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==CA12k_Nov2014_V4 + Suppressor Oligos in vitro Capture Sequencing Analysis== *[[Matt:LabNotes/2015-4-28 | Capture Experiment]] *39 Suppressor oligos designed for probes targeting soft masked regions and had high counts in V4 in vitro capture data **[[Matt:LabNotes/2015-3-19#Conclusion | Deciding which probes to suppress]] **Sequences on Google probe order form: 3/24/2015 and [[Media:CA12kNov2014_V4_SoftMaskedProbes.txt | CA12kNov2014_V4_SoftMaskedProbes.txt]] ===Check Sequencing Quality=== /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 | 650px]] [[File:MC20150502-CA12kNov2014supp-V4cDNA_qualstats.png | 650px]] *Looks good! No trimming necessary ===Mapping Reads to Probelist=== *Used bowtie2 reference file from [[Matt:LabNotes/2015-3-19#Mapping_MiSeq_reads_to_Oligo_Sequences]] 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=== [[Media:CountReadsPer_Gene_Probe.txt | 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=== [[File:20150508_With_vs_Without_Suppressor_Scatterplot.JPG|650px]] *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 [[Media:20150508_With_vs_Without_Suppressor.xlsx | Excel analysis]] ===Design Additional Suppressor Oligos=== *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 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe (Gene_targetsequence)''' | align="center" style="background:#f0f0f0;"|'''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 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe (Gene_targetsequence)''' | align="center" style="background:#f0f0f0;"|'''Count''' | align="center" style="background:#f0f0f0;"|'''Supp Oligo''' | align="center" style="background:#f0f0f0;"|'''Left Tm''' | align="center" style="background:#f0f0f0;"|'''Right Tm''' | align="center" style="background:#f0f0f0;"|'''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 |}
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