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==MONOD Round 2== ===Target identification=== *Data use: **Cancer data: same as Round 1 **Placenta data: ENCODE RRBS (BC_Placenta_UHN00189); WGBS GSE39775 (I downloaded the raw reads and repeated the mapping to hg19) **Whole blood data: Round 1 data, plus 32 UCLA_SZ BSPP330k data. ./[[Media:find_fetal_DMS_v2.txt|find_fetal_DMS_v2.pl]] > fetal_DMS_v2.txt ./[[Media:find_DMS_PANC_GBM_v2.txt|find_DMS_PANC_GBM_v2.pl]] > PANC_GBM_DMS_v2.txt ./[[Media:extract_clusters.txt|extract_clusters.pl]] fetal_DMS_v2.txt > [[Media:fetal_DMS_clusters_v2.txt|fetal_DMS_clusters_v2.txt]] ./[[Media:extract_clusters.txt|extract_clusters.pl]] PANC_GBM_DMS_v2.txt > [[Media:PANC_GBM_DMS_clusters_v2.txt|PANC_GBM_DMS_clusters_v2.txt]] ===Probe design=== *I designed probes for both the plus and minus strands to achieve a better coverage. In addition, I added a requirement that no CpG is allowed in H1/H2 annealing region to avoid methylation dependent capturing bias. Gap size: 125-175bp. **GBM_PC probes: [[Media:jobFile_GBM_PC_DMR_150_minus.txt|jobFile_GBM_PC_DMR_150_minus.pl]]; [[Media:jobFile_GBM_PC_DMR_150_plus.txt|jobFile_GBM_PC_DMR_150_plus.pl]] **Fetal probes: [[Media:jobFile_fetalDMR_150_minus.txt|jobFile_fetalDMR_150_minus.pl]]; [[Media:jobFile_fetalDMR_150_plus.txt|jobFile_fetalDMR_150_plus.pl]] *Probe filtering and selection. **Fetal probes: 173,034 probes were designed for 54,660 targets (39,517 targets failed). After filtering probes that have less than 4 CpG sites in the captured regions, I got 159,874 probes. I kept all probes in chr8,9,13,18,21,22; for the other chromosomes, randomly sample enough to fill the order. **GBM_PC probes: 23,707 probes were designed for 13,181 targets. After removing probes that either overlap with the fetal probe set or having fewer than 4 CpG sites in the captured region, I obtained 18,104 probes. *In total, I included 18,104 probes for GBM_PANC, 65343 for the fetal DMR in the order. I used the 8bp UMI. These oligos are in the DMR540k set F, and should be amplified by the V6 primer set. ===Capture & sequencing=== *Noi performed the [[Noi/NOTES/2014-3-2|experiment]], and the sequencing libraries were sequenced in the 140310_MiSeq run. ===Data analysis=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''# reads''' | align="center" style="background:#f0f0f0;"|'''# mapped reads''' | align="center" style="background:#f0f0f0;"|'''% mapping''' | align="center" style="background:#f0f0f0;"|'''# on-target reads''' | align="center" style="background:#f0f0f0;"|'''% on-target''' | align="center" style="background:#f0f0f0;"|'''# unique reads''' | align="center" style="background:#f0f0f0;"|'''Clonal rate''' |- | 1,346,769||937,785||69.60%||35724||3.8%||35521||0.6% |- | 1,092,716||716,930||65.60%||28539||4.0%||28441||0.3% |- | 1,172,030||786,617||67.10%||27690||3.5%||27641||0.2% |- | 1,291,158||864,157||66.90%||34947||4.0%||34781||0.5% |- | 1,110,997||687,345||61.90%||27167||4.0%||27045||0.4% |- | 1,243,264||811,064||65.20%||32016||3.9%||31963||0.2% |- | 1,425,464||880,798||61.80%||36412||4.1%||36209||0.6% |- | 2,172,772||1,476,496||68.00%||64276||4.4%||63603||1.0% |- | 1,345,928||757,309||56.30%||26866||3.5%||25971||3.3% |- | 1,307,927||808,708||61.80%||33887||4.2%||33216||2.0% |- | 1,906,290||1,070,550||56.20%||39475||3.7%||36852||6.6% |- | |}
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