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===Filter Out Bad Probes=== *Align probes to reference genome and mRNA perl [[Media:Probes2fasta.txt | Probes2fasta.pl]] < outputFile_0gap.txt > outputFile_0gap.fa /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/RNAseq/Data/CommonFiles/refMrna.ndx -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_refMrna.out & /home/kunzhang/softwares/Novocraft/novocraft/novoalign -d /home/kunzhang/softwares/Novocraft/novocraft/human_g1k_v37 -f outputFile_0gap.fa -F FA -r ALL > outputFile_0gap_novoalign_human_g1k_v37.out & *Filter out probes that map to multiple locations in genome or don't map to refMrna perl [[Media:CleanupProbelist.txt | CleanupProbelist.pl]] *3,553 probes **389 genes **2,813 exons ====Filter Probes with Low Coverage based on Brain RNA-seq Data from Rui/Blue==== *Must still filter out probes that target regions with low coverage based on RNA-seq data **Sequencing data for transcripts from tissue Brodmann Area 8,10,17,21,22,41 /media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR/ samtools merge ~/scratch/FISSEQ_cDNA_ProbeDesign/BrainBulkTissueRNA_mappedhg19.bam RL-BA10-t-N707-15May14_S7_mapped/RL-BA10-t-N707-15May14_S7_Aligned.sorted.bam RL-BA17-t-N703-15May14_S3_mapped/RL-BA17-t-N703-15May14_S3_Aligned.sorted.bam RL-BA21-t-N705-15May14_S5_mapped/RL-BA21-t-N705-15May14_S5_Aligned.sorted.bam RL-BA22-t-N709-15May14_S2_mapped/RL-BA22-t-N709-15May14_S2_Aligned.sorted.bam RL-BA41-t-N711-15May14_S4_mapped/RL-BA41-t-N711-15May14_S4_Aligned.sorted.bam RL-BA8-sec9-t-N701-15May14_S1_mapped/RL-BA8-sec9-t-N701-15May14_S1_Aligned_chr12_16.sorted.bam samtools sort BrainBulkTissueRNA_mappedhg19.bam BrainBulkTissueRNA_mappedhg19.sorted samtools index BrainBulkTissueRNA_mappedhg19.sorted.bam BrainBulkTissueRNA_mappedhg19.sorted.bam.bai *Convert probe outputFile_0gap_filtered.txt (from CleanupProbelist.pl) to BED file: [[Media:ConvertProbesHg38_2_BedHg19.txt | ConvertProbesHg38_2_BedHg19.pl]] **Also converts hg38 positions to hg19 to match RNA-seq data ***Uses position alignments to human_g1k_v37 to get hg19 coordinates ***Checked new coordinates with UCSC Genome Browser for genes on forward and reverse strands - both were correct coverageBed -abam BrainBulkTissueRNA_mappedhg19.sorted.bam -b outputFile_0gap_hg19.bed > outputFile_BrainBulkTissueRNA.cov *Rank Order plot of Probes and the number of reads that overlap with each probe target region **621 of 3,553 probes had 0 overlap [[File:RankOrderProbesOverlapReads.JPG]] *Filter out probes with <10 reads overlapping target region [[Media:FilterProbelist_LowReadCounts.txt | FilterProbelist_LowReadCounts.pl]] **Result: [[Media:OutputFile_0gap_10reads.txt | outputFile_0gap_10reads.txt]]
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