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==CA12k Capture Analysis (MiSeq_130325)== *Here is improved analysis of the MiSeq_130325 data **Previously used Bowtie2 custom parameters, which now I know are not optimal **Previously aligned to hg19 and now aligned to probelist used to order the probes **The old analysis can be found in the commented out section at the bottom of the "edit" page or [[Matt:LabNotes/MiSeq_130325_Analysis]] ===Building bowtie2 index for CA12k probes=== *First converted probelist to fasta file with H1, H2,and 100bp linking region rearranged using [[Media:CAProbelist_to_fasta.txt | CAProbelist_to_fasta.pl]]: Feb2013_200bp_probes_to_order.txt -> CAprobes_to_order_H1H2.fa **Fasta reference made to match reads: RevComp(H1) + RevComp(H2) + RevComp(Barcode) *Build index: genome-miner:$ bowtie2-build CAprobes_to_order_H1H2.fa CAprobes_H1H2 ===Processing reads=== *Shorten Read1 reads to 146bp to match reference length with h1h2_first146_only.pl (nothing fancy, just substr($_,0,146) for base call and base quality strings) D1-cDNA-RNaseA_S2_L001_R1_001.fastq -> cDNA-RNaseA_R1_146shortened.fastq D1-cDNAwRNaseA_S3_L001_R1_001.fastq -> cDNAwRNaseA_R1_146shortened.fastq D1-gDNA_S1_L001_R1_001.fastq -> gDNA_R1_146shortened.fastq ===Map reads with default Bowtie2=== bowtie2 --phred33 -x CAprobes_H1H2 -q gDNA_R1_146shortened.fastq > gDNA_R1_146shortened_CAprobesH1H2.sam & 4476041 reads; of these: 4476041 (100.00%) were unpaired; of these: 129983 (2.90%) aligned 0 times 4322633 (96.57%) aligned exactly 1 time 23425 (0.52%) aligned >1 times 97.10% overall alignment rate bowtie2 --phred33 -x CAprobes_H1H2 -q cDNA-RNaseA_R1_146shortened.fastq > cDNA-RNaseA_R1_146shortened_CAprobesH1H2.sam 2> cDNA-RNase_stderr.txt & 2852904 reads; of these: 2852904 (100.00%) were unpaired; of these: 81850 (2.87%) aligned 0 times 2740392 (96.06%) aligned exactly 1 time 30662 (1.07%) aligned >1 times 97.13% overall alignment rate bowtie2 --phred33 -x CAprobes_H1H2 -q cDNAwRNaseA_R1_146shortened.fastq > cDNAwRNaseA_R1_146shortened_CAprobesH1H2.sam 2> cDNAwRNase_stderr.txt & 2091417 reads; of these: 2091417 (100.00%) were unpaired; of these: 55021 (2.63%) aligned 0 times 2010743 (96.14%) aligned exactly 1 time 25653 (1.23%) aligned >1 times 97.37% overall alignment rate ===Sort and filter (MAPQ>=8) aligned reads=== *MAPQ=8 corresponds to 16% chance alignment is wrong, but manual checking showed that alignments with MAPQ=8 matched well samtools view -bS gDNA_R1_146shortened_CAprobesH1H2.sam | samtools sort - gDNA_R1_146shortened_CAprobesH1H2_sorted samtools view -h -F 4 -q 8 gDNA_R1_146shortened_CAprobesH1H2_sorted.bam > gDNA_R1_146shortened_CAprobesH1H2_sorted_filtered.sam samtools view -bS cDNA-RNaseA_R1_146shortened_CAprobesH1H2.sam | samtools sort - cDNA-RNaseA_R1_146shortened_CAprobesH1H2_sorted samtools view -h -F 4 -q 8 cDNA-RNaseA_R1_146shortened_CAprobesH1H2_sorted.bam > cDNA-RNaseA_R1_146shortened_CAprobesH1H2_sorted_filtered.sam samtools view -bS cDNAwRNaseA_R1_146shortened_CAprobesH1H2.sam | samtools sort - cDNAwRNaseA_R1_146shortened_CAprobesH1H2_sorted samtools view -h -F 4 -q 8 cDNAwRNaseA_R1_146shortened_CAprobesH1H2_sorted.bam > cDNAwRNaseA_R1_146shortened_CAprobesH1H2_sorted_filtered.sam ===Count reads aligned for each probe=== *Used [[Media:Count_reads_from_bowtie2_SAM.txt | Count_reads_from_bowtie2_SAM.pl]] to count number of appearances of each probe in column 3 (Reference Name) of sam file **CountofcDNA-RNaseA_CAprobesH1H2_filtered.txt -> total count for all probes: 2,554,856 **CountofcDNAwRNaseA_CAprobesH1H2_filtered.txt -> total count for all probes: 1,881,008 **CountofgDNA_CAprobesH1H2_filtered.txt -> total count for all probes: 3,976,822 ===Histograms and Results=== *Only used cDNA-RNaseA since previous regression analysis showed with or without RNaseA were essentially the same *"Good" probes were the set of probes that had a capture efficiency within one order of magnitude *"Brain optimized" probes were the set of probes that captured [[File:CA12k_gDNA_Capture.JPG]][[File:CA12k_cDNA-RNaseA_Capture.JPG]] *3,141 Unique "good" genes *2,615 Unique "brain optimized" genes *4,185 "Brain optimized" and "good" probes **1,963 "Brain optimized" and "good" genes **1,849 of which are brain genes **189 of which are stem cell genes Details of genes/probes can be found in [[Media:CA12k_Capture_Analysis.xlsx | CA12k_Capture_Analysis.xlsx]] ==Comparison of CA12k to Agi26k Results Summary== {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''CA12k''' | align="center" style="background:#f0f0f0;"|'''Agi26k0gap''' | align="center" style="background:#f0f0f0;"|'''Agi26k20gap''' |- | Good probes|| 7,900 || 6,596 || 5,403 |- | Good genes|| 3,141 || 2,628 || 2,529 |- | Brain-optimized probes|| 6,412 || 8,640 || 8,438 |- | Brain-optimized genes|| 2,615 || 3,140 || 3,133 |- | Good & Brain-optimized probes|| 4,185 || 4,822 || 4,006 |- | Good & Brain-optimized genes|| 1,963 || 2,075 || 2,075 |- | Good & Brain-optimized brain genes|| 1,849 || 2,002 || 1,996 |- | Good & Brain-optimized stem cell genes|| 189 || 138 || 151 |} <!-- 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) [[Media:FilteringGoodProbes_matched_to_Genes.xlsx|Counts matched with probes from:]] *[[Media:probe2padlockFISSEQ_Feb2013_0bp.txt|probe2padlockFISSEQ_Feb2013_0bp.pl]] [[Media:probe2padlockFISSEQ_Feb2013_0bp_stemCellGenes.txt|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 <br/> [[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 [[Media:CustomArray_Probe_Analysis_Summaryv4.docx|Final Summary]]-->
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