Matt:LabNotes/2016-7-13
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TB12k_Apr2016_V7 in vitro Capture Sequencing Analysis[edit]
- Sequencing library prep
- Compare to CA12k_Nov2014_V4
- mzcai@genome-miner2:~/scratch/TB12kApr2016_V7_CaptureAnalysis$
Check Sequencing Quality[edit]
/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
Build Bowtie2 reference file[edit]
- 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
- Also remove "-p#" from end of gene names that have it
- Build index
bowtie2-build TB12k_Apr2016_V7_H1H2.fa TB12k_Apr2016_V7_H1H2
Mapping Reads to Probelist[edit]
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[edit]
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";