Matt:LabNotes/2017-1-3

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CA12k_Oct2016_V4 in vitro Capture Sequencing Analysis=[edit]

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]

 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]

CountReadsPer_Gene_Probe.pl

  • 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