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=TB12k_Apr2016_V7 in vitro Capture Sequencing Analysis= *[[Matt:LabNotes/2016-6-20|Sequencing library prep]] *[[Matt:LabNotes/2015-7-12|Compare to CA12k_Nov2014_V4]] *mzcai@genome-miner2:~/scratch/TB12kApr2016_V7_CaptureAnalysis$ ===Check Sequencing Quality=== /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/src/fastx_quality_stats/fastx_quality_stats -Q33 -i MC20160628-TB12kApr16V7-gDNA-1_S17_L001_R1_001.fastq -o MC20160628-TB12kApr16V7-gDNA_qualstats.txt /home/kunzhang/softwares/fastx_toolkit-0.0.13.2/scripts/fastq_quality_boxplot_graph.sh -i MC20160628-TB12kApr16V7-gDNA_qualstats.txt -o MC20160628-TB12kApr16V7-gDNA_qualstats.png MC20160628-TB12kApr16V7-gDNA [[File:MC20160628-TB12kApr16V7-gDNA_qualstats.png | 650px]] ===Build Bowtie2 reference file=== *Working in mzcai@genome-miner2:~/scratch/TB12kApr2016_V7_CaptureAnalysis$ *Use TB12kApr2016_Probelist2Fasta.pl to convert outputFile_0gap_FULL200_MouseBrain_V7.txt to TB12k_Apr2016_V7_H1H2.fa **Also remove "-p#" from end of gene names that have it ***This suffix was used to generate different barcodes for the same gene during probe design *Build index bowtie2-build TB12k_Apr2016_V7_H1H2.fa TB12k_Apr2016_V7_H1H2 ===Mapping Reads to Probelist=== bowtie2 --phred33 -x TB12k_Apr2016_V7_H1H2 -q MC20160628-TB12kApr16V7-gDNA-1_S17_L001_R1_001.fastq > TB12kApr2016_V7gDNA_R1_H1H2.sam 2> TB12kApr2016_V7gDNA_stderr.txt & 1029831 reads; of these: 1029831 (100.00%) were unpaired; of these: 209569 (20.35%) aligned 0 times 820262 (79.65%) aligned exactly 1 time 0 (0.00%) aligned >1 times 79.65% overall alignment rate samtools view -bS TB12kApr2016_V7gDNA_R1_H1H2.sam | samtools sort - TB12kApr2016_V7gDNA_R1_H1H2_sorted samtools view -h -F 4 TB12kApr2016_V7gDNA_R1_H1H2_sorted.bam > TB12kApr2016_V7gDNA_R1_H1H2_sorted_filtered.sam ===Counting Reads for each Probe=== [[Media:CountReadsPer_Gene_Probe.txt | CountReadsPer_Gene_Probe.pl]] my $samfile = "TB12kApr2016_V7gDNA_R1_H1H2_sorted_filtered.sam"; my $probecountfile = "TB12kApr2016_V7gDNA_R1_H1H2_sorted_filtered_Probecounts.txt"; my $genecountfile = "TB12kApr2016_V7gDNA_R1_H1H2_sorted_filtered_Genecounts.txt"; ===Results=== ===Conclusions=== [[Media:.xlsx | Excel analysis]]
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