Matt:LabNotes/2017-1-3: Difference between revisions

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*Probelist: [[Media:padlockFile_0gap_HumanBrain_V4.txt]]  
*Probelist: [[Media:padlockFile_0gap_HumanBrain_V4.txt]]  
**[[Media:CA12kOct2016V4_Probelist2Fasta.txt | CA12kOct2016V4_Probelist2Fasta.pl]] to generate ref file: CA12k_Oct2016_V4_H1H2.fa
**[[Media:CA12kOct2016V4_Probelist2Fasta.txt | CA12kOct2016V4_Probelist2Fasta.pl]] to generate ref file: CA12k_Oct2016_V4_H1H2.fa
  bowtie2-build CA12k_Oct2016_V4_H1H2.fa CA12k_Oct2016_V4_H1H2
  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
<!--
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  bowtie2 --phred33 -x /media/LTS_15T/MC_LTS/InSitu_MiSeq_150312_Analysis/CA12k_Nov2014_V4_H1H2 -q MC20150522-CA12kNov14suppv2-cDNAdT-2_S6_L001_R1_001.fastq > MC20150522-CA12kNov14suppv2-cDNAdT_H1H2.sam 2> MC20150522-CA12kNov14suppv2-cDNAdT_stderr.txt &
  352826 reads; of these:
  352826 (100.00%) were unpaired; of these:
    79183 (22.44%) aligned 0 times
    272540 (77.24%) aligned exactly 1 time
    1103 (0.31%) aligned >1 times
  77.56% overall alignment rate
  bowtie2 --phred33 -x /media/LTS_15T/MC_LTS/InSitu_MiSeq_150312_Analysis/CA12k_Nov2014_V4_H1H2 -q MC20150522-CA12kNov14suppv2-cDNARan-3_S11_L001_R1_001.fastq > MC20150522-CA12kNov14suppv2-cDNARan_H1H2.sam 2> MC20150522-CA12kNov14suppv2-cDNARan_stderr.txt &
  2223465 reads; of these:
  2223465 (100.00%) were unpaired; of these:
    168135 (7.56%) aligned 0 times
    2054429 (92.40%) aligned exactly 1 time
    901 (0.04%) aligned >1 times
  92.44% overall alignment rate
  bowtie2 --phred33 -x /media/LTS_15T/MC_LTS/InSitu_MiSeq_150312_Analysis/CA12k_Nov2014_V4_H1H2 -q MC20150522-CA12kNov14suppv2-gDNA-1_S5_L001_R1_001.fastq > MC20150522-CA12kNov14suppv2-gDNA_H1H2.sam 2> MC20150522-CA12kNov14suppv2-gDNA_stderr.txt &
  267387 reads; of these:
  267387 (100.00%) were unpaired; of these:
    28205 (10.55%) aligned 0 times
    239156 (89.44%) aligned exactly 1 time
    26 (0.01%) aligned >1 times
  89.45% overall alignment rate
   samtools view -bS MC20150522-CA12kNov14suppv2-cDNAdT_H1H2.sam | samtools sort - CA12kNov14suppv2-cDNAdT_H1H2_sorted
   samtools view -bS MC20150522-CA12kNov14suppv2-cDNAdT_H1H2.sam | samtools sort - CA12kNov14suppv2-cDNAdT_H1H2_sorted
   samtools view -h -F 4 CA12kNov14suppv2-cDNAdT_H1H2_sorted.bam > CA12kNov14suppv2-cDNAdT_H1H2_sorted_filtered.sam
   samtools view -h -F 4 CA12kNov14suppv2-cDNAdT_H1H2_sorted.bam > CA12kNov14suppv2-cDNAdT_H1H2_sorted_filtered.sam

Revision as of 00:58, 12 January 2017

CA12k_Oct2016_V4 in vitro Capture Sequencing Analysis=

Check Sequencing Quality

  • 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

Convert Probelist to Fasta File

 bowtie2-build CA12k_Oct2016_V4_H1H2.fa CA12k_Oct2016_V4_H1H2
 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