Matt:LabNotes/2015-5-8: Difference between revisions

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[[Media:20150508_With_vs_Without_Suppressor.xlsx | Excel analysis]]
[[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
**Take top 5 and design suppressor oligos that anneal to 10bp of H1 arm, has a 2bp gap, and then anneal to whole H2 arm
{| {{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
|}

Revision as of 01:52, 9 May 2015

CA12k_Nov2014_V4 + Suppressor Oligos in vitro Capture Sequencing Analysis

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 File:MC20150502-CA12kNov2014supp-V4cDNA qualstats.png

  • Looks good! No trimming necessary

Mapping Reads to Probelist

 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

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

  • 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

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
    • Take top 5 and design suppressor oligos that anneal to 10bp of H1 arm, has a 2bp gap, and then anneal to whole H2 arm
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