_Probecounts.txt
  • CA12kNov14suppv2-gDNA_H1H2_sorted_filtered.sam ->
    • CA12kNov14suppv2-gDNA_H1H2_sorted_filtered_Genecounts.txt
    • CA12kNov14suppv2-gDNA_H1H2_sorted_filtered_Probecounts.txt
  • CA12kNov14suppv2-NegCtrl_H1H2_sorted_filtered.sam ->
    • CA12kNov14suppv2-NegCtrl_H1H2_sorted_filtered_Genecounts.txt
    • CA12kNov14suppv2-NegCtrl_H1H2_sorted_filtered_Probecounts.txt

Comparison[edit]

File:20150712 With vs Without Suppressorv2 Scatterplot.PNG

  • Orange line is y=x
  • Blue line is trendline
  • The 4 genes suppressed by v2 design are labeled
    • Being left of y=x indicates suppressor oligos decreased the number of padlock probes capturing the gene/target

Conclusions[edit]

  • Suppressorv2 is effective at decreasing capture counts of most captured genes
  • Good correlation between dT and Ran primer counts
    • 6 genes did not appear in dT dataset but could be due to lower sequencing depth of that library (those genes should have poly-A tail)
  • NegCtrl correlates better with gDNA than RNA so probably a little DNA contamination in BA8 sample
    • Low enough that probably negligible
      • eg. the cDNA Ran primer library was taken out of PCR before the NegCtrl curve started to go up

Excel analysis