Matt:LabNotes/2017-1-3
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CA12k_Oct2016_V4 in vitro Capture Sequencing Analysis=[edit]
- For reference doing analysis
- in vitro capture experiment
- Indx1: T4
- Indx2: SplintR
- Indx3: Ampligase
Check Sequencing Quality[edit]
- Currently in genomeMiner:~/scratch/CA12kOct2016_V4_CaptureAnalysis/Matt
/media/Home_Raid1/kunzhang/softwares/fastx_toolkit-0.0.13.2/src/fastx_quality_stats/fastx_quality_stats -Q33 -i Index1_S1_L001_R1_001.fastq -o MC20161215_CA12kOct20161215_V4_T4_Indx1_qualstats.txt /media/Home_Raid1/kunzhang/softwares/fastx_toolkit-0.0.13.2/src/fastx_quality_stats/fastx_quality_stats -Q33 -i Index2_S2_L001_R1_001.fastq -o MC20161215_CA12kOct20161215_V4_SplintR_Indx2_qualstats.txt /media/Home_Raid1/kunzhang/softwares/fastx_toolkit-0.0.13.2/src/fastx_quality_stats/fastx_quality_stats -Q33 -i Index3_S3_L001_R1_001.fastq -o MC20161215_CA12kOct20161215_V4_Amp_Indx3_qualstats.txt
/media/Home_Raid1/kunzhang/softwares/fastx_toolkit-0.0.13.2/scripts/fastq_quality_boxplot_graph.sh -i MC20161215_CA12kOct20161215_V4_T4_Indx1_qualstats.txt -o MC20161215_CA12kOct20161215_V4_T4_Indx1_qualstats.png -t CA12kOct2016_V4_T4 /media/Home_Raid1/kunzhang/softwares/fastx_toolkit-0.0.13.2/scripts/fastq_quality_boxplot_graph.sh -i MC20161215_CA12kOct20161215_V4_SplintR_Indx2_qualstats.txt -o MC20161215_CA12kOct20161215_V4_SplintR_Indx2_qualstats.png -t CA12kOct2016_V4_SplintR /media/Home_Raid1/kunzhang/softwares/fastx_toolkit-0.0.13.2/scripts/fastq_quality_boxplot_graph.sh -i MC20161215_CA12kOct20161215_V4_Amp_Indx3_qualstats.txt -o MC20161215_CA12kOct20161215_V4_Amp_Indx3_qualstats.png -t CA12kOct2016_V4_Ampligase
File:MC20161215 CA12kOct20161215 V4 T4 Indx1 qualstats.png File:MC20161215 CA12kOct20161215 V4 SplintR Indx2 qualstats.png File:MC20161215 CA12kOct20161215 V4 Amp Indx3 qualstats.png
- Acceptable quality scores up to 65bp read, after 65bp quality drops
Mapping Reads to Probelist[edit]
Convert Probelist to Fasta File[edit]
- In Dropbox\GradZhangLab\CA12k_Oct2016\HumanBrain_V4\invitroCapture
- Probelist: Media:padlockFile_0gap_HumanBrain_V4.txt
- CA12kOct2016V4_Probelist2Fasta.pl to generate ref file: CA12k_Oct2016_V4_H1H2.fa
bowtie2-build CA12k_Oct2016_V4_H1H2.fa CA12k_Oct2016_V4_H1H2
Align[edit]
bowtie2 --phred33 -x CA12k_Oct2016_V4_H1H2 -q Index1_S1_L001_R1_001.fastq > MC20161215_CA12kOct20161215_V4_T4_H1H2.sam 2> MC20161215_CA12kOct20161215_V4_T4_stderr.txt & 828460 reads; of these: 828460 (100.00%) were unpaired; of these: 621272 (74.99%) aligned 0 times 207179 (25.01%) aligned exactly 1 time 9 (0.00%) aligned >1 times 25.01% overall alignment rate bowtie2 --phred33 -x CA12k_Oct2016_V4_H1H2 -q Index2_S2_L001_R1_001.fastq > MC20161215_CA12kOct20161215_V4_SplintR_H1H2.sam 2> MC20161215_CA12kOct20161215_V4_SplintR_stderr.txt & 778774 reads; of these: 778774 (100.00%) were unpaired; of these: 699239 (89.79%) aligned 0 times 79525 (10.21%) aligned exactly 1 time 10 (0.00%) aligned >1 times 10.21% overall alignment rate bowtie2 --phred33 -x CA12k_Oct2016_V4_H1H2 -q Index3_S3_L001_R1_001.fastq > MC20161215_CA12kOct20161215_V4_Ampligase_H1H2.sam 2> MC20161215_CA12kOct20161215_V4_Ampligase_stderr.txt & 633888 reads; of these: 633888 (100.00%) were unpaired; of these: 342474 (54.03%) aligned 0 times 291410 (45.97%) aligned exactly 1 time 4 (0.00%) aligned >1 times 45.97% overall alignment rate
Try Trimming to only good quality[edit]
- Marginal improvement in alignment rate
fastx_trimmer -Q33 -l 50 -i Index2_S2_L001_R1_001.fastq -o Index2_50bp.fastq bowtie2 --phred33 -x CA12k_Oct2016_V4_H1H2 -q Index2_50bp.fastq > MC20161215_CA12kOct20161215_V4_SplintR_50bp.sam 2> MC20161215_CA12kOct20161215_V4_SplintR_50bp_stderr.txt & 778774 reads; of these: 778774 (100.00%) were unpaired; of these: 559938 (71.90%) aligned 0 times 212096 (27.23%) aligned exactly 1 time 6740 (0.87%) aligned >1 times 28.10% overall alignment rate
samtools view -bS MC20161215_CA12kOct20161215_V4_T4_H1H2.sam | samtools sort -o CA12kOct20161215_V4_T4_H1H2_sorted.bam samtools view -h -F 4 CA12kOct20161215_V4_T4_H1H2_sorted.bam > CA12kOct20161215_V4_T4_H1H2_sorted_filtered.sam
samtools view -bS MC20161215_CA12kOct20161215_V4_SplintR_H1H2.sam | samtools sort -o CA12kOct20161215_V4_SplintR_H1H2_sorted.bam samtools view -h -F 4 CA12kOct20161215_V4_SplintR_H1H2_sorted.bam > CA12kOct20161215_V4_SplintR_H1H2_sorted_filtered.sam
samtools view -bS MC20161215_CA12kOct20161215_V4_Ampligase_H1H2.sam | samtools sort -o CA12kOct20161215_V4_Ampligase_H1H2_sorted.bam samtools view -h -F 4 CA12kOct20161215_V4_Ampligase_H1H2_sorted.bam > CA12kOct20161215_V4_Ampligase_H1H2_sorted_filtered.sam
Counting Reads for each Probe[edit]
- CA12kOct20161215_V4_T4_H1H2_sorted_filtered.sam ->
- CA12kOct20161215_V4_T4_H1H2_sorted_filtered_Genecounts.txt
- CA12kOct20161215_V4_T4_H1H2_sorted_filtered_Probecounts.txt
- CA12kOct20161215_V4_SplintR_H1H2_sorted_filtered.sam ->
- CA12kOct20161215_V4_SplintR_H1H2_sorted_filtered_Genecounts.txt
- CA12kOct20161215_V4_SplintR_H1H2_sorted_filtered_Probecounts.txt
- CA12kOct20161215_V4_Ampligase_H1H2_sorted_filtered.sam ->
- CA12kOct20161215_V4_Ampligase_H1H2_sorted_filtered_Genecounts.txt
- CA12kOct20161215_V4_Ampligase_H1H2_sorted_filtered_Probecounts.txt