Kun:LabNotes/MONOD/2014-1-17
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MONOD Round 2[edit]
Target identification[edit]
- 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.
./find_fetal_DMS_v2.pl > fetal_DMS_v2.txt ./find_DMS_PANC_GBM_v2.pl > PANC_GBM_DMS_v2.txt
./extract_clusters.pl fetal_DMS_v2.txt > fetal_DMS_clusters_v2.txt ./extract_clusters.pl PANC_GBM_DMS_v2.txt > PANC_GBM_DMS_clusters_v2.txt
Probe design[edit]
- 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: jobFile_GBM_PC_DMR_150_minus.pl; jobFile_GBM_PC_DMR_150_plus.pl
- Fetal probes: jobFile_fetalDMR_150_minus.pl; 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[edit]
- Noi performed the experiment, and the sequencing libraries were sequenced in the 140310_MiSeq run.
Data analysis[edit]
# reads | # mapped reads | % mapping | # on-target reads | % on-target | # unique reads | 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% |