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==Agi26k Capture Analysis (MiSeq_130729)== *Analysis here is mostly similar to [[Matt:LabNotes/2013-8-20|CA12k Capture (MiSeq_130325) Analysis]] ===Building bowtie2 index for Agi26k probes=== *For 0gap probes converted probelist to fasta file with H1, H2,and barcode region rearranged similar to CA12kprobes [[Media:Agi26k0gapProbelist_to_fasta.txt | Agi26k0gapProbelist_to_fasta.pl]]: FISSEQ_Probes_Dec2012.txt -> Agi26k0gapprobes_to_order_H1H2.fa **Fasta reference made to match reads: RevComp(H1) + RevComp(H2) + RevComp(Barcode) **Had to delete probes 12964-26142 since they are 20gap probes *For 20gap probes did something similar but had to include 20bp target region in between H1 and H2 [[Media:Agi26k20gapProbelist_to_fasta_corrected.txt | Agi26k20gapProbelist_to_fasta_corrected.pl]]: FISSEQ_Probes_Dec2012.txt -> Agi26k20gapprobes_to_order_H1H2_corrected.fa **Target sequence was found by using H1 sequence to search through outputFile_20gap.txt and outputFile_20gap_stem.txt (the two files output from ppDesigner when designing probes for genes and stem cell genes) **Fasta reference made to match reads: RevComp(H1) + target + RevComp(H2) + RevComp(Barcode) **Had to delete probes 0-12963 since they are 0gap probes *Build index: genome-miner:$ bowtie2-build Agi26k0gapprobes_to_order_H1H2.fa Agi26k0gap_H1H2 genome-miner:$ bowtie2-build Agi26k20gapprobes_to_order_H1H2_corrected.fa Agi26k20gap_H1targetH2 ===Reads=== MZC-Agi26k-0cDNA-Jul11_CCTATT_L001_R1_001.fastq<br> MZC-Agi26k-0gDNA-Jul11_ACTACG_L001_R1_001.fastq<br> MZC-Agi26k-20cDNA-Jul11_CGTGTT_L001_R1_001.fastq<br> MZC-Agi26k-20gDNA-Jul11_ACGGGT_L001_R1_001.fastq<br> ===Map reads with default Bowtie2=== bowtie2 --phred33 -x Agi26k0gap_H1H2 -q MZC-Agi26k-0gDNA-Jul11_ACTACG_L001_R1_001.fastq > Agi26k-0gDNA_R1_H1H2.sam 2> Agi26k-0gDNA_stderr.txt & 4535752 reads; of these: 4535752 (100.00%) were unpaired; of these: 81030 (1.79%) aligned 0 times 4433426 (97.74%) aligned exactly 1 time 21296 (0.47%) aligned >1 times 98.21% overall alignment rate bowtie2 --phred33 -x Agi26k0gap_H1H2 -q MZC-Agi26k-0cDNA-Jul11_CCTATT_L001_R1_001.fastq > Agi26k-0cDNA_R1_H1H2.sam 2> Agi26k-0cDNA_stderr.txt & 4981156 reads; of these: 4981156 (100.00%) were unpaired; of these: 84336 (1.69%) aligned 0 times 4836874 (97.10%) aligned exactly 1 time 59946 (1.20%) aligned >1 times 98.31% overall alignment rate bowtie2 --phred33 -x Agi26k20gap_H1targetH2 -q MZC-Agi26k-20gDNA-Jul11_ACGGGT_L001_R1_001.fastq > Agi26k-20gDNA_R1_H1targetH2.sam 2> Agi26k-20gDNA_stderr.txt & 3789673 reads; of these: 3789673 (100.00%) were unpaired; of these: 948660 (25.03%) aligned 0 times 2819628 (74.40%) aligned exactly 1 time 21385 (0.56%) aligned >1 times 74.97% overall alignment rate bowtie2 --phred33 -x Agi26k20gap_H1targetH2 -q MZC-Agi26k-20cDNA-Jul11_CGTGTT_L001_R1_001.fastq > Agi26k-20cDNA_R1_H1targetH2.sam 2> Agi26k-20cDNA_stderr.txt & 5808711 reads; of these: 5808711 (100.00%) were unpaired; of these: 873070 (15.03%) aligned 0 times 4880686 (84.02%) aligned exactly 1 time 54955 (0.95%) aligned >1 times 84.97% 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 Agi26k-0gDNA_R1_H1H2.sam | samtools sort - Agi26k-0gDNA_R1_H1H2_sorted samtools view -h -F 4 -q 8 Agi26k-0gDNA_R1_H1H2_sorted.bam > Agi26k-0gDNA_R1_H1H2_sorted_filtered.sam samtools view -bS Agi26k-0cDNA_R1_H1H2.sam | samtools sort - Agi26k-0cDNA_R1_H1H2_sorted samtools view -h -F 4 -q 8 Agi26k-0cDNA_R1_H1H2_sorted.bam > Agi26k-0cDNA_R1_H1H2_sorted_filtered.sam samtools view -bS Agi26k-20gDNA_R1_H1targetH2.sam | samtools sort - Agi26k-20gDNA_R1_H1targetH2_sorted samtools view -h -F 4 -q 8 Agi26k-20gDNA_R1_H1targetH2_sorted.bam > Agi26k-20gDNA_R1_H1targetH2_sorted_filtered.sam samtools view -bS Agi26k-20cDNA_R1_H1targetH2.sam | samtools sort - Agi26k-20cDNA_R1_H1targetH2_sorted samtools view -h -F 4 -q 8 Agi26k-20cDNA_R1_H1targetH2_sorted.bam > Agi26k-20cDNA_R1_H1targetH2_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 **CountofgDNA_Agi26k0gapH1H2_filtered.txt -> total count for all probes: 4,414,961 **CountofcDNA_Agi26k0gapH1H2_filtered.txt -> total count for all probes: 4,850,618 **CountofgDNA_Agi26k20gapH1targetH2_filtered.txt -> total count for all probes: 1,710,730 **CountofcDNA_Agi26k20gapH1targetH2_filtered.txt -> total count for all probes: 4,298,273 ===Histograms and Results=== *"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 Details of genes/probes can be found in [[Media:Agi26k_Capture_Analysis.xlsx | Agi26k_Capture_Analysis.xlsx]] ====Agi26k0gap==== [[File:Agi26k0gap_gDNA_Capture.JPG]][[File:Agi26k0gap_cDNA_Capture.JPG]] *2,628 Unique "good" genes *3,140 Unique "brain optimized" genes *4,822 "Brain optimized" and "good" probes **2,075 "Brain optimized" and "good" genes **2,002 of which are brain genes **138 of which are stem cell genes ====Agi26k20gap==== [[File:Agi26k20gap_gDNA_Capture.JPG]][[File:Agi26k20gap_cDNA_Capture.JPG]] *2,529 Unique "good" genes *3,133 Unique "brain optimized" genes *4,006 "Brain optimized" and "good" probes **2,075 "Brain optimized" and "good" genes **1,996 of which are brain genes **151 of which are stem cell genes ==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 |}
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