Matt:LabNotes/2014-11-1
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Design FISSEQ Padlock Probeset for in situ cDNA Capture (New Genelist)[edit]
Get Genes[edit]
- Took all HUGO gene names from new genelist: TopDiffExpGenes_1000hNucDataset_10282014.xlsx
- Removed all genes with names that had: "-", "AC###.#", "AS", "RP", "RNA", "MT", "LINC" ...
- Result: 409 genes
Build Target File for ppDesigner[edit]
Biomart: Get Exon Locations for Genes on Reference Genome[edit]
- Use 409 HUGO names as input
- GRCh38/hg38 reference genome
- Unfortunately when try to output 'Associated Gene Name' (aka HUGO Official Gene Symbol) Biomart gave error so had to output Ensembl Gene IDs
- Some LRG (locus reference genome) gene names and HSCHR (haplotype variants) outputted so will filter those out
- Output parameters:
- Ensembl Gene ID
- Ensembl Transcript ID
- Chromosome
- Exon Rank
- Exon Start (bp)
- Exon End (bp)
- Strand
Create Hash to convert Ensembl to HUGO[edit]
- Using media:TopDiffExpGenes_1000hNucDataset_10282014.xlsx created hash table for converting Ensembl IDs to HUGO since Biomart output had to be ENSG
- Hash naturally removes duplicates but also checked to see if there was gene names that didn't match 1-to-1
- ENSG000000236922 matched to LINC01378 and AC092661.1
- ENSG000000228918 matched to LINC01344 and GS1-122H1.2
- Hash naturally removes duplicates but also checked to see if there was gene names that didn't match 1-to-1
Create Target File v2: Target consitutive exons (no crossing exon boundaries)[edit]
- Changed the algorithm to only select intervals that don't contain boundaries of exons (like constitutive exons but doesn't have to be present in all transcripts)
- Ordered all boundaries from least to great regardless if start or stop
- In another array of equal size kept track of whether it was start or stop boundary
- Selected intervals that on the left have a start boundary and on the right has a stop boundary with no boundaries in the middle
- Ordered all boundaries from least to great regardless if start or stop
- CreateTargetFile_v2.pl
- input: biomart_export_newGenes.txt
- output: target_file_newGenes.txt
- Tested with one gene and two genes consisting of multiple overlapping exons
- Runs with warnings but behaves correctly
- Hash for converting Ensembl to HUGO missed a few genes due to multiple Ensembl Gene IDs for same HUGO IDs so had to do them manually
- KCNMB2
- NBPF15
- NEFL
- sortTargetFilesByChr.pl
- Removes 25bp from each end of target
- Switch strand from + to - and - to +
- Also runs with warnings that 'given' and 'when' are experimental
Run ppDesigner[edit]
ppDesignerCommands.sh
Consolidate OutputFiles and Convert to 0gap[edit]
- Concatenate output files into 1 file: outputFile_1gap.txt
cat outputFile_chr*.txt > outputFile_1gap.txt
- ConvertToZeroGapProbe.pl
- 4,593 probes designed
- 402 genes
- 3,599 exons
Filter Out Bad Probes[edit]
- Align probes to reference genome and mRNA
perl 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 CleanupProbelist.pl
- 3,553 probes
- 389 genes
- 2,813 exons
Filter Probes with Low Coverage based on Brain RNA-seq Data from Rui/Blue[edit]
- 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: 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
- Also converts hg38 positions to hg19 to match RNA-seq data
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
FilterProbelist_LowReadCounts.pl
- Result: outputFile_0gap_10reads.txt
RevComp Probes for potential RNA capture[edit]
- RevComp() each arm and then swap arms (columns 5 and 7)
- RevComp() target region (column 11)
RevComp_Probes.pl
- Result: outputFile_0gap_10reads_RevComp.txt
- For each outputFile_0gap_10reads.txt and outputFile_0gap_10reads_RevComp.txt
- 1,993 probes
- 1,570 exons
- 348 genes