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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]]
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