Matt:LabNotes/MiSeq 130325 Analysis
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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