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=5fC DIP-seq= '''HL102''' Dipseq-Input-Control-Sep2 : s1, s2 Dipseq-Input-KD-Sep2 : s3, s4 Dipseq-IP-Control-Sep2 : s5, s6 Dipseq-IP-KD-Sep2 : s7, s8 ==Mapping== bowtie -n 2 --best --strata -l 28 -k 1 -m 1 -p 12 --phred64-quals --un $f.unmappable.txt /media/1TB_store1/Rui_Scratch/iGenome/Mus_musculus/UCSC/mm9/Sequence/BowtieIndex/genome $f $f.bowtie.map 2> $f.log & **Each lane was mapped separately, data was then concatenated. '''Mapping rates''' {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Unique Hit''' | align="center" style="background:#f0f0f0;"|'''No Hit''' | align="center" style="background:#f0f0f0;"|'''>1 Hits''' | align="center" style="background:#f0f0f0;"|'''TOTAL''' | align="center" style="background:#f0f0f0;"|'''%Unique Hit''' | align="center" style="background:#f0f0f0;"|'''%No Hit''' | align="center" style="background:#f0f0f0;"|'''%>1 Hits''' |- | s1||24626804||1084945||7643512||33355261||73.83%||3.25%||22.92% |- | s2||25108666||1082767||7818626||34010059||73.83%||3.18%||22.99% |- | s3||25759355||1188545||8379585||35327485||72.92%||3.36%||23.72% |- | s4||21717915||1067567||7093600||29879082||72.69%||3.57%||23.74% |- | s5||19239845||1244633||7924574||28409052||67.72%||4.38%||27.89% |- | s6||23397892||1180981||9638993||34217866||68.38%||3.45%||28.17% |- | s7||23974239||1228065||9301572||34503876||69.48%||3.56%||26.96% |- | s8||22722261||1188036||8854346||32764643||69.35%||3.63%||27.02% |} *Note that Input has more hits than IP. cat s_1_sequence.txt.gz.fastq.bowtie.map s_2_sequence.txt.gz.fastq.bowtie.map > 5fC_dIP-seq_Input_Ctrl.bowtie.map & cat s_3_sequence.txt.gz.fastq.bowtie.map s_4_sequence.txt.gz.fastq.bowtie.map > 5fC_dIP-seq_Input_KD.bowtie.map & cat s_5_sequence.txt.gz.fastq.bowtie.map s_6_sequence.txt.gz.fastq.bowtie.map > 5fC_dIP-seq_IP_Ctrl.bowtie.map & cat s_7_sequence.txt.gz.fastq.bowtie.map s_8_sequence.txt.gz.fastq.bowtie.map > 5fC_dIP-seq_IP_KD.bowtie.map & ==MACS== Downloads and installed MACS debian package onto meangenemachine http://liulab.dfci.harvard.edu/MACS/ Followed instructions to install PeakSplitter_v1.0 (copied to /usr/local/bin) (**Results from peak splitter was not used because no p-value given for each peak) ===Control=== macs14 -t 5fC_dIP-seq_IP_Ctrl.bowtie.map -c 5fC_dIP-seq_Input_Ctrl.bowtie.map -f BOWTIE -g mm -n Control-Dipseq -w --call-subpeaks > macs14_ctrol & All peaks called with p-value (based on Poisson distribution with local lambda) and FDR (%) [[File:Control-Dipseq_peaks.xls]] Negative peaks were called by switching input and ip [[File:Control-Dipseq_negative_peaks.xls]] Total peaks = 512; Total negative peaks = 25 (5.08% FDR for this method) - '''I should down-sample hits so that input and ip have equal number of hits.''' File for viewing peaks (FDR<5%) on genome browser: [[File:Control-Dipseq_peaks.BED.txt]] ===Knockdown=== macs14 -t 5fC_dIP-seq_IP_KD.bowtie.map -c 5fC_dIP-seq_Input_KD.bowtie.map -f BOWTIE -g mm -n KD-Dipseq -w --call-subpeaks > macs14_kd & All peaks called with p-value (based on Poisson distribution with local lambda) and FDR (%) [[File:KD-Dipseq_peaks.xls]] Negative peaks were called by switching input and ip [[File:KD-Dipseq_negative_peaks.xls]] Total peaks = 317; Total negative peaks = 23 (7.57% FDR for this method) File for viewing peaks (FDR<5%) on genome browser: [[File:KD-Dipseq_peaks.BED.txt]] ==Enrichment in genomic regions== (A) Gene regions were taken from iGenome data which Rui downloaded: /media/1TB_store1/Rui_Scratch/iGenome/Mus_musculus/UCSC/mm9/Annotation/Genes [[File:refFlat.txt.gz]] File was parsed into five regions files using this script : [[File:parseRefFlat.txt]] mm9_ucsc_genes.3utr.bed - regions from coding start end to transcription end N = 28,108 sort -u | wc -l : 24,052 mm9_ucsc_genes.5utr.bed - regions from transcription start to coding start N = 28,108 sort -u | wc -l : 24,143 mm9_ucsc_genes.tss.bed - regions from -800 to +200 bp of transcription start N = 28,108 sort -u | wc -l : 23,652 mm9_ucsc_genes.exons.bed - coding regions N = 267,543 sort -u | wc -l : 208,522 mm9_ucsc_genes.introns.bed - non-coding regions (between every exons) N = 239,435 sort -u | wc -l : 186,730 **Performed sort -u on all files. (B) CpGIslands-regions.bed : downloaded from UCSC Table Browser for mm9 (C) mouseESp300binding_LICR_regions.bed : downloaded from UCSC Table Browser for mm9 (D) REST-binding_sites.bed (Repressor Element 1 Silencing Transcription Factor binding sites) : downloaded from UCSC Table Browser for mm9 ===Criteria for mapping to genomic regions=== *Count frequency of overlaps with a genomic region: For peaks: Total # bp overlapping with peaks For randomized regions: Total # bp overlapping with random regions ** Note Pastor et al required for larger regions that entire HERGs/MERGs fall within a larger genomic region. ===Criteria for randomized regions=== *Randomized regions conserve # peaks, chromosome distribution, and peak widths distribution. *Performed 1000 randomizations (Imputations). *Script: [[File:imputeRegionEnrichment.txt]] ===Results=== '''Control sample''' Total bp = 400,486bp {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Region_File''' | align="center" style="background:#f0f0f0;"|'''Test_counts''' | align="center" style="background:#f0f0f0;"|'''Imputed_ave''' | align="center" style="background:#f0f0f0;"|'''Imputed_std''' | align="center" style="background:#f0f0f0;"|'''Empirical_p (greater than)''' | align="center" style="background:#f0f0f0;"|'''Empirical_p (less than)''' |- | CpGIslands-regions.bed||5632||1754.636||973.5852||'''0'''||1 |- | mm9_ucsc_genes.3utr.bed||6782||12116.25||3978.4707||0.929||'''0.071''' |- | mm9_ucsc_genes.5utr.bed||22232||16190.186||4545.6284||'''0.087'''||0.913 |- | mm9_ucsc_genes.exons.bed||3856||9942.256||2401.9812||0.999||'''0.001''' |- | mm9_ucsc_genes.introns.bed||143652||149212.281||11603.9548||0.688||0.312 |- | mm9_ucsc_genes.tss.bed||4479||3729.626||1575.1404||0.296||0.704 |- | mouseESp300binding_LICR_regions.bed||6007||3879.494||1621.2735||0.112||0.888 |- | REST-binding_sites.bed||37||28.687||57.2239||0.238||0.762 |} *Control is '''enriched''' in CpG Islands and 5'UTR. *Control is '''depleted''' in 3'UTR and exons. '''Knock down''' Total bp = 220,304bp {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Region_File''' | align="center" style="background:#f0f0f0;"|'''Test_counts''' | align="center" style="background:#f0f0f0;"|'''Imputed_ave''' | align="center" style="background:#f0f0f0;"|'''Imputed_std''' | align="center" style="background:#f0f0f0;"|'''Empirical_p (greater than)''' | align="center" style="background:#f0f0f0;"|'''Empirical_p (less than)''' |- | CpGIslands-regions.bed||121||950.83||760.1284||0.876||0.124 |- | mm9_ucsc_genes.3utr.bed||4465||6457.113||2854.0275||0.742||0.258 |- | mm9_ucsc_genes.5utr.bed||5055||8971.008||3276.3609||0.891||0.109 |- | mm9_ucsc_genes.exons.bed||436||5460.863||1840.9045||1||'''0''' |- | mm9_ucsc_genes.introns.bed||86823||81022.857||8769.8664||0.248||0.752 |- | mm9_ucsc_genes.tss.bed||0||2064.625||1219.295||1||'''0''' |- | mouseESp300binding_LICR_regions.bed||1278||2084.766||1255.2735||0.707||0.293 |- | REST-binding_sites.bed||26||17.38||46.7585||0.169||0.831 |} *Knockdown is '''depleted''' at exons and tss. ==Reads balancing== head -42637737 5fC_dIP-seq_Input_Ctrl.bowtie.map > sampled_5fC_dIP-seq_Input_Ctrl.bowtie.map head -42637737 5fC_dIP-seq_Input_KD.bowtie.map > sampled_5fC_dIP-seq_Input_KD.bowtie.map head -42637737 5fC_dIP-seq_IP_KD.bowtie.map > sampled_5fC_dIP-seq_IP_KD.bowtie.map ** 5fC_dIP-seq_IP_Ctrl.bowtie.map had the fewest hits ** '''Call peaks with macs14''' nohup macs14 -t 5fC_dIP-seq_IP_Ctrl.bowtie.map -c sampled_5fC_dIP-seq_Input_Ctrl.bowtie.map -f BOWTIE -g mm -n Control-Dipseq -w --call-subpeaks > macs14_ctrol_balanced & nohup macs14 -t sampled_5fC_dIP-seq_IP_KD.bowtie.map -c sampled_5fC_dIP-seq_Input_KD.bowtie.map -f BOWTIE -g mm -n KD-Dipseq -w --call-subpeaks > macs14_kd_balanced & '''Peaks called''' Positive Negative Result file: Control 354 24 [[File:Control_Dipseq_balanced_peaks.xls]] Knockdown 301 21 [[File:KD_Dipseq_balanced_peaks.xls]] FDR < 5% BED file: Control [[File:Control_Dipseq_balanced_peaks.BED.txt]] Total 107,302 bp Knockdown [[File:KD_Dipseq_balanced_peaks.BED.txt]] Total 124,348 bp {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Region_File''' | align="center" style="background:#f0f0f0;"|'''Control_Test_counts''' | align="center" style="background:#f0f0f0;"|'''Imputed_ave''' | align="center" style="background:#f0f0f0;"|'''Imputed_std''' | align="center" style="background:#f0f0f0;"|'''Empirical_p (greater than)''' | align="center" style="background:#f0f0f0;"|'''Empirical_p (less than)''' |- | CpGIslands-regions.bed||4991||453.284||533.1481||'''0'''||1 |- | mm9_ucsc_genes.3utr.bed||162||3183.955||2565.3198||0.935||'''0.065''' |- | mm9_ucsc_genes.5utr.bed||5601||4194.234||2735.7066||0.236||0.764 |- | mm9_ucsc_genes.exons.bed||748||2570.128||1355.5966||0.954||'''0.0459999999999999''' |- | mm9_ucsc_genes.introns.bed||45261||38383.062||7101.3337||0.161||0.839 |- | mm9_ucsc_genes.tss.bed||2002||949.66||869.7907||0.123||0.877 |- | mouseESp300binding_LICR_regions.bed||1187||1065.255||915.3372||0.387||0.613 |- | REST-binding_sites.bed||10||7.649||40.1048||'''0.085'''||0.915 |} {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Region_File''' | align="center" style="background:#f0f0f0;"|'''KD_Test_counts''' | align="center" style="background:#f0f0f0;"|'''Imputed_ave''' | align="center" style="background:#f0f0f0;"|'''Imputed_std''' | align="center" style="background:#f0f0f0;"|'''Empirical_p (greater than)''' | align="center" style="background:#f0f0f0;"|'''Empirical_p (less than)''' |- | CpGIslands-regions.bed||0||465.608||533.0195||1||'''0''' |- | mm9_ucsc_genes.3utr.bed||2560||3584.865||2209.6552||0.64||0.36 |- | mm9_ucsc_genes.5utr.bed||1268||5157.965||2703.5931||0.952||'''0.0479999999999999''' |- | mm9_ucsc_genes.exons.bed||192||3080.453||1407.9471||0.999||'''0.001''' |- | mm9_ucsc_genes.introns.bed||39030||45091.197||6652.1637||0.811||0.189 |- | mm9_ucsc_genes.tss.bed||0||1169.726||903.6226||1||'''0''' |- | mouseESp300binding_LICR_regions.bed||584||1142.344||904.8301||0.71||0.29 |- | REST-binding_sites.bed||11||10.553||40.473||0.116||0.884 |} ==Discussion== [[File:5fC_Figure1.pdf]] A) Genome browser snap shot of wig files around a called peak. B) Pie charts showing base pairs genomic distribution of all significant peaks (FDR<5%) with annotated genomic regions. C) Using wig files from input and IP, the mean occupancy of reads a 100bp sliding window around each of ~19K promoters were calculated. The mean values from input were subtracted from mean values of IP. Genes were separated into 5 groups based on the expression level of replicate #1 in the [GSE13805 | http://www.wip.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE13805] study on mouse ESCs. * More peaks were found in knockdown than in wildtype, this supports that the knocked-down protein is responsible for converting 5fC into something else. * In both control and knockdown, enrichment of peaks were in introns or other genomic regions and depletion of peaks (p<0.05) were in exons. * In wildtype, occupancy of 5fC around promoters were not correlated with gene activity. * In knockdown, occupancy of 5fC around promoters appears to be correlated with gene activity. Higher densities of 5fC were found towards the 5'UTR region of the gene than upstream of TSS.
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