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=RRBS Data Analysis to Encode Project (Fastq)= ==RRBS Data Analysis to Encode Project (Bed/MethylFreq)== ===Data Download=== Fastq and bed files can be downloaded from Encode Project. 101 RRBS data (bed files) were downloaded. 2,646,999 CpG loci were covered by 101 RRBS data while 866,979 CpG loci (32.8%) were detected in at least 80% samples. * File Download cd /home/shg047/oasis/monod/rrbs_encode wget http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethylRrbs/files.txt wget http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethylRrbs/md5sum.txt perl fastqDownloadRRBSEncode.pl files.txt *bed11 to bedGraph cd /home/shg047/oasis/monod/rrbs_encode for i in `ls *bed.gz` do zcat $i | grep -v "^track" |sort -k1,1 -k2,2n | awk '$5>9 {print $1"\t"$2"\t"$3"\t"$11}'> $i.bedGraph done *Pearson Correlation cd /home/shg047/oasis/monod/rrbs_encode ==Aim 2: Haib39bioChain== #methylation haplotype block (MHB) calling with RRBS dataset ===Background=== #Basic:Single-end 40bp reads #Encode: http://genome.ucsc.edu/ENCODE/downloads.html #Encode|RRBS1: http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethylRrbs/ #Encode|RRBS2: http://genome.ucsc.edu/cgi-bin/hgTrackUi?hgsid=437674359_aUhx08DjchWwtBjyCYv61EB7Yy8S&c=chr1&g=wgEncodeHaibMethylRrbs #Encode|Methy450K: http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethyl450/ #Encode|software: https://www.encodeproject.org/software/ ===Method=== # Never Download Encode Data from UCSC. Encode Mainpage is great: https://www.encodeproject.org/search/?type=Experiment # Download 39 biochain RRBS dataset from Encode Project (Fastq). Alignment with Bismark and merge all the BAM files # make the haplotype calling with previous perl script. Finally MHB calling were down with Dr. Zhang perl script. # Methyfreq based MHB calling were conducted with MethBed files download from UCSC. ===Result=== Summary Excel [https://docs.google.com/spreadsheets/d/1FGm4_BuInVmjZolUlhFZHc56AckrrBKf4dQ8XmFS4hs/edit#gid=0 Haib Dataset manifest ] *Alignment: Maybe walltime time is too short , not all the samples were aligned completely, so I extend the walltime to 72 hours. #PBS -q glean #PBS -l nodes=1:ppn=8 #PBS -l walltime=72:00:00 bismark --bowtie2 --phred64-quals --fastq -L 30 -N 1 --multicore 2 /home/shg047/db/hg19/meth/bismark ../fastq_trim/HOT197_trimmed.fq.gz -o ../bam2 *With above setting, the alignment time usage is about 16 hours *The time is propotional to the size of the fastq file {| style="font-size:80%;" | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''N(reads)''' | align="center" style="background:#f0f0f0;"|'''N(mapped)''' | align="center" style="background:#f0f0f0;"|'''P(mapping)''' | align="center" style="background:#f0f0f0;"|'''N(C)''' | align="center" style="background:#f0f0f0;"|'''N(MCPG)''' | align="center" style="background:#f0f0f0;"|'''N(MCHG)''' | align="center" style="background:#f0f0f0;"|'''N(MCHH)''' | align="center" style="background:#f0f0f0;"|'''N(UCPG)''' | align="center" style="background:#f0f0f0;"|'''N(UCHG)''' | align="center" style="background:#f0f0f0;"|'''N(UCHH)''' | align="center" style="background:#f0f0f0;"|'''P(MCPG)''' | align="center" style="background:#f0f0f0;"|'''P(MCHG)''' | align="center" style="background:#f0f0f0;"|'''P(MCHH)''' |- | ENCFF000LVN||42665329||31372051||73.50%||227374577||9979920||566174||2568709||16516656||50531408||147211710||37.70%||1.10%||1.70% |- | ENCFF000LWL||37938401||25076517||66.10%||232580318||11488185||403775||1382726||44058434||51038500||124208698||20.70%||0.80%||1.10% |- | ENCFF000LVR||36779630||25226017||68.60%||216621540||12232832||438455||1846136||33041219||46480917||122581981||27.00%||0.90%||1.50% |- | ENCFF000LVW||36169867||22446797||62.10%||224690704||12356273||372366||871608||41484525||51598506||118007426||22.90%||0.70%||0.70% |- | ENCFF000LVB||35375019||22874896||64.70%||211937214||13517127||359167||1121727||31366464||47860817||117711912||30.10%||0.70%||0.90% |- | ENCFF000LUP||34214415||20212548||59.10%||221862905||16119238||340947||580628||49637185||51949984||103234923||24.50%||0.70%||0.60% |- | ENCFF000LWA||33924878||20895342||61.60%||226298397||14274674||319602||583229||52752860||53011846||105356186||21.30%||0.60%||0.60% |- | ENCFF000LUV||33798173||23173663||68.60%||249630261||10489904||225941||428583||62372541||57063134||119050158||14.40%||0.40%||0.40% |- | ENCFF000LWY||33715116||19999502||59.30%||213287381||13105506||316152||583822||46433586||48748868||104099447||22.00%||0.60%||0.60% |- | ENCFF000LWW||33642793||20282649||60.30%||216267059||15823449||340126||638093||43292443||49869268||106303680||26.80%||0.70%||0.60% |- | ENCFF000LWP||32858930||20654452||62.90%||219544115||13223974||319601||599593||48455363||50129553||106816031||21.40%||0.60%||0.60% |- | ENCFF000LUU||32815340||17866807||54.40%||196534662||12582324||421909||799252||48097172||45287260||89346745||20.70%||0.90%||0.90% |- | ENCFF000LVF||31276946||22130843||70.80%||241339808||11057578||348695||664774||64178792||55276052||109813917||14.70%||0.60%||0.60% |- | ENCFF000LXB||29681695||14796658||49.90%||160388755||8182288||279840||480680||41788931||37024968||72632048||16.40%||0.80%||0.70% |- | ENCFF000LWK||29349446||12860133||43.80%||137008343||8665157||302283||567461||29580229||31441673||66451540||22.70%||1.00%||0.80% |- | ENCFF000LWE||29211774||11162094||38.20%||115410335||8071486||277005||511615||23863725||26859483||55827021||25.30%||1.00%||0.90% |- | ENCFF000LVK||27335110||16938233||62.00%||193589572||12327753||322954||495345||51995628||43692466||84755426||19.20%||0.70%||0.60% |- | ENCFF000LVO||26190075||19276422||73.60%||150096025||7012551||300245||1332640||19959469||31819734||89671386||26.00%||0.90%||1.50% |- | ENCFF000LVA||25660341||16009728||62.40%||152217865||12748317||546338||1806399||26835875||36223859||74057077||32.20%||1.50%||2.40% |- | ENCFF000LWD||25467744||15459206||60.70%||149205383||11335949||535669||1829440||30226946||34383379||70894000||27.30%||1.50%||2.50% |- | ENCFF000LVU||23511285||15078397||64.10%||141659239||8631088||272002||734661||21915485||33328412||76777591||28.30%||0.80%||0.90% |- | ENCFF000LWO||22844877||14206467||62.20%||153005363||9813445||247506||461786||33745829||35223721||73513076||22.50%||0.70%||0.60% |- | ENCFF000LVI||22656883||13335953||58.90%||143634615||13714133||389619||650547||32241351||34451974||62186991||29.80%||1.10%||1.00% |- | ENCFF000LUQ||22247066||13650824||61.40%||150112632||9777330||287617||504659||38167142||34898963||66476921||20.40%||0.80%||0.80% |- | ENCFF000LUT||22240097||14849726||66.80%||162779228||9543204||285378||485629||41824570||38682843||71957604||18.60%||0.70%||0.70% |- | ENCFF000LWH||21943620||12872848||58.70%||137662143||11585659||414164||781429||29526002||32473470||62881419||28.20%||1.30%||1.20% |- | ENCFF000LVE||21473578||14519161||67.60%||157450743||7444292||221900||417095||40943018||36372568||72051870||15.40%||0.60%||0.60% |- | ENCFF000LWX||21208447||13218301||62.30%||145215571||9668358||223882||356827||34200783||33840207||66925514||22.00%||0.70%||0.50% |- | ENCFF000LVV||21080863||13197949||62.60%||142640926||10137634||255425||440789||32877645||33833167||65096266||23.60%||0.70%||0.70% |- | ENCFF000MLE||20338545||12207095||60.00%||110465581||11201397||457519||1827464||19226614||25845153||51907434||36.80%||1.70%||3.40% |- | ENCFF000LWS||20241908||11560661||57.10%||126245157||11387405||245297||424016||29669508||30429870||54089061||27.70%||0.80%||0.80% |- | ENCFF000LVZ||20058311||11004008||54.90%||120506134||9791631||242794||448332||25470114||28547035||56006228||27.80%||0.80%||0.80% |- | ENCFF000LWT||19407909||10887950||56.10%||119134111||9343415||203527||331921||25331949||28315688||55607611||26.90%||0.70%||0.60% |- | ENCFF000MLD||19184685||14418868||75.20%||162604971||6716952||305536||587608||54429999||38467374||62097502||11.00%||0.80%||0.90% |- | ENCFF000MLP||18102015||10032177||55.40%||109233809||10521402||215592||370895||25497472||26468867||46159581||29.20%||0.80%||0.80% |- | ENCFF000LVJ||17856591||11324104||63.40%||102317536||8591993||307833||1097735||17653908||24511494||50154573||32.70%||1.20%||2.10% |- | ENCFF000MLJ||16177211||8675428||53.60%||94678187||9430914||200777||318563||22135878||23265164||39326891||29.90%||0.90%||0.80% |- | ENCFF000LUN||10913594||3643074||33.40%||40450477||3011262||76408||126647||9141529||9549119||18545512||24.80%||0.80%||0.70% |- | ENCFF000MLM||5127779||2931330||57.20%||36675584||1281379||40172||75178||10725366||8687156||15866333||10.70%||0.50%||0.50% |- | |} *MHB regions identification #!/bin/csh #PBS -N bam2MHB #PBS -q pdafm #PBS -l nodes=1:ppn=16 #PBS -l walltime=72:00:00 #PBS -o bam2MHB.log #PBS -e bam2MHB.err #PBS -V #PBS -M shihcheng.guo@gmail.com #PBS -m abe #PBS -A k4zhang-group cd /home/shg047/oasis/Haib/sortBam # samtools cat -h header.sam -o haib.merge.bam *sort.bam samtools sort -@ 16 haib.encode.merge.bam -o haib.merge.sort.bam samtools index haib.merge.sort.bam bedtools genomecov -bg -split -ibam haib.merge.sort.bam > haib.merge.bam.pool.bed awk '$4>9 { print $1"\t"$2"\t"$3}' haib.merge.bam.pool.bed | bedtools merge -d 10 -i - > haib.RD10.genomecov.bed awk '$3-$2>80 {print $1"\t"$2"\t"$3"\t"$3-$2+1}' haib.RD10.genomecov.bed > haib.RD10_80up.genomecov.bed *Statistic: haib.RD10_80up.genomecov.bed cat '''haib.RD10_80up.genomecov.bed'''|awk '{sum+=$4} END { print "N = ", NR, "Sum = ", sum, " Average = ",sum/NR}' N = 120994 Sum = 17702479 Average = 146.309 *haploinfo to MHB cd /home/shg047/oasis/Haib/sortBam /home/shg047/oasis/Haib/mhb/haib.RD10_80up.genomecov.bed /home/shg047/oasis/Haib/mhb/haib.merge.sort.bam /home/shg047/oasis/Haib/hapInfo2mld_blocks.pl ../mergedBam2hapInfo.pl ./haib.RD10_80up.genomecov.bed haib.merge.sort.bam > Haib.merge.RD10_80up.hapinfo.txt # get hapinfo ../hapInfo2mld_block.pl ./Haib.merge.RD10_80up.hapinfo.txt 0.5 > Haib.merge_RD10_80up.mld_blocks_r2-0.5.bed *MHB identified with different threshold: R-square from 0.1-0.9 /home/shg047/oasis/Haib/mhb/Haib.merge_RD10_80up.mld_blocks_r2-0.1.bed /home/shg047/oasis/Haib/mhb/Haib.merge_RD10_80up.mld_blocks_r2-0.2.bed /home/shg047/oasis/Haib/mhb/Haib.merge_RD10_80up.mld_blocks_r2-0.3.bed /home/shg047/oasis/Haib/mhb/Haib.merge_RD10_80up.mld_blocks_r2-0.4.bed /home/shg047/oasis/Haib/mhb/Haib.merge_RD10_80up.mld_blocks_r2-0.5.bed /home/shg047/oasis/Haib/mhb/Haib.merge_RD10_80up.mld_blocks_r2-0.6.bed /home/shg047/oasis/Haib/mhb/Haib.merge_RD10_80up.mld_blocks_r2-0.7.bed {|style="font-size:80%;" |align="center" style="background:#f0f0f0;"|''' R-square threshold''' |align="center" style="background:#f0f0f0;"|''' MHB counts''' |- | 0.1||14933 |- | 0.2||13367 |- | 0.3||11667 |- | 0.4||9754 |- | 0.5||8155 |- | 0.6||7683 |- | 0.7||7445 |- |} bedtools intersect -wa -u /home/shg047/oasis/Haib/mhb/Haib.merge_RD10_80up.mld_blocks_r2-0.5.bed *MHB calling based on RRBS Haib biochain data cd /home/shg047/oasis/Haib/sortBam samtools cat -h header.sam -o haib.encode.merge.bam *sort.bam samtools sort haib.encode.merge.bam -o haib.encode.merge.sort.bam samtools index haib.encode.merge.sort.bam bedtools genomecov -bg -split -ibam haib.encode.merge.sort.bam > haib.encode.merge.bam.pool.bed awk '$4>9 { print $1"\t"$2"\t"$3}' haib.encode.merge.bam.pool.bed | bedtools merge -d 10 -i - > haib.encode.RD10.genomecov.bed awk '$3-$2>80 {print $1"\t"$2"\t"$3"\t"$3-$2+1}' haib.encode.RD10.genomecov.bed > haib.encode.RD10_80up.genomecov.bed *haploinfo to MHB ../hapInfo2mld_block.pl ./Haib.merge.RD10_80up.hapinfo.txt 0.5 > Haib.merge_RD10_80up.mld_blocks_r2-0.5.bed
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