Matt:LabNotes/2015-5-8
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CA12k_Nov2014_V4 + Suppressor Oligos in vitro Capture Sequencing Analysis[edit]
- Capture Experiment
- 39 Suppressor oligos designed for probes targeting soft masked regions and had high counts in V4 in vitro capture data
- Deciding which probes to suppress
- Sequences on Google probe order form: 3/24/2015 and CA12kNov2014_V4_SoftMaskedProbes.txt
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]
- 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[edit]
- 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
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 |