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== 141112 HiSeqRapidRun == * WGBS libraries were captured with biotin probes by Noi: [[Noi/NOTES/2014-11-4]] === Mapping results === {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Library ID''' | align="center" style="background:#f0f0f0;"|'''Total sequenced bases''' | align="center" style="background:#f0f0f0;"|'''Total trimmed bases''' | align="center" style="background:#f0f0f0;"|'''Total mapped bases''' | align="center" style="background:#f0f0f0;"|'''%trimmed''' | align="center" style="background:#f0f0f0;"|'''%mapped''' |- | SeqCap-pCancerSetA-Nov4_12||1,557,796,200||1,490,698,729||1,183,722,483||4.31%||79.41% |- | SeqCap-pCancerSetA-Nov4_13||1,956,384,000||1,875,643,861||1,441,463,344||4.13%||76.85% |- | SeqCap-pCancerSetA-Nov4_14||1,604,724,200||1,533,952,230||1,236,147,785||4.41%||80.59% |- | SeqCap-pCancerSetA-Nov4_15||1,656,779,800||1,582,578,941||1,278,421,106||4.48%||80.78% |- | SeqCap-pCancerSetA-Nov4_16||1,551,344,600||1,482,228,220||1,172,067,971||4.46%||79.07% |- | SeqCap-pCancerSetA-Nov4_18||1,544,614,600||1,475,609,077||1,188,780,824||4.47%||80.56% |- | SeqCap-pCancerSetA-Nov4_19||1,937,986,600||1,868,857,625||1,466,874,474||3.57%||78.49% |- | SeqCap-pCancerSetA-Nov4_2||1,926,683,000||1,840,697,493||1,484,110,129||4.46%||80.63% |- | SeqCap-pCancerSetA-Nov4_4||2,527,810,600||2,414,697,940||1,897,794,294||4.47%||78.59% |- | SeqCap-pCancerSetA-Nov4_5||1,685,759,800||1,617,249,202||1,293,881,116||4.06%||80.01% |- | SeqCap-pCancerSetA-Nov4_6||1,974,733,600||1,891,689,068||1,507,964,542||4.21%||79.72% |- | SeqCap-pCancerSetA-Nov4_7||1,896,482,800||1,815,259,906||1,391,697,180||4.28%||76.67% |- | SeqCap-pNormalSetA-Nov4_12||2,074,019,600||1,975,408,680||1,571,662,041||4.75%||79.56% |- | SeqCap-pNormalSetA-Nov4_13||1,740,938,000||1,658,485,066||1,318,975,132||4.74%||79.53% |- | SeqCap-pNormalSetA-Nov4_14||1,867,843,400||1,781,077,564||1,442,362,287||4.65%||80.98% |- | SeqCap-pNormalSetA-Nov4_15||2,428,342,400||2,310,196,699||1,966,339,089||4.87%||85.12% |- | SeqCap-pNormalSetA-Nov4_16||1,922,011,000||1,827,491,168||1,448,437,222||4.92%||79.26% |- | SeqCap-pNormalSetA-Nov4_18||1,750,909,200||1,663,264,442||1,302,849,113||5.01%||78.33% |- | SeqCap-pNormalSetA-Nov4_19||1,714,868,600||1,634,401,979||1,295,380,595||4.69%||79.26% |- | SeqCap-pNormalSetA-Nov4_2||1,723,048,400||1,647,260,004||1,359,456,644||4.40%||82.53% |- | SeqCap-pNormalSetA-Nov4_4||2,111,874,400||2,013,677,234||1,563,440,813||4.65%||77.64% |- | SeqCap-pNormalSetA-Nov4_5||1,505,598,000||1,436,744,421||1,126,814,306||4.57%||78.43% |- | SeqCap-pNormalSetA-Nov4_6||1,731,991,400||1,653,646,386||1,314,038,490||4.52%||79.46% |- | SeqCap-pNormalSetA-Nov4_7||1,953,762,600||1,863,167,423||1,495,812,111||4.64%||80.28% |} ===Scripts=== * Go.trimReads.sh <nowiki>reads_dir="/oasis/tscc/scratch/ddiep/Working/141117_RRBS/Reads" cur_dir=`pwd` cope="/home/ddiep/softwares/cope-src-v1.1.3/src/cope -o connect.fq -2 left1.fq -3 left2.fq -m 0" #THIS ONE for PE trim="/home/ddiep/softwares/trim_galore_latest/trim_galore --phred33 --paired --dont_gzip -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -a2 AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT" #cd $reads_dir #FILES=`ls *R1_001.fastq` cd $cur_dir for f in Sample_NP-SeqCap-pCancerSetA-Nov4_2 Sample_NP-SeqCap-pNormalSetA-Nov4_12 Sample_NP-SeqCap-pNormalSetA-Nov4_13 do n=`echo $f | sed 's/Sample_NP-//g'` echo "#!/bin/csh" > $n.job echo "#PBS -l nodes=1:ppn=2" >> $n.job echo "#PBS -l walltime=3:00:00" >> $n.job echo "#PBS -o $n.log" >> $n.job echo "#PBS -e $n.err" >> $n.job echo "#PBS -V" >> $n.job echo "#PBS -M diep.hue.dinh@gmail.com" >> $n.job echo "#PBS -m abe" >> $n.job echo "#PBS -A k4zhang-group" >> $n.job echo "cd /state/partition1/\$USER/\$PBS_JOBID" >> $n.job echo "zcat $reads_dir/$f/*_R1_*gz > $n.R1.fastq" >> $n.job echo "zcat $reads_dir/$f/*_R2_*gz > $n.R2.fastq" >> $n.job echo "$trim $n.R1.fastq $n.R2.fastq" >> $n.job echo "cp *fq $cur_dir/" >> $n.job echo "cp *txt $cur_dir/" >> $n.job qsub -q hotel $n.job done </nowiki> * Go.Map.sh <nowiki>#===Change the following paths===# cur_dir=`pwd` scripts_dir="/oasis/tscc/scratch/ddiep/BisReadMapper/src" reads_dir="$cur_dir/Trimmed_reads" # reference files ref_dir="/oasis/tscc/scratch/ddiep" ref_fai="$ref_dir/bisHg19_plusLambda/hg19_lambda.fa.fai" template_fwd="$ref_dir/bisHg19_plusLambda/hg19_lambda.bis.CT" template_rev="$ref_dir/bisHg19_plusLambda/hg19_lambda.bis.GA" # softwares bwa="/home/ddiep/softwares/bwa-0.7.5a/bwa" #=== List FASTQ to process ===# cd $reads_dir FILES=`ls *val_1.fq` #FILES="s_1_1_ILMN_Indx01.cope.trimmed.fq" cd $cur_dir #===Begin===# for f in ${FILES} do n=`echo $f | sed 's/.R1_val_1.fq//g'` mkdir $cur_dir/$n cd $cur_dir/$n #1) Run mapper: echo "#!/bin/csh" > $n.job echo "#PBS -l nodes=1:ppn=4" >> $n.job echo "#PBS -l walltime=14:00:00" >> $n.job echo "#PBS -o $n.log" >> $n.job echo "#PBS -e $n.err" >> $n.job echo "#PBS -V" >> $n.job echo "#PBS -M diep.hue.dinh@gmail.com" >> $n.job echo "#PBS -m abe" >> $n.job echo "#PBS -A k4zhang-group" >> $n.job echo "cd /state/partition1/\$USER/\$PBS_JOBID" >> $n.job echo "cat $reads_dir/${n}.R1_val_1.fq $reads_dir/${n}.R2_val_2.fq > reads.fq" >> $n.job echo "$scripts_dir/BisReadMapper.pl -r reads.fq -W $template_fwd -C $template_rev -g $ref_fai -a $bwa -b 64 -p 4 -n $n > $n.status" >> $n.job echo "cp *sam $cur_dir/$n" >> $n.job echo "cp *status $cur_dir/$n" >> $n.job echo "ls *sorted.sam > $cur_dir/list_sams" >> $n.job echo "$scripts_dir/BamExtractor.pl -i $cur_dir/list_sams -s $cur_dir/list_paths_Hg19 -o $n -r none -v no -b no -p no -d 5" >> $n.job echo "cp *bam $cur_dir/$n" >> $n.job echo "cp *methylFreq $cur_dir/$n" >> $n.job qsub -q hotel $n.job done #===End===# </nowiki> ===Assessment of capturing performance=== {| class="wikitable" | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''total_bases_coverred''' | align="center" style="background:#f0f0f0;"|'''total_bases''' | align="center" style="background:#f0f0f0;"|'''on_target_bases_covered''' | align="center" style="background:#f0f0f0;"|'''on_target_bases''' | align="center" style="background:#f0f0f0;"|'''Specificity''' | align="center" style="background:#f0f0f0;"|'''target_coverred''' | align="center" style="background:#f0f0f0;"|'''Enrichment_factor''' |- | SeqCap-pCancerSetA-Nov4_12||138,931,528||1,168,877,279||5,883,919||450,558,243||38.5%||20.0%||63 |- | SeqCap-pCancerSetA-Nov4_13||369,134,874||1,424,314,151||21,209,194||614,835,055||43.2%||72.2%||77 |- | SeqCap-pCancerSetA-Nov4_14||302,700,391||1,203,488,680||13,952,014||489,036,528||40.6%||47.5%||69 |- | SeqCap-pCancerSetA-Nov4_15||277,562,818||1,250,329,814||12,577,032||511,943,961||40.9%||42.8%||70 |- | SeqCap-pCancerSetA-Nov4_16||274,256,170||1,164,394,023||12,418,863||474,550,198||40.8%||42.3%||70 |- | SeqCap-pCancerSetA-Nov4_18||403,128,602||1,170,296,929||23,073,184||461,126,140||39.4%||78.5%||66 |- | SeqCap-pCancerSetA-Nov4_19||385,167,143||1,417,434,873||22,614,122||614,480,251||43.4%||77.0%||77 |- | SeqCap-pCancerSetA-Nov4_2||354,784,810||1,514,708,612||19,490,111||751,025,646||49.6%||66.3%||99 |- | SeqCap-pCancerSetA-Nov4_4||352,108,468||2,000,885,721||23,609,719||1,141,652,594||57.1%||80.3%||134 |- | SeqCap-pCancerSetA-Nov4_5||196,108,966||1,300,234,129||8,246,274||563,004,318||43.3%||28.1%||77 |- | SeqCap-pCancerSetA-Nov4_6||450,502,450||1,502,032,451||28,135,196||645,300,745||43.0%||95.7%||76 |- | SeqCap-pCancerSetA-Nov4_7||226,755,475||1,432,933,860||10,016,683||629,536,922||43.9%||34.1%||79 |- | SeqCap-pNormalSetA-Nov4_12||368,272,742||1,450,833,379||32,791,421||853,947,465||58.9%||111.6%||145 |- | SeqCap-pNormalSetA-Nov4_13||330,611,335||1,291,478,405||29,764,564||756,289,960||58.6%||101.3%||143 |- | SeqCap-pNormalSetA-Nov4_14||360,000,998||1,364,998,815||32,082,346||801,469,734||58.7%||109.2%||144 |- | SeqCap-pNormalSetA-Nov4_15||375,193,438||1,970,841,697||30,241,737||1,295,408,500||65.7%||102.9%||194 |- | SeqCap-pNormalSetA-Nov4_16||365,138,307||1,417,821,628||31,787,247||825,520,233||58.2%||108.2%||141 |- | SeqCap-pNormalSetA-Nov4_18||326,952,891||1,305,603,890||28,937,598||760,701,025||58.3%||98.5%||141 |- | SeqCap-pNormalSetA-Nov4_19||312,924,926||1,271,134,775||27,505,559||749,645,411||59.0%||93.6%||145 |- | SeqCap-pNormalSetA-Nov4_2||353,552,883||1,275,196,941||31,331,422||731,603,678||57.4%||106.6%||136 |- | SeqCap-pNormalSetA-Nov4_4||354,621,865||1,456,339,179||31,829,571||867,560,025||59.6%||108.3%||149 |- | SeqCap-pNormalSetA-Nov4_5||291,891,632||1,066,253,299||27,061,797||618,610,388||58.0%||92.1%||140 |- | SeqCap-pNormalSetA-Nov4_6||333,830,242||1,234,362,498||31,357,436||723,278,371||58.6%||106.7%||143 |- | SeqCap-pNormalSetA-Nov4_7||377,475,888||1,451,628,039||32,325,421||843,343,133||58.1%||110.0%||140 |- | |} === Assessing the complexity of each library === * Count the number of starting positions in each BAM file in non-overlapping 50 bp bins * Normalize each bin count by per million mapped reads * Plot the density for each file. Y-axis is the frequency of each count, X-axis is the log2(normalized counts). * Normal samples have greater complexity because most bins have fewer counts whereas cancer have more bins with higher counts (more reads covering same bins). * Cancer samples [[File:141112_HiSeqRapidRun_cancer_samples_50bp_coverage.png]] * Normal samples [[File:141112_HiSeqRapidRun_normal_samples_50bp_coverage.png]]
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