Matt:LabNotes/2015-5-8
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CA12k_Nov2014_V4 + Suppressor Oligos in vitro Capture Sequencing Analysis
- 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
/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
- 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
- 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