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=CA12k_Nov2014_V4 + Suppressor Oligos v2 in vitro Capture Sequencing Analysis== *[[Matt:LabNotes/2015-5-8 | Previous experiment]] with 39 suppressor oligos failed to suppress the most "efficient" probes so [[Matt:LabNotes/2015-5-8#Design_Additional_Suppressor_Oligos | designed 5 more oligos]] **This set of 39 + 5 suppressor oligos is referred to in my notes as suppv2 or CA12kNov2014_V4suppv2 *[[Matt:LabNotes/2015-5-18 | Capture Experiment using RNA extracted from BA8 by Blue]] **Gel of sequencing library showed odd bands in Negative Control indicating possible DNA contamination in RNA **Also compared poly dT vs random nonamer RT priming in tube ===Check Sequencing Quality=== /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 | 650px]] [[File:MC20150522-CA12kNov14suppv2-cDNARan_qualstats.png | 650px]] [[File:MC20150522-CA12kNov14suppv2-gDNA_qualstats.png | 650px]] [[File:MC20150522-CA12kNov14suppv2-NegCtrl_qualstats.png | 650px]] *All have acceptable quality scores *NegCtrl is slightly worse than the other ===Mapping Reads to Probelist=== *Used bowtie2 reference file from [[Matt:LabNotes/2015-3-19#Mapping_MiSeq_reads_to_Oligo_Sequences]] 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=== [[Media:CountReadsPer_Gene_Probe.txt | 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=== [[File:20150712_With_vs_Without_Suppressorv2_Scatterplot.PNG|650px]] *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==== *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 [[Media:20150712_With_vs_Without_Suppressorv2.xlsx | Excel analysis]]
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