Matt:LabNotes/MiSeq 130325 Analysis

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Revision as of 17:41, 8 April 2013 by >Mzcai (Created page with "1. Starting with 3 R1_001.fastq MiSeq reads clip the reads to the first 46 nt with h1h2_first46_only.pl 2. Run each of the 3 46shortened.fastq files through Bowtie2 * ./bowti...")
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1. Starting with 3 R1_001.fastq MiSeq reads clip the reads to the first 46 nt with h1h2_first46_only.pl

2. Run each of the 3 46shortened.fastq files through Bowtie2

  • ./bowtie2 -k 1 --phred33 --mp 1,0 --rdg 0,1 --rfg 0,1 -x /media/Ext12T/GenomeDB/UCSC/Homo_sapiens/hg19/Sequence/Bowtie2Index/genome -q /home/kunzhang/seqStore/130325_MiSeq/gDNA_R1_46shortened.fastq > /home/kunzhang/seqStore/130325_MiSeq/gDNA_outputv4.txt
 4476041 reads; of these:
 4476041 (100.00%) were unpaired; of these:
   180684 (4.04%) aligned 0 times
   4295357 (95.96%) aligned exactly 1 time
   0 (0.00%) aligned >1 times

95.96% overall alignment rate

  • ./bowtie2 -k 1 --phred33 --mp 1,0 --rdg 0,1 --rfg 0,1 -x /media/Ext12T/GenomeDB/UCSC/Homo_sapiens/hg19/Sequence/Bowtie2Index/genome -q /home/kunzhang/seqStore/130325_MiSeq/cDNA-RNaseA_R1_46shortened.fastq > /home/kunzhang/seqStore/130325_MiSeq/cDNA-RNaseA_outputv4.txt
 2852904 reads; of these:
 2852904 (100.00%) were unpaired; of these:
   132404 (4.64%) aligned 0 times
   2720500 (95.36%) aligned exactly 1 time
   0 (0.00%) aligned >1 times

95.36% overall alignment rate

  • ./bowtie2 -k 1 --phred33 --mp 1,0 --rdg 0,1 --rfg 0,1 -x /media/Ext12T/GenomeDB/UCSC/Homo_sapiens/hg19/Sequence/Bowtie2Index/genome -q /home/kunzhang/seqStore/130325_MiSeq/cDNAwRNaseA_R1_46shortened.fastq > /home/kunzhang/seqStore/130325_MiSeq/cDNAwRNaseA_outputv4.txt
 2091417 reads; of these:
 2091417 (100.00%) were unpaired; of these:
   99778 (4.77%) aligned 0 times
   1991639 (95.23%) aligned exactly 1 time
   0 (0.00%) aligned >1 times

95.23% overall alignment rate 3. Turn 200bp ordered probe oligo sequences into CAprobes.fastq and run through Bowtie2 to get probes_output.txt

  • ./bowtie2 -k 1 --phred33 --mp 1,0 --rdg 0,1 --rfg 0,1 -x /media/Ext12T/GenomeDB/UCSC/Homo_sapiens/hg19/Sequence/Bowtie2Index/genome -q /home/kunzhang/seqStore/130325_MiSeq/CAprobes.fastq > /home/kunzhang/seqStore/130325_MiSeq/probes_output.txt
 12355 reads; of these:
 12355 (100.00%) were unpaired; of these:
   0 (0.00%) aligned 0 times
   12355 (100.00%) aligned exactly 1 time
   0 (0.00%) aligned >1 times

100.00% overall alignment rate 4. Count reads for each probe by matching the alignments in outputv4.txt (SAM files) to probes_output.txt

  • Count_reads_from_bowtie2_SAM_gDNA.pl (536,927 reads not matched)
  • Count_reads_from_bowtie2_SAM_cDNA-RNaseA.pl (222,592 reads not matched)
  • Count_reads_from_bowtie2_SAM_cDNAwRNaseA.pl (164,310 reads not matched)

Counts matched with probes from:

 *probe2padlockFISSEQ_Feb2013_0bp.pl    probe2padlockFISSEQ_Feb2013_0bp_stemCellGenes.pl
 *Also has filtered probes leaving: 
 **"good" probes (gDNA count between 100 and 1000)
 **Brain optimized probes (cDNA-RNaseA count between 10 and 1000 or just greater than 10)

5. Plot the counts
File:InSitu MiSeq CAgoodprobes.jpg File:InSitu MiSeq CAbrainoptimizedprobes.jpg File:InSitu MiSeq CAcDNA vs cDNA.jpg

6. Summarize results

  • "Good" probes have gDNA counts between 100 and 1000
    • 7,736 "good" probes from 12,355 total
    • 3,099 unique genes
  • Brain optimized probes are "good" probes that also have cDNA-RNaseA counts between 10 and 1000
    • 5,270 brain optimized probes from 12,355 total
    • 1,619 unique genes

Final Summary