Matt:LabNotes/2015-7-12
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CA12k_Nov2014_V4 + Suppressor Oligos v2 in vitro Capture Sequencing Analysis=[edit]
- Previous experiment with 39 suppressor oligos failed to suppress the most "efficient" probes so designed 5 more oligos
- This set of 39 + 5 suppressor oligos is referred to in my notes as suppv2 or CA12kNov2014_V4suppv2
- 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[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]
- 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[edit]
- 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
- Low enough that probably negligible